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Okamoto K, Mihara Y, Ogasawara S, Murakami T, Ohmori S, Mori T, Umata T, Kawasaki Y, Hirano K, Yano H, Tsuneoka M. Interaction Between PHF8 and a Segment of KDM2A, Which Is Controlled by the Phosphorylation Status at a Specific Serine in an Intrinsically Disordered Region of KDM2A, Regulates rRNA Transcription and Cell Proliferation in a Breast Cancer Cell Line. Biomolecules 2025; 15:661. [PMID: 40427554 PMCID: PMC12109296 DOI: 10.3390/biom15050661] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2025] [Revised: 04/24/2025] [Accepted: 04/29/2025] [Indexed: 05/29/2025] Open
Abstract
Mild starvation due to low concentrations of an inhibitor of glycolysis, 2-deoxy-D-glucose, activates AMP-activated protein kinase (AMPK) and lysine-specific demethylase 2A (KDM2A) to reduce rRNA transcription and cell proliferation in breast cancer cells. However, the mechanisms of how AMPK regulates KDM2A are unknown. Here, we found that PHD finger protein 8 (PHF8) interacted with KDM2A and contributed to the reduction in rRNA transcription and cell proliferation by 2-deoxy-D-glucose in a breast cancer cell line, MCF-7. We analyzed how KDM2A bound PHF8 in detail and found that PHF8 interacted with KDM2A via two regions of KDM2A. One of the regions contained an intrinsically disordered region (IDR). IDRs can show rapidly switchable protein-protein interactions. Deletion of the PHF8-binding region activated KDM2A to reduce rRNA transcription, and 2-deoxy-D-glucose reduced the interaction between PHF8 and the KDM2A fragment containing the PHF8-binding region. A 2-deoxy-D-glucose or AMPK activator dephosphorylated KDM2A at Ser731, which is located on the N-terminal side of the PHF8-binding region. Replacement of Ser731 by Ala decreased binding of PHF8 to the KDM2A fragment that contains the PHF8-binding region and Ser731 and reduced rRNA transcription and cell proliferation. These results suggest that the mode of interaction between KDM2A and PHF8 is regulated via dephosphorylation of KDM2A through AMPK to control rRNA transcription, and control of the phosphorylation state of Ser731 would be a novel target for breast cancer therapy.
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Affiliation(s)
- Kengo Okamoto
- Faculty of Agriculture, Department of Applied Biological Science, Takasaki University of Health and Welfare, Takasaki 370-0033, Gunma, Japan;
| | - Yutaro Mihara
- Department of Pathology, School of Medicine, Kurume University, Kurume 830-0011, Fukuoka, Japan; (Y.M.); (S.O.); (H.Y.)
| | - Sachiko Ogasawara
- Department of Pathology, School of Medicine, Kurume University, Kurume 830-0011, Fukuoka, Japan; (Y.M.); (S.O.); (H.Y.)
| | - Takashi Murakami
- Department of Microbiology, Faculty of Medicine, Saitama Medical University, Iruma 350-1241, Saitama, Japan;
| | - Sinya Ohmori
- Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki 370-0033, Gunma, Japan; (S.O.); (T.M.); (Y.K.); (K.H.)
| | - Tetsuya Mori
- Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki 370-0033, Gunma, Japan; (S.O.); (T.M.); (Y.K.); (K.H.)
| | - Toshiyuki Umata
- Radioisotope Research Center, Facility for Education and Research Support, University of Occupational and Environmental Health, Kitakyushu 807-8555, Fukuoka, Japan;
| | - Yuki Kawasaki
- Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki 370-0033, Gunma, Japan; (S.O.); (T.M.); (Y.K.); (K.H.)
| | - Kazuya Hirano
- Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki 370-0033, Gunma, Japan; (S.O.); (T.M.); (Y.K.); (K.H.)
| | - Hirohisa Yano
- Department of Pathology, School of Medicine, Kurume University, Kurume 830-0011, Fukuoka, Japan; (Y.M.); (S.O.); (H.Y.)
| | - Makoto Tsuneoka
- Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki 370-0033, Gunma, Japan; (S.O.); (T.M.); (Y.K.); (K.H.)
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Rodriguez‐Algarra F, Whittaker E, del Castillo del Rio S, Rakyan VK. Assessing Human Ribosomal DNA Variation and Its Association With Phenotypic Outcomes. Bioessays 2025; 47:e202400232. [PMID: 39834111 PMCID: PMC11931683 DOI: 10.1002/bies.202400232] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2024] [Revised: 12/18/2024] [Accepted: 01/07/2025] [Indexed: 01/22/2025]
Abstract
Although genome-scale analyses have provided insights into the connection between genetic variability and complex human phenotypes, much trait variation is still not fully understood. Genetic variation within repetitive elements, such as the multi-copy, multi-locus ribosomal DNA (rDNA), has emerged as a potential contributor to trait variation. Whereas rDNA was long believed to be largely uniform within a species, recent studies have revealed substantial variability in the locus, both within and across individuals. This variation, which takes the form of copy number, structural arrangement, and sequence differences, has been found to be associated with human phenotypes. This review summarizes what is currently known about human rDNA variation, its causes, and its association with phenotypic outcomes, highlighting the technical challenges the field faces and the solutions proposed to address them. Finally, we suggest experimental approaches that can help clarify the elusive mechanisms underlying the phenotypic consequences of rDNA variation.
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Affiliation(s)
| | - Elliott Whittaker
- The Blizard InstituteSchool of Medicine and DentistryQueen Mary University of LondonLondonUK
| | | | - Vardhman K. Rakyan
- The Blizard InstituteSchool of Medicine and DentistryQueen Mary University of LondonLondonUK
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Hall AN, Morton EA, Walters R, Cuperus JT, Queitsch C. Phenotypic tolerance for rDNA copy number variation within the natural range of C. elegans. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.21.644675. [PMID: 40196474 PMCID: PMC11974728 DOI: 10.1101/2025.03.21.644675] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/09/2025]
Abstract
The genes for ribosomal RNA (rRNA) are encoded by ribosomal DNA (rDNA), whose structure is notable for being present in arrays of tens to thousands of tandemly repeated copies in eukaryotic genomes. The exact number of rDNA copies per genome is highly variable within a species, with differences between individuals measuring in potentially hundreds of copies and megabases of DNA. The extent to which natural variation in rDNA copy number impacts whole-organism phenotypes such as fitness and lifespan is poorly understood, in part due to difficulties in manipulating such large and repetitive tracts of DNA even in model organisms. Here, we used the natural resource of copy number variation in C. elegans wild isolates to generate new tools and investigated the phenotypic consequences of this variation. Specifically, we generated a panel of recombinant inbred lines (RILs) using a laboratory strain derivative with ∼130 haploid rDNA copies and a wild isolate with ∼417 haploid rDNA copies, one of the highest validated C. elegans rDNA copy number arrays. We find that rDNA copy number is stable in the RILs, rejecting prior hypotheses that predicted copy number instability and copy number reversion. To isolate effects of rDNA copy number on phenotype, we produced a series of near isogenic lines (NILs) with rDNA copy numbers representing the high and low end of the rDNA copy number spectrum in C. elegans wild isolates. We find no correlation between rDNA copy number and phenotypes of rRNA abundance, competitive fitness, early life fertility, lifespan, or global transcriptome under standard laboratory conditions. These findings demonstrate a remarkable ability of C. elegans to tolerate substantial variation in a locus critical to fundamental cell function. Our study provides strain resources for future investigations into the boundaries of this tolerance.
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Tran TTQ, Do TH, Pham TT, Luu PTT, Pham OM, Nguyen UQ, Vuong LD, Nguyen QN, Mai TV, Ho SV, Nguyen TT, Vo LTT. Hypermethylation at 45S rDNA promoter in cancers. PLoS One 2025; 20:e0311085. [PMID: 39775079 PMCID: PMC11706406 DOI: 10.1371/journal.pone.0311085] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2024] [Accepted: 09/11/2024] [Indexed: 01/11/2025] Open
Abstract
The ribosomal genes (rDNA genes) encode 47S rRNA which accounts for up to 80% of all cellular RNA. At any given time, no more than 50% of rDNA genes are actively transcribed, and the other half is silent by forming heterochromatin structures through DNA methylation. In cancer cells, upregulation of ribosome biogenesis has been recognized as a hallmark feature, thus, the reduced methylation of rDNA promoter has been thought to support conformational changes of chromatin accessibility and the subsequent increase in rDNA transcription. However, an increase in the heterochromatin state through rDNA hypermethylation can be a protective mechanism teetering on the brink of a threshold where cancer cells rarely successfully proliferate. Hence, clarifying hypo- or hypermethylation of rDNA will unravel its additional cellular functions, including organization of genome architecture and regulation of gene expression, in response to growth signaling, cellular stressors, and carcinogenesis. Using the bisulfite-based quantitative real-time methylation-specific PCR (qMSP) method after ensuring unbiased amplification and complete bisulfite conversion of the minuscule DNA amount of 1 ng, we established that the rDNA promoter was significantly hypermethylated in 107 breast, 65 lung, and 135 colon tumour tissue samples (46.81%, 51.02% and 96.60%, respectively) as compared with their corresponding adjacent normal samples (26.84%, 38.26% and 77.52%, respectively; p < 0.0001). An excessive DNA input of 1 μg resulted in double-stranded rDNA remaining unconverted even after bisulfite conversion, hence the dramatic drop in the single-stranded DNA that strictly required for bisulfite conversion, and leading to an underestimation of rDNA promoter methylation, in other words, a faulty hypomethylation status of the rDNA promoter. Our results are in line with the hypothesis that an increase in rDNA methylation is a natural pathway protecting rDNA repeats that are extremely sensitive to DNA damage in cancer cells.
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Affiliation(s)
- Trang Thi Quynh Tran
- Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam
- VNU Institute of Microbiology and Biotechnology
| | - Trang Hien Do
- Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam
| | - Tung The Pham
- Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam
| | - Phương Thi Thu Luu
- Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam
| | - Oanh Minh Pham
- Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam
| | | | | | | | | | - Son Van Ho
- Department of Chemistry, 175 Hospital, Ho Chi Minh City, Vietnam
| | - Than Thi Nguyen
- Department of Chemistry, 175 Hospital, Ho Chi Minh City, Vietnam
| | - Lan Thi Thuong Vo
- Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam
- VNU Institute of Microbiology and Biotechnology
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Poma P, Rigogliuso S, Labbozzetta M, Nicosia A, Costa S, Ragusa MA, Notarbartolo M. Epigenetic and Cellular Reprogramming of Doxorubicin-Resistant MCF-7 Cells Treated with Curcumin. Int J Mol Sci 2024; 25:13416. [PMID: 39769180 PMCID: PMC11679585 DOI: 10.3390/ijms252413416] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2024] [Revised: 12/10/2024] [Accepted: 12/11/2024] [Indexed: 01/11/2025] Open
Abstract
The MCF-7R breast cancer cell line, developed by treating the parental MCF-7 cells with increasing doses of doxorubicin, serves as a model for studying acquired multidrug resistance (MDR). MDR is a major challenge in cancer therapy, often driven by overexpression of the efflux pump P-glycoprotein (P-gp) and epigenetic modifications. While many P-gp inhibitors show promise in vitro, their nonspecific effects on the efflux pump limit in vivo application. Curcumin, a natural compound with pleiotropic action, is a nontoxic P-gp inhibitor capable of modulating multiple pathways. To explore curcumin's molecular effects on MCF-7R cells, we analyzed the expression of genes involved in DNA methylation and transcription regulation, including ABCB1/MDR1. Reduced representation bisulfite sequencing further unveiled key epigenetic changes induced by curcumin. Our findings indicate that curcumin treatment not only modulates critical cellular processes, such as ribosome biogenesis and cytoskeletal dynamics, but also reverses the resistant phenotype, toward that of sensitive cells. This study highlights curcumin's potential as an adjuvant therapy to overcome chemoresistance, offering new avenues for pharmacological strategies targeting epigenetic regulation to re-sensitize resistant cancer cells.
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Affiliation(s)
- Paola Poma
- Department of Biological Chemical and Pharmaceutical Science and Technology (STEBICEF), University of Palermo, 90128 Palermo, Italy; (P.P.); (S.R.); (M.L.); (S.C.); (M.N.)
| | - Salvatrice Rigogliuso
- Department of Biological Chemical and Pharmaceutical Science and Technology (STEBICEF), University of Palermo, 90128 Palermo, Italy; (P.P.); (S.R.); (M.L.); (S.C.); (M.N.)
| | - Manuela Labbozzetta
- Department of Biological Chemical and Pharmaceutical Science and Technology (STEBICEF), University of Palermo, 90128 Palermo, Italy; (P.P.); (S.R.); (M.L.); (S.C.); (M.N.)
| | - Aldo Nicosia
- Institute for Biomedical Research and Innovation—National Research Council (IRIB-CNR), 90146 Palermo, Italy;
| | - Salvatore Costa
- Department of Biological Chemical and Pharmaceutical Science and Technology (STEBICEF), University of Palermo, 90128 Palermo, Italy; (P.P.); (S.R.); (M.L.); (S.C.); (M.N.)
| | - Maria Antonietta Ragusa
- Department of Biological Chemical and Pharmaceutical Science and Technology (STEBICEF), University of Palermo, 90128 Palermo, Italy; (P.P.); (S.R.); (M.L.); (S.C.); (M.N.)
| | - Monica Notarbartolo
- Department of Biological Chemical and Pharmaceutical Science and Technology (STEBICEF), University of Palermo, 90128 Palermo, Italy; (P.P.); (S.R.); (M.L.); (S.C.); (M.N.)
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6
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Blokhina Y, Buchwalter A. Modeling the consequences of age-linked rDNA hypermethylation with dCas9-directed DNA methylation in human cells. PLoS One 2024; 19:e0310626. [PMID: 39666677 PMCID: PMC11637357 DOI: 10.1371/journal.pone.0310626] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2024] [Accepted: 09/03/2024] [Indexed: 12/14/2024] Open
Abstract
Ribosomal DNA (rDNA) genes encode the structural RNAs of the ribosome and are present in hundreds of copies in mammalian genomes. Age-linked DNA hypermethylation throughout the rDNA constitutes a robust "methylation clock" that accurately reports age, yet the consequences of hypermethylation on rDNA function are unknown. We confirmed that pervasive hypermethylation of rDNA occurs during mammalian aging and senescence while rDNA copy number remains stable. We found that DNA methylation is exclusively found on the promoters and gene bodies of inactive rDNA. To model the effects of age-linked methylation on rDNA function, we directed de novo DNA methylation to the rDNA promoter or gene body with a nuclease-dead Cas9 (dCas9)-DNA methyltransferase fusion enzyme in human cells. Hypermethylation at each target site had no detectable effect on rRNA transcription, nucleolar morphology, or cellular growth rate. Instead, human UBF and Pol I remain bound to rDNA promoters in the presence of increased DNA methylation. These data suggest that promoter methylation is not sufficient to impair transcription of the human rDNA and imply that the human rDNA transcription machinery may be resilient to age-linked rDNA hypermethylation.
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Affiliation(s)
- Yana Blokhina
- Cardiovascular Research Institute and Department of Physiology, University of California, San Francisco, San Francisco, California, United States of America
| | - Abigail Buchwalter
- Cardiovascular Research Institute and Department of Physiology, University of California, San Francisco, San Francisco, California, United States of America
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7
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Li HY, Wang M, Jiang X, Jing Y, Wu Z, He Y, Yan K, Sun S, Ma S, Ji Z, Wang S, Belmonte JC, Qu J, Zhang W, Wei T, Liu GH. CRISPR screening uncovers nucleolar RPL22 as a heterochromatin destabilizer and senescence driver. Nucleic Acids Res 2024; 52:11481-11499. [PMID: 39258545 PMCID: PMC11514463 DOI: 10.1093/nar/gkae740] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Revised: 08/02/2024] [Accepted: 08/16/2024] [Indexed: 09/12/2024] Open
Abstract
Dysfunction of the ribosome manifests during cellular senescence and contributes to tissue aging, functional decline, and development of aging-related disorders in ways that have remained enigmatic. Here, we conducted a comprehensive CRISPR-based loss-of-function (LOF) screen of ribosome-associated genes (RAGs) in human mesenchymal progenitor cells (hMPCs). Through this approach, we identified ribosomal protein L22 (RPL22) as the foremost RAG whose deficiency mitigates the effects of cellular senescence. Consequently, absence of RPL22 delays hMPCs from becoming senescent, while an excess of RPL22 accelerates the senescence process. Mechanistically, we found in senescent hMPCs, RPL22 accumulates within the nucleolus. This accumulation triggers a cascade of events, including heterochromatin decompaction with concomitant degradation of key heterochromatin proteins, specifically heterochromatin protein 1γ (HP1γ) and heterochromatin protein KRAB-associated protein 1 (KAP1). Subsequently, RPL22-dependent breakdown of heterochromatin stimulates the transcription of ribosomal RNAs (rRNAs), triggering cellular senescence. In summary, our findings unveil a novel role for nucleolar RPL22 as a destabilizer of heterochromatin and a driver of cellular senescence, shedding new light on the intricate mechanisms underlying the aging process.
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Affiliation(s)
- Hong-Yu Li
- Key Laboratory of Biomacromolecules (CAS), National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Min Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Division of Life Sciences and Medicine, School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
| | - Xiaoyu Jiang
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Yaobin Jing
- International center for Aging and Cancer, Hainan Academy of Medical Sciences, Hainan Medical University, Haikou 571199, China
| | - Zeming Wu
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Yifang He
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Kaowen Yan
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Shuhui Sun
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Shuai Ma
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Aging Biomarker Consortium, Beijing 100101, China
| | - Zhejun Ji
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Si Wang
- Advanced Innovation Center for Human Brain Protection, and National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing, China
- Aging Translational Medicine Center, Beijing Municipal Geriatric Medical Research Center, Xuanwu Hospital, Capital Medical University, Beijing, China
- Aging Biomarker Consortium, Beijing 100101, China
| | | | - Jing Qu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Aging Biomarker Consortium, Beijing 100101, China
| | - Weiqi Zhang
- CAS key laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences and China National Center for Bioinformation, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Aging Biomarker Consortium, Beijing 100101, China
| | - Taotao Wei
- Key Laboratory of Biomacromolecules (CAS), National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Guang-Hui Liu
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- International center for Aging and Cancer, Hainan Academy of Medical Sciences, Hainan Medical University, Haikou 571199, China
- Advanced Innovation Center for Human Brain Protection, and National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing, China
- Aging Translational Medicine Center, Beijing Municipal Geriatric Medical Research Center, Xuanwu Hospital, Capital Medical University, Beijing, China
- Aging Biomarker Consortium, Beijing 100101, China
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Bi X. Hmo1: A versatile member of the high mobility group box family of chromosomal architecture proteins. World J Biol Chem 2024; 15:97938. [PMID: 39156122 PMCID: PMC11325855 DOI: 10.4331/wjbc.v15.i1.97938] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/13/2024] [Revised: 07/21/2024] [Accepted: 08/01/2024] [Indexed: 08/08/2024] Open
Abstract
Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures, which is facilitated by linker histone H1. Formation of chromatin compacts and protects the genome, but also hinders DNA transactions. Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions. The high mobility group box (HMGB) proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion. They play a major role in chromatin dynamics. The Saccharomyces cerevisiae (yeast hereafter) HMGB protein Hmo1 contains two HMGB motifs. However, unlike a canonical HMGB protein that has an acidic C-terminus, Hmo1 ends with a lysine rich, basic, C-terminus, resembling linker histone H1. Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions. For instance, Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones. Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome. This minireview reviews the functions of Hmo1 and the underlying mechanisms, highlighting recent discoveries.
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Affiliation(s)
- Xin Bi
- Department of Biology, University of Rochester, Rochester, NY 14627, United States
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9
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Elguweidi A, Crease T. Copy number and sequence variation in rDNA of Daphnia pulex from natural populations: insights from whole-genome sequencing. G3 (BETHESDA, MD.) 2024; 14:jkae105. [PMID: 38771699 PMCID: PMC11228840 DOI: 10.1093/g3journal/jkae105] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/17/2024] [Revised: 02/17/2024] [Accepted: 05/10/2024] [Indexed: 05/23/2024]
Abstract
Ribosomal DNA (rDNA) has a vital role in ribosome biogenesis as it contains the genes that encode ribosomal RNA (rRNA) separated by intergenic spacers (IGSs). The rRNA genes occur in hundreds to tens of thousands of copies per haploid genome in eukaryotes and are generally highly conserved with low variation within species. Due to the repetitive nature and large size of rDNA arrays, detecting intraindividual variation can be difficult. In this study, we use whole-genome sequences of 169 Daphnia pulex individuals from 10 natural populations to measure the copy number and sequence variation in rDNA. This revealed that variation in rDNA copy number between individuals spans an order of magnitude. We further observed a substantial level of sequence variation within individual genomes. As expected, single-nucleotide polymorphisms occurred in regions of lower functional constraint such as the IGS and expansion segments of the rRNA genes. The presence of strong linkage disequilibrium among variants facilitated identification of haplotypes within each population. Although there was evidence of recombination among haplotypes from different populations, it is insufficient to eliminate linkage disequilibrium within populations. Estimating copy number and haplotype diversity within individuals revealed that the level of intraindividual sequence variation is not strongly correlated with copy number. The observed patterns of variation highlight a complex evolutionary history of rDNA in D. pulex. Future research should explore the functional implications of rDNA copy number and sequence variation on organismal phenotypes.
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Affiliation(s)
- Abir Elguweidi
- Department of Integrative Biology, University of Guelph, Guelph, ON, N1G2W1, Canada
| | - Teresa Crease
- Department of Integrative Biology, University of Guelph, Guelph, ON, N1G2W1, Canada
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10
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Correll CC, Rudloff U, Schmit JD, Ball DA, Karpova TS, Balzer E, Dundr M. Crossing boundaries of light microscopy resolution discerns novel assemblies in the nucleolus. Histochem Cell Biol 2024; 162:161-183. [PMID: 38758429 PMCID: PMC11330670 DOI: 10.1007/s00418-024-02297-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/03/2024] [Indexed: 05/18/2024]
Abstract
The nucleolus is the largest membraneless organelle and nuclear body in mammalian cells. It is primarily involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and accounts for the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the substructural mechanistic principles of the nucleolar function in preribosome biogenesis have only recently begun to emerge. Here, we provide a new perspective using advanced super-resolution microscopy and single-molecule MINFLUX nanoscopy on the mechanistic principles governing ribosomal RNA-seeded nucleolar formation and the resulting tripartite suborganization of the nucleolus driven, in part, by liquid-liquid phase separation. With recent advances in the cryogenic electron microscopy (cryoEM) structural analysis of ribosome biogenesis intermediates, we highlight the current understanding of the step-wise assembly of preribosomal subunits in the nucleolus. Finally, we address how novel anticancer drug candidates target early steps in ribosome biogenesis to exploit these essential dependencies for growth arrest and tumor control.
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Affiliation(s)
- Carl C Correll
- Center for Proteomics and Molecular Therapeutics and Biochemistry and Molecular Biology, Chicago Medical School, Rosalind Franklin University of Medicine & Science, North Chicago, IL, 60064, USA
| | - Udo Rudloff
- Rare Tumor Initiative, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Jeremy D Schmit
- Department of Physics, Kansas State University, Manhattan, KS, 66506, USA
| | - David A Ball
- Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Tatiana S Karpova
- Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Eric Balzer
- Nikon Instruments Inc., Melville, NY, 11747, USA
| | - Miroslav Dundr
- Rare Tumor Initiative, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
- Center for Cancer Cell Biology, Chicago Medical School, Rosalind Franklin University of Medicine & Science, North Chicago, IL, 60064, USA.
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11
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Diamantopoulos MA, Georgoulia KK, Levis P, Kotronopoulos G, Stravodimos K, Kontos CK, Avgeris M, Scorilas A. 28S rRNA-Derived Fragments Represent an Independent Molecular Predictor of Short-Term Relapse in Prostate Cancer. Int J Mol Sci 2023; 25:239. [PMID: 38203408 PMCID: PMC10779029 DOI: 10.3390/ijms25010239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2023] [Revised: 12/19/2023] [Accepted: 12/20/2023] [Indexed: 01/12/2024] Open
Abstract
Prostate cancer (PCa) is a global health concern, being a leading cause of cancer-related mortality among males. Early detection and accurate prognosis are crucial for effective management. This study delves into the diagnostic and prognostic potential of 28S rRNA-derived fragments (rRFs) in PCa. Total RNA extracted from 89 PCa and 53 benign prostate hyperplasia (BPH) tissue specimens. After 3'-end polyadenylation, we performed reverse transcription to create first-strand cDNA. Using an in-house quantitative real-time PCR (qPCR) assay, we quantified 28S rRF levels. Post-treatment biochemical relapse served as the clinical endpoint event for survival analysis, which we validated internally through bootstrap analysis. Our results revealed downregulated 28S rRF levels in PCa compared to BPH patients. Additionally, we observed a significant positive correlation between 28S rRF levels and higher Gleason scores and tumor stages. Furthermore, PCa patients with elevated 28S rRF expression had a significantly higher risk of post-treatment disease relapse independently of clinicopathological data. In conclusion, our study demonstrates, for the first time, the prognostic value of 28S rRF in prostate adenocarcinoma. Elevated 28S rRF levels independently predict short-term PCa relapse and enhance risk stratification. This establishes 28S rRF as a potential novel molecular marker for PCa prognosis.
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Affiliation(s)
- Marios A. Diamantopoulos
- Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15701 Athens, Greece; (M.A.D.); (K.K.G.); (C.K.K.); (M.A.)
| | - Konstantina K. Georgoulia
- Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15701 Athens, Greece; (M.A.D.); (K.K.G.); (C.K.K.); (M.A.)
| | - Panagiotis Levis
- First Department of Urology, “Laiko” General Hospital, School of Medicine, National and Kapodistrian University of Athens, 11527 Athens, Greece; (P.L.); (G.K.); (K.S.)
| | - Georgios Kotronopoulos
- First Department of Urology, “Laiko” General Hospital, School of Medicine, National and Kapodistrian University of Athens, 11527 Athens, Greece; (P.L.); (G.K.); (K.S.)
| | - Konstantinos Stravodimos
- First Department of Urology, “Laiko” General Hospital, School of Medicine, National and Kapodistrian University of Athens, 11527 Athens, Greece; (P.L.); (G.K.); (K.S.)
| | - Christos K. Kontos
- Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15701 Athens, Greece; (M.A.D.); (K.K.G.); (C.K.K.); (M.A.)
| | - Margaritis Avgeris
- Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15701 Athens, Greece; (M.A.D.); (K.K.G.); (C.K.K.); (M.A.)
- Laboratory of Clinical Biochemistry-Molecular Diagnostics, Second Department of Pediatrics, “P. & A. Kyriakou” Children’s Hospital, School of Medicine, National and Kapodistrian University of Athens, 11527 Athens, Greece
| | - Andreas Scorilas
- Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15701 Athens, Greece; (M.A.D.); (K.K.G.); (C.K.K.); (M.A.)
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12
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Blokhina Y, Buchwalter A. Modeling the consequences of age-linked rDNA hypermethylation with dCas9-directed DNA methylation in human cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.18.562830. [PMID: 37904963 PMCID: PMC10614900 DOI: 10.1101/2023.10.18.562830] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/02/2023]
Abstract
Ribosomal DNA (rDNA) genes encode the structural RNAs of the ribosome and are present in hundreds of copies in mammalian genomes. Age-linked DNA hypermethylation throughout the rDNA constitutes a robust "methylation clock" that accurately reports age, yet the consequences of hypermethylation on rDNA function are unknown. We confirmed that pervasive hypermethylation of rDNA occurs during mammalian aging and senescence while rDNA copy number remains stable. We found that DNA methylation is exclusively found on the promoters and gene bodies of inactive rDNA. To model the effects of age-linked methylation on rDNA function, we directed de novo DNA methylation to the rDNA promoter or gene body with a nuclease-dead Cas9 (dCas9) - DNA methyltransferase fusion enzyme in human cells. Hypermethylation at each target site had no detectable effect on rRNA transcription, nucleolar morphology, or cellular growth rate. Instead, human UBF and Pol I remain bound to rDNA promoters in the presence of increased DNA methylation. These data suggest that promoter methylation is not sufficient to impair transcription of the human rDNA and imply that the human rDNA transcription machinery may be resilient to age-linked rDNA hypermethylation.
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Affiliation(s)
- Yana Blokhina
- Cardiovascular Research Institute and Department of Physiology, University of California, San Francisco
- present address: NewLimit, South San Francisco, CA
| | - Abigail Buchwalter
- Cardiovascular Research Institute and Department of Physiology, University of California, San Francisco
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13
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Moss T, LeDoux MS, Crane-Robinson C. HMG-boxes, ribosomopathies and neurodegenerative disease. Front Genet 2023; 14:1225832. [PMID: 37600660 PMCID: PMC10435976 DOI: 10.3389/fgene.2023.1225832] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2023] [Accepted: 07/19/2023] [Indexed: 08/22/2023] Open
Abstract
The UBTF E210K neuroregression syndrome is a predominantly neurological disorder caused by recurrent de novo dominant variants in Upstream Binding Factor, that is, essential for transcription of the ribosomal RNA genes. This unusual form of ribosomopathy is characterized by a slow decline in cognition, behavior, and sensorimotor functioning during the critical period of development. UBTF (or UBF) is a multi-HMGB-box protein that acts both as an epigenetic factor to establish "open" chromatin on the ribosomal genes and as a basal transcription factor in their RNA Polymerase I transcription. Here we review the possible mechanistic connections between the UBTF variants, ribosomal RNA gene transcription and the neuroregression syndrome, and suggest that DNA topology may play an important role.
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Affiliation(s)
- Tom Moss
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, QC, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, QC, Canada
| | - Mark S. LeDoux
- Department of Psychology, University of Memphis, Memphis, TN, United States
- Veracity Neuroscience LLC, Memphis, TN, United States
| | - Colyn Crane-Robinson
- Biophysics Laboratories, School of Biology, University of Portsmouth, Portsmouth, United Kingdom
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14
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Morton EA, Hall AN, Cuperus JT, Queitsch C. Substantial rDNA copy number reductions alter timing of development and produce variable tissue-specific phenotypes in C. elegans. Genetics 2023; 224:iyad039. [PMID: 36919976 PMCID: PMC10474940 DOI: 10.1093/genetics/iyad039] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2023] [Revised: 03/01/2023] [Accepted: 03/02/2023] [Indexed: 03/16/2023] Open
Abstract
The genes that encode ribosomal RNAs are present in several hundred copies in most eukaryotes. These vast arrays of repetitive ribosomal DNA (rDNA) have been implicated not just in ribosome biogenesis, but also aging, cancer, genome stability, and global gene expression. rDNA copy number is highly variable among and within species; this variability is thought to associate with traits relevant to human health and disease. Here we investigate the phenotypic consequences of multicellular life at the lower bounds of rDNA copy number. We use the model Caenorhabditis elegans, which has previously been found to complete embryogenesis using only maternally provided ribosomes. We find that individuals with rDNA copy number reduced to ∼5% of wild type are capable of further development with variable penetrance. Such individuals are sterile and exhibit severe morphological defects, particularly in post-embryonically dividing tissues such as germline and vulva. Developmental completion and fertility are supported by an rDNA copy number ∼10% of wild type, with substantially delayed development. Worms with rDNA copy number reduced to ∼33% of wild type display a subtle developmental timing defect that was absent in worms with higher copy numbers. Our results support the hypothesis that rDNA requirements vary across tissues and indicate that the minimum rDNA copy number for fertile adulthood is substantially less than the lowest naturally observed total copy number. The phenotype of individuals with severely reduced rDNA copy number is highly variable in penetrance and presentation, highlighting the need for continued investigation into the biological consequences of rDNA copy number variation.
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Affiliation(s)
| | - Ashley N Hall
- Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI 53726, USA
- Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA
| | - Josh T Cuperus
- Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
| | - Christine Queitsch
- Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
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15
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Liskovykh M, Petrov NS, Noskov VN, Masumoto H, Earnshaw WC, Schlessinger D, Shabalina SA, Larionov V, Kouprina N. Actively transcribed rDNA and distal junction (DJ) sequence are involved in association of NORs with nucleoli. Cell Mol Life Sci 2023; 80:121. [PMID: 37043028 PMCID: PMC10097779 DOI: 10.1007/s00018-023-04770-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 03/27/2023] [Accepted: 03/28/2023] [Indexed: 04/13/2023]
Abstract
Although they are organelles without a limiting membrane, nucleoli have an exclusive structure, built upon the rDNA-rich acrocentric short arms of five human chromosomes (nucleolar organizer regions or NORs). This has raised the question: what are the structural features of a chromosome required for its inclusion in a nucleolus? Previous work has suggested that sequences adjacent to the tandemly repeated rDNA repeat units (DJ, distal junction sequence) may be involved, and we have extended such studies by addressing several issues related to the requirements for the association of NORs with nucleoli. We exploited both a set of somatic cell hybrids containing individual human acrocentric chromosomes and a set of Human Artificial Chromosomes (HACs) carrying different parts of a NOR, including an rDNA unit or DJ or PJ (proximal junction) sequence. Association of NORs with nucleoli was increased when constituent rDNA was transcribed and may be also affected by the status of heterochromatin blocks formed next to the rDNA arrays. Furthermore, our data suggest that a relatively small size DJ region, highly conserved in evolution, is also involved, along with the rDNA repeats, in the localization of p-arms of acrocentric chromosomes in nucleoli. Thus, we infer a cooperative action of rDNA sequence-stimulated by its activity-and sequences distal to rDNA contributing to incorporation into nucleoli. Analysis of NOR sequences also identified LncRNA_038958 in the DJ, a candidate transcript with the region of the suggested promoter that is located close to the DJ/rDNA boundary and contains CTCF binding sites. This LncRNA may affect RNA Polymerase I and/or nucleolar activity. Our findings provide the basis for future studies to determine which RNAs and proteins interact critically with NOR sequences to organize the higher-order structure of nucleoli and their function in normal cells and pathological states.
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Affiliation(s)
- Mikhail Liskovykh
- Developmental Therapeutics Branch, National Cancer Institute, NIH, Bethesda, MD, 20892, USA.
| | - Nikolai S Petrov
- Developmental Therapeutics Branch, National Cancer Institute, NIH, Bethesda, MD, 20892, USA
| | - Vladimir N Noskov
- Developmental Therapeutics Branch, National Cancer Institute, NIH, Bethesda, MD, 20892, USA
| | - Hiroshi Masumoto
- Laboratory of Chromosome Engineering, Department of Frontier Research and Development, Kazusa DNA Research Institute, Kisarazu, Chiba, 292-0818, Japan
| | - William C Earnshaw
- Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3JR, Scotland, UK
| | - David Schlessinger
- National Institute on Aging, Laboratory of Genetics and Genomics, NIH, Baltimore, MD, 21224, USA
| | - Svetlana A Shabalina
- National Center for Biotechnology Information, National Library of Medicine, NIH, Bethesda, MD, 20892, USA
| | - Vladimir Larionov
- Developmental Therapeutics Branch, National Cancer Institute, NIH, Bethesda, MD, 20892, USA.
| | - Natalay Kouprina
- Developmental Therapeutics Branch, National Cancer Institute, NIH, Bethesda, MD, 20892, USA.
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16
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Peng H, Mirouze M, Bucher E. Extrachromosomal circular DNA: A neglected nucleic acid molecule in plants. CURRENT OPINION IN PLANT BIOLOGY 2022; 69:102263. [PMID: 35872391 DOI: 10.1016/j.pbi.2022.102263] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 06/15/2022] [Accepted: 06/19/2022] [Indexed: 06/15/2023]
Abstract
Throughout the years, most plant genomic studies were focused on nuclear chromosomes. Extrachromosomal circular DNA (eccDNA) has largely been neglected for decades since its discovery in 1965. While initial research showed that eccDNAs can originate from highly repetitive sequences, recent findings show that many regions of the genome can contribute to the eccDNA pool. Currently, the biological functions of eccDNAs, if any, are a mystery but recent studies have indicated that they can be regulated by different genomic loci and contribute to stress response and adaptation. In this review, we outline current relevant technological developments facilitating eccDNA identification and the latest discoveries about eccDNAs in plants. Finally, we explore the probable functions and future research directions that could be undertaken with respect to different eccDNA sources.
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Affiliation(s)
- Haoran Peng
- Crop Genome Dynamics Group, Agroscope Changins, 1260, Nyon, Switzerland; Department of Botany and Plant Biology, Section of Biology, Faculty of Science, University of Geneva, 1211, Geneva, Switzerland
| | - Marie Mirouze
- Institut de Recherche pour le Développement (IRD), EMR269 MANGO, Université de Perpignan, 66860 Perpignan, France; Laboratory of Plant Genome and Development, Université de Perpignan, 66860, Perpignan, France.
| | - Etienne Bucher
- Crop Genome Dynamics Group, Agroscope Changins, 1260, Nyon, Switzerland.
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17
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Shih HT, Chen WY, Wang HY, Chao T, Huang HD, Chou CH, Chang ZF. DNMT3b protects centromere integrity by restricting R-loop-mediated DNA damage. Cell Death Dis 2022; 13:546. [PMID: 35688824 PMCID: PMC9187704 DOI: 10.1038/s41419-022-04989-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Revised: 05/26/2022] [Accepted: 05/30/2022] [Indexed: 01/21/2023]
Abstract
This study used DNA methyltransferase 3b (DNMT3b) knockout cells and the functional loss of DNMT3b mutation in immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) cells to understand how DNMT3b dysfunction causes genome instability. We demonstrated that R-loops contribute to DNA damages in DNMT3b knockout and ICF cells. More prominent DNA damage signal in DNMT3b knockout cells was due to the loss of DNMT3b expression and the acquirement of p53 mutation. Genome-wide ChIP-sequencing mapped DNA damage sites at satellite repetitive DNA sequences including (peri-)centromere regions. However, the steady-state levels of (peri-)centromeric R-loops were reduced in DNMT3b knockout and ICF cells. Our analysis indicates that XPG and XPF endonucleases-mediated cleavages remove (peri-)centromeric R-loops to generate DNA beaks, causing chromosome instability. DNMT3b dysfunctions clearly increase R-loops susceptibility to the cleavage process. Finally, we showed that DNA double-strand breaks (DSBs) in centromere are probably repaired by error-prone end-joining pathway in ICF cells. Thus, DNMT3 dysfunctions undermine the integrity of centromere by R-loop-mediated DNA damages and repair.
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Affiliation(s)
- Hsueh-Tzu Shih
- Institute of Molecular Medicine, National Taiwan University, Taipei, 10051, Taiwan
- Center of Precision Medicine, College of Medicine, National Taiwan University, Taipei, 10051, Taiwan
| | - Wei-Yi Chen
- Institute of Biochemistry and Molecular Biology, National Yang Ming Chiao Tung University, Taipei, 11221, Taiwan
- Cancer Progression Research Center, National Yang Ming Chiao Tung University, Taipei, 11221, Taiwan
| | - Hsin-Yen Wang
- Institute of Molecular Medicine, National Taiwan University, Taipei, 10051, Taiwan
| | - Tung Chao
- Institute of Molecular Medicine, National Taiwan University, Taipei, 10051, Taiwan
| | - Hsien-Da Huang
- Warshel Institute for Computational Biology, The Chinese University of Hong Kong, Longgang District, 518172, Shenzhen, China
- School of Life and Health Sciences, The Chinese University of Hong Kong, Longgang District, 518172, Shenzhen, China
- School of Medicine, The Chinese University of Hong Kong, Longgang District, 518172, Shenzhen, China
| | - Chih-Hung Chou
- Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, 30010, Taiwan
- Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B), National Yang Ming Chiao Tung University, Hsinchu, 30010, Taiwan
| | - Zee-Fen Chang
- Institute of Molecular Medicine, National Taiwan University, Taipei, 10051, Taiwan.
- Center of Precision Medicine, College of Medicine, National Taiwan University, Taipei, 10051, Taiwan.
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18
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Hall AN, Morton E, Queitsch C. First discovered, long out of sight, finally visible: ribosomal DNA. Trends Genet 2022; 38:587-597. [PMID: 35272860 PMCID: PMC10132741 DOI: 10.1016/j.tig.2022.02.005] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Revised: 02/08/2022] [Accepted: 02/09/2022] [Indexed: 10/18/2022]
Abstract
With the advent of long-read sequencing, previously unresolvable genomic elements are being revisited in an effort to generate fully complete reference genomes. One such element is ribosomal DNA (rDNA), the highly conserved genomic region that encodes rRNAs. Genomic structure and content of the rDNA are variable in both prokarya and eukarya, posing interesting questions about the biology of rDNA. Here, we consider the types of variation observed in rDNA - including locus structure and number, copy number, and sequence variation - and their known phenotypic consequences. With recent advances in long-read sequencing technology, incorporating the full rDNA sequence into reference genomes is within reach. This knowledge will have important implications for understanding rDNA biology within the context of cell physiology and whole-organism phenotypes.
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Affiliation(s)
- Ashley N Hall
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Elizabeth Morton
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Christine Queitsch
- Department of Genome Sciences, University of Washington, Seattle, WA, USA.
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19
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Structural insights into nuclear transcription by eukaryotic DNA-dependent RNA polymerases. Nat Rev Mol Cell Biol 2022; 23:603-622. [PMID: 35505252 DOI: 10.1038/s41580-022-00476-9] [Citation(s) in RCA: 61] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/18/2022] [Indexed: 02/07/2023]
Abstract
The eukaryotic transcription apparatus synthesizes a staggering diversity of RNA molecules. The labour of nuclear gene transcription is, therefore, divided among multiple DNA-dependent RNA polymerases. RNA polymerase I (Pol I) transcribes ribosomal RNA, Pol II synthesizes messenger RNAs and various non-coding RNAs (including long non-coding RNAs, microRNAs and small nuclear RNAs) and Pol III produces transfer RNAs and other short RNA molecules. Pol I, Pol II and Pol III are large, multisubunit protein complexes that associate with a multitude of additional factors to synthesize transcripts that largely differ in size, structure and abundance. The three transcription machineries share common characteristics, but differ widely in various aspects, such as numbers of RNA polymerase subunits, regulatory elements and accessory factors, which allows them to specialize in transcribing their specific RNAs. Common to the three RNA polymerases is that the transcription process consists of three major steps: transcription initiation, transcript elongation and transcription termination. In this Review, we outline the common principles and differences between the Pol I, Pol II and Pol III transcription machineries and discuss key structural and functional insights obtained into the three stages of their transcription processes.
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20
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Diamantopoulos MA, Georgoulia KK, Scorilas A. Identification and expression analysis of ten novel small non-coding RNAs (sncRNAs) in cancer cells using a high-throughput sequencing approach. Gene 2022; 809:146025. [PMID: 34710527 DOI: 10.1016/j.gene.2021.146025] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Revised: 09/13/2021] [Accepted: 10/14/2021] [Indexed: 01/18/2023]
Abstract
Non-coding RNAs are characterized as RNA molecules, which lack the capacity to encode protein structures and appear to include a level of internal signals. Moreover, they control various stages of gene expression, thus controlling the cell physiology and development. In this study, we implemented a high-throughput sequencing approach based on the primary semi-conductor technology and computational tools, in order to identity novel small non-coding RNAs. Fourteen human cancer cell lines were cultured, and RNA samples were enriched for small RNAs following semi-conductor next generation sequencing (NGS). Bioinformatics analysis of NGS data revealed the existence of several classes of ncRNAs using the miRDeep* and CPSS 2.0 software. To investigate the existence of the predicted non-coding RNA sequences in cDNA pools of cell lines, a developed qPCR-based assay was implemented. The structure of each novel small ncRNA was visualized, using the RNAfold algorithm. Our results support the existence of twenty (20) putative new small ncRNAs, ten (10) of which have had their expression experimentally validated and presented differential profiles in cancerous and normal cells. A deeper comprehension of the ncRNAs interactive network and its role in cancer can therefore be translated into a wide range of clinical applications. Despite this progress, further scientific research from different perspectives and in different fields is needed, so that the riddle of the human transcriptome can be solved.
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Affiliation(s)
- Marios A Diamantopoulos
- Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Greece
| | - Konstantina K Georgoulia
- Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Greece
| | - Andreas Scorilas
- Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Greece
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21
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Wang M, Bissonnette N, Dudemaine PL, Zhao X, Ibeagha-Awemu EM. Whole Genome DNA Methylation Variations in Mammary Gland Tissues from Holstein Cattle Producing Milk with Various Fat and Protein Contents. Genes (Basel) 2021; 12:1727. [PMID: 34828333 PMCID: PMC8618717 DOI: 10.3390/genes12111727] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Revised: 10/22/2021] [Accepted: 10/22/2021] [Indexed: 12/20/2022] Open
Abstract
Milk fat and protein contents are among key elements of milk quality, and they are attracting more attention in response to consumers' demand for high-quality dairy products. To investigate the potential regulatory roles of DNA methylation underlying milk component yield, whole genome bisulfite sequencing was employed to profile the global DNA methylation patterns of mammary gland tissues from 17 Canada Holstein cows with various milk fat and protein contents. A total of 706, 2420 and 1645 differentially methylated CpG sites (DMCs) were found between high vs. low milk fat (HMF vs. LMF), high vs. low milk protein (HMP vs. LMP), and high vs. low milk fat and protein (HMFP vs. LMFP) groups, respectively (q value < 0.1). Twenty-seven, 56 and 67 genes harboring DMCs in gene regions (denoted DMC genes) were identified for HMF vs. LMF, HMP vs. LMP and HMFP vs. LMFP, respectively. DMC genes from HMP vs. LMP and HMFP vs. LMFP comparisons were significantly overrepresented in GO terms related to aerobic electron transport chain and/or mitochondrial ATP (adenosine triphosphate) synthesis coupled electron transport. A total of 83 (HMF vs. LMF), 708 (HMP vs. LMP) and 408 (HMFP vs. LMFP) DMCs were co-located with 87, 147 and 158 quantitative trait loci (QTL) for milk component and yield traits, respectively. In conclusion, the identified methylation changes are potentially involved in the regulation of milk fat and protein yields, as well as the variation in reported co-located QTLs.
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Affiliation(s)
- Mengqi Wang
- Sherbrooke Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, QC J1M 0C8, Canada; (M.W.); (N.B.); (P.-L.D.)
| | - Nathalie Bissonnette
- Sherbrooke Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, QC J1M 0C8, Canada; (M.W.); (N.B.); (P.-L.D.)
| | - Pier-Luc Dudemaine
- Sherbrooke Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, QC J1M 0C8, Canada; (M.W.); (N.B.); (P.-L.D.)
| | - Xin Zhao
- Department of Animal Science, McGill University, Ste-Anne-De-Bellevue, QC H9X 3V9, Canada;
| | - Eveline M. Ibeagha-Awemu
- Sherbrooke Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, QC J1M 0C8, Canada; (M.W.); (N.B.); (P.-L.D.)
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22
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Huang X, Zhang X, Zong L, Gao Q, Zhang C, Wei R, Guan Y, Huang L, Zhang L, Lyu G, Tao W. Gene body methylation safeguards ribosomal DNA transcription by preventing PHF6-mediated enrichment of repressive histone mark H4K20me3. J Biol Chem 2021; 297:101195. [PMID: 34520760 PMCID: PMC8511956 DOI: 10.1016/j.jbc.2021.101195] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2021] [Revised: 09/04/2021] [Accepted: 09/10/2021] [Indexed: 02/04/2023] Open
Abstract
DNA methylation shows complex correlations with gene expression, and the role of promoter hypermethylation in repressing gene transcription has been well addressed. Emerging evidence indicates that gene body methylation promotes transcription; however, the underlying mechanisms remain to be further investigated. Here, using methylated DNA immunoprecipitation sequencing (MeDIP-seq), bisulfite genomic sequencing, and immunofluorescent labeling, we show that gene body methylation is indeed positively correlated with rRNA gene (rDNA) transcription. Mechanistically, gene body methylation is largely maintained by DNA methyltransferase 1 (DNMT1), deficiency or downregulation of which during myoblast differentiation or nutrient deprivation results in decreased gene body methylation levels, leading to increased gene body occupancy of plant homeodomain (PHD) finger protein 6 (PHF6). PHF6 binds to hypomethylated rDNA gene bodies where it recruits histone methyltransferase SUV4-20H2 to establish the repressive histone modification, H4K20me3, ultimately inhibiting rDNA transcription. These findings demonstrate that DNMT1-mediated gene body methylation safeguards rDNA transcription by preventing enrichment of repressive histone modifications, suggesting that gene body methylation serves to maintain gene expression in response to developmental and/or environmental stresses.
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Affiliation(s)
- Xiaoke Huang
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Xuebin Zhang
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Le Zong
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Qianqian Gao
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Chao Zhang
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Ran Wei
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Yiting Guan
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Li Huang
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Lijun Zhang
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Guoliang Lyu
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China.
| | - Wei Tao
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China.
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Bouchla A, Thomopoulos TP, Papageorgiou SG, Apostolopoulou C, Loucari C, Mpazani E, Pappa V. Predicting outcome in higher-risk myelodysplastic syndrome patients treated with azacitidine. Epigenomics 2021; 13:1129-1143. [PMID: 34291653 DOI: 10.2217/epi-2021-0124] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
5-Azacitidine (5-AZA) is widely used for the treatment of higher-risk myelodysplastic syndromes. However, response and survival rates vary considerably, while indicated treatment duration remains undefined. For these reasons, factors determining response and survival are of major importance. Clinical, morphological, flow cytometry, cytogenetic and molecular factors are discussed in this review. Biomarkers predictive of response and prognosis, as well as their link to the mode of action of 5-AZA are also addressed, shifting the focus from clinical practice to investigational research. Their use could further improve prognostic classification of 5-AZA treated higher-risk myelodysplastic syndromes in the near future.
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Affiliation(s)
- Anthi Bouchla
- Second Department of Internal Medicine & Research Unit Hematology Unit, University General Hospital Attikon, Rimini, 12462 Chaidari, Athens, Greece
| | - Thomas P Thomopoulos
- Second Department of Internal Medicine & Research Unit Hematology Unit, University General Hospital Attikon, Rimini, 12462 Chaidari, Athens, Greece
| | - Sotirios G Papageorgiou
- Second Department of Internal Medicine & Research Unit Hematology Unit, University General Hospital Attikon, Rimini, 12462 Chaidari, Athens, Greece
| | - Christina Apostolopoulou
- Second Department of Internal Medicine & Research Unit Hematology Unit, University General Hospital Attikon, Rimini, 12462 Chaidari, Athens, Greece
| | - Constantinos Loucari
- Second Department of Internal Medicine & Research Unit Hematology Unit, University General Hospital Attikon, Rimini, 12462 Chaidari, Athens, Greece
| | - Efthimia Mpazani
- Second Department of Internal Medicine & Research Unit Hematology Unit, University General Hospital Attikon, Rimini, 12462 Chaidari, Athens, Greece
| | - Vasiliki Pappa
- Second Department of Internal Medicine & Research Unit Hematology Unit, University General Hospital Attikon, Rimini, 12462 Chaidari, Athens, Greece
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24
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Nuclear Organization during Hepatogenesis in Zebrafish Requires Uhrf1. Genes (Basel) 2021; 12:genes12071081. [PMID: 34356097 PMCID: PMC8304062 DOI: 10.3390/genes12071081] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2021] [Revised: 07/08/2021] [Accepted: 07/12/2021] [Indexed: 01/07/2023] Open
Abstract
Acquisition of cellular fate during development is initiated and maintained by well-coordinated patterns of gene expression that are dictated by the epigenetic landscape and genome organization in the nucleus. While the epigenetic marks that mediate developmental gene expression patterns during organogenesis have been well studied, less is known about how epigenetic marks influence nuclear organization during development. This study examines the relationship between nuclear structure, chromatin accessibility, DNA methylation, and gene expression during hepatic outgrowth in zebrafish larvae. We investigate the relationship between these features using mutants that lack DNA methylation. Hepatocyte nuclear morphology was established coincident with hepatocyte differentiation at 80 h post-fertilization (hpf), and nuclear shape and size continued to change until the conclusion of outgrowth and morphogenesis at 120 hpf. Integrating ATAC-Seq analysis with DNA methylation profiling of zebrafish livers at 120 hpf showed that closed and highly methylated chromatin occupies most transposable elements and that open chromatin correlated with gene expression. DNA hypomethylation, due to mutation of genes encoding ubiquitin-like, containing PHD and RING Finger Domains 1 (uhrf1) and DNA methyltransferase (dnmt1), did not block hepatocyte differentiation, but had dramatic effects on nuclear organization. Hepatocytes in uhrf1 mutants have large, deformed nuclei with multiple nucleoli, downregulation of nucleolar genes, and a complete lack of the nuclear lamina. Loss of lamin B2 staining was phenocopied by dnmt1 mutation. Together, these data show that hepatocyte nuclear morphogenesis coincides with organ morphogenesis and outgrowth, and that DNA methylation directs chromatin organization, and, in turn, hepatocyte nuclear shape and size during liver development.
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25
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Commuting to Work: Nucleolar Long Non-Coding RNA Control Ribosome Biogenesis from Near and Far. Noncoding RNA 2021; 7:ncrna7030042. [PMID: 34287370 PMCID: PMC8293466 DOI: 10.3390/ncrna7030042] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2021] [Revised: 07/09/2021] [Accepted: 07/11/2021] [Indexed: 12/26/2022] Open
Abstract
Gene expression is an essential process for cellular growth, proliferation, and differentiation. The transcription of protein-coding genes and non-coding loci depends on RNA polymerases. Interestingly, numerous loci encode long non-coding (lnc)RNA transcripts that are transcribed by RNA polymerase II (RNAPII) and fine-tune the RNA metabolism. The nucleolus is a prime example of how different lncRNA species concomitantly regulate gene expression by facilitating the production and processing of ribosomal (r)RNA for ribosome biogenesis. Here, we summarise the current findings on how RNAPII influences nucleolar structure and function. We describe how RNAPII-dependent lncRNA can both promote nucleolar integrity and inhibit ribosomal (r)RNA synthesis by modulating the availability of rRNA synthesis factors in trans. Surprisingly, some lncRNA transcripts can directly originate from nucleolar loci and function in cis. The nucleolar intergenic spacer (IGS), for example, encodes nucleolar transcripts that counteract spurious rRNA synthesis in unperturbed cells. In response to DNA damage, RNAPII-dependent lncRNA originates directly at broken ribosomal (r)DNA loci and is processed into small ncRNA, possibly to modulate DNA repair. Thus, lncRNA-mediated regulation of nucleolar biology occurs by several modes of action and is more direct than anticipated, pointing to an intimate crosstalk of RNA metabolic events.
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26
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The genomic structure of a human chromosome 22 nucleolar organizer region determined by TAR cloning. Sci Rep 2021; 11:2997. [PMID: 33542373 PMCID: PMC7862453 DOI: 10.1038/s41598-021-82565-x] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Accepted: 01/18/2021] [Indexed: 12/13/2022] Open
Abstract
The rDNA clusters and flanking sequences on human chromosomes 13, 14, 15, 21 and 22 represent large gaps in the current genomic assembly. The organization and the degree of divergence of the human rDNA units within an individual nucleolar organizer region (NOR) are only partially known. To address this lacuna, we previously applied transformation-associated recombination (TAR) cloning to isolate individual rDNA units from chromosome 21. That approach revealed an unexpectedly high level of heterogeneity in human rDNA, raising the possibility of corresponding variations in ribosome dynamics. We have now applied the same strategy to analyze an entire rDNA array end-to-end from a copy of chromosome 22. Sequencing of TAR isolates provided the entire NOR sequence, including proximal and distal junctions that may be involved in nucleolar function. Comparison of the newly sequenced rDNAs to reference sequence for chromosomes 22 and 21 revealed variants that are shared in human rDNA in individuals from different ethnic groups, many of them at high frequency. Analysis infers comparable intra- and inter-individual divergence of rDNA units on the same and different chromosomes, supporting the concerted evolution of rDNA units. The results provide a route to investigate further the role of rDNA variation in nucleolar formation and in the empirical associations of nucleoli with pathology.
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27
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Goffová I, Fajkus J. The rDNA Loci-Intersections of Replication, Transcription, and Repair Pathways. Int J Mol Sci 2021; 22:1302. [PMID: 33525595 PMCID: PMC7865372 DOI: 10.3390/ijms22031302] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Revised: 01/25/2021] [Accepted: 01/25/2021] [Indexed: 12/28/2022] Open
Abstract
Genes encoding ribosomal RNA (rDNA) are essential for cell survival and are particularly sensitive to factors leading to genomic instability. Their repetitive character makes them prone to inappropriate recombinational events arising from collision of transcriptional and replication machineries, resulting in unstable rDNA copy numbers. In this review, we summarize current knowledge on the structure and organization of rDNA, its role in sensing changes in the genome, and its linkage to aging. We also review recent findings on the main factors involved in chromatin assembly and DNA repair in the maintenance of rDNA stability in the model plants Arabidopsis thaliana and the moss Physcomitrella patens, providing a view across the plant evolutionary tree.
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Affiliation(s)
- Ivana Goffová
- Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, CZ-61137 Brno, Czech Republic;
- Chromatin Molecular Complexes, Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, CZ-62500 Brno, Czech Republic
| | - Jiří Fajkus
- Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, CZ-61137 Brno, Czech Republic;
- Chromatin Molecular Complexes, Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, CZ-62500 Brno, Czech Republic
- Department of Cell Biology and Radiobiology, Institute of Biophysics of the Czech Academy of Sciences, CZ-61265 Brno, Czech Republic
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28
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Lafita-Navarro MC, Venkateswaran N, Kilgore JA, Kanji S, Han J, Barnes S, Williams NS, Buszczak M, Burma S, Conacci-Sorrell M. Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells. PLoS Genet 2020; 16:e1009117. [PMID: 33201894 PMCID: PMC7707548 DOI: 10.1371/journal.pgen.1009117] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 12/01/2020] [Accepted: 09/14/2020] [Indexed: 12/15/2022] Open
Abstract
Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.
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Affiliation(s)
- M. Carmen Lafita-Navarro
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Niranjan Venkateswaran
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Jessica A. Kilgore
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Suman Kanji
- Department of Neurosurgery, University of Texas Health Science Center, San Antonio, Texas, United States of America
| | - Jungsoo Han
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Spencer Barnes
- Bioinformatics Core Facility, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Noelle S. Williams
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Michael Buszczak
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Sandeep Burma
- Department of Neurosurgery, University of Texas Health Science Center, San Antonio, Texas, United States of America
- Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, United States of America
| | - Maralice Conacci-Sorrell
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States of America
- Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
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Dichotomous Impact of Myc on rRNA Gene Activation and Silencing in B Cell Lymphomagenesis. Cancers (Basel) 2020; 12:cancers12103009. [PMID: 33081395 PMCID: PMC7656300 DOI: 10.3390/cancers12103009] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Accepted: 10/14/2020] [Indexed: 01/20/2023] Open
Abstract
Simple Summary B cell lymphomas mostly arise from malignant transformation of mature B cells and are typically driven by elevated levels of the oncoprotein Myc. Myc is a transcription factor regulating many protein-coding genes as well as the multicopy genes encoding ribosomal RNA (rRNA). The aim of this study was to understand, how Myc impacts rRNA genes in the course of B cell lymphomagenesis. Using a transgenic mouse model, we found that Myc and rRNA gene expression strongly increase upon tumor formation. Surprisingly, Myc also facilitates epigenetic silencing of a fraction of rRNA genes, thereby safeguarding genomic integrity in lymphoma cells. Together, the results show that Myc balances high activity and stability of rRNA genes. Perturbation of this equilibrium may be used as a therapeutic strategy. Abstract A major transcriptional output of cells is ribosomal RNA (rRNA), synthesized by RNA polymerase I (Pol I) from multicopy rRNA genes (rDNA). Constitutive silencing of an rDNA fraction by promoter CpG methylation contributes to the stabilization of these otherwise highly active loci. In cancers driven by the oncoprotein Myc, excessive Myc directly stimulates rDNA transcription. However, it is not clear when during carcinogenesis this mechanism emerges, and how Myc-driven rDNA activation affects epigenetic silencing. Here, we have used the Eµ-Myc mouse model to investigate rDNA transcription and epigenetic regulation in Myc-driven B cell lymphomagenesis. We have developed a refined cytometric strategy to isolate B cells from the tumor initiation, promotion, and progression phases, and found a substantial increase of both Myc and rRNA gene expression only in established lymphoma. Surprisingly, promoter CpG methylation and the machinery for rDNA silencing were also strongly up-regulated in the tumor progression state. The data indicate a dichotomous role of oncogenic Myc in rDNA regulation, boosting transcription as well as reinforcing repression of silent repeats, which may provide a novel angle on perturbing Myc function in cancer cells.
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30
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Zhou H, Li L, Wang Q, Hu Y, Zhao W, Gautam M, Li L. H3K9 Demethylation-Induced R-Loop Accumulation Is Linked to Disorganized Nucleoli. Front Genet 2020; 11:43. [PMID: 32117455 PMCID: PMC7025566 DOI: 10.3389/fgene.2020.00043] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2019] [Accepted: 01/15/2020] [Indexed: 11/24/2022] Open
Abstract
The nucleolar structure and integrity are important for a range of cellular functions of the nucleoli. It has been shown that cells lacking histone H3 Lysine 9 (H3K9) methylation form fragmented nucleoli. However, the molecular mechanism involved remains poorly understood. Here, we present evidence suggesting that loss of H3K9 dimethylation (H3K9me2) triggers R-loop accumulation at the rDNA locus, which further leads to the multilobed nucleoli. We reveal that suppression of H3K9 methyltransferase G9a by the inhibitor BIX 01294 causes R-loop accumulation at the rDNA region as well as inducing formation of multiple nucleoli. SiRNA-mediated knockdown of RNase H1 which can hydrolyze the RNA chain in R-loops causes an increase in R-loop formation, which in turn results in multiple nucleoli in one nucleus, whereas H3K9me2 levels are not affected by R-loop accumulation. Inhibition of RNA polymerase I transcription elongation by small molecule inhibitors induces a substantial decrease in H3K9me2 levels, accumulation of R-loops at rDNA sites, and nucleolus fragmentation. These results provide a mechanistic insight into the role of H3K9me2 in the structural integrity and organization of nucleoli via regulating R-loop accumulation.
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Affiliation(s)
- Hong Zhou
- State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
| | - Le Li
- State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
| | - Qing Wang
- State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
| | - Yan Hu
- State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
| | - Weiwei Zhao
- State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
| | - Mayank Gautam
- State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
| | - Lijia Li
- State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China
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31
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Singh PB, Newman AG. On the relations of phase separation and Hi-C maps to epigenetics. ROYAL SOCIETY OPEN SCIENCE 2020; 7:191976. [PMID: 32257349 PMCID: PMC7062049 DOI: 10.1098/rsos.191976] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/14/2019] [Accepted: 02/03/2020] [Indexed: 05/10/2023]
Abstract
The relationship between compartmentalization of the genome and epigenetics is long and hoary. In 1928, Heitz defined heterochromatin as the largest differentiated chromatin compartment in eukaryotic nuclei. Müller's discovery of position-effect variegation in 1930 went on to show that heterochromatin is a cytologically visible state of heritable (epigenetic) gene repression. Current insights into compartmentalization have come from a high-throughput top-down approach where contact frequency (Hi-C) maps revealed the presence of compartmental domains that segregate the genome into heterochromatin and euchromatin. It has been argued that the compartmentalization seen in Hi-C maps is owing to the physiochemical process of phase separation. Oddly, the insights provided by these experimental and conceptual advances have remained largely silent on how Hi-C maps and phase separation relate to epigenetics. Addressing this issue directly in mammals, we have made use of a bottom-up approach starting with the hallmarks of constitutive heterochromatin, heterochromatin protein 1 (HP1) and its binding partner the H3K9me2/3 determinant of the histone code. They are key epigenetic regulators in eukaryotes. Both hallmarks are also found outside mammalian constitutive heterochromatin as constituents of larger (0.1-5 Mb) heterochromatin-like domains and smaller (less than 100 kb) complexes. The well-documented ability of HP1 proteins to function as bridges between H3K9me2/3-marked nucleosomes contributes to polymer-polymer phase separation that packages epigenetically heritable chromatin states during interphase. Contacts mediated by HP1 'bridging' are likely to have been detected in Hi-C maps, as evidenced by the B4 heterochromatic subcompartment that emerges from contacts between large KRAB-ZNF heterochromatin-like domains. Further, mutational analyses have revealed a finer, innate, compartmentalization in Hi-C experiments that probably reflect contacts involving smaller domains/complexes. Proteins that bridge (modified) DNA and histones in nucleosomal fibres-where the HP1-H3K9me2/3 interaction represents the most evolutionarily conserved paradigm-could drive and generate the fundamental compartmentalization of the interphase nucleus. This has implications for the mechanism(s) that maintains cellular identity, be it a terminally differentiated fibroblast or a pluripotent embryonic stem cell.
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Affiliation(s)
- Prim B. Singh
- Nazarbayev University School of Medicine, 5/1 Kerei, Zhanibek Khandar Street, Nur-Sultan Z05K4F4, Kazakhstan
- Epigenetics Laboratory, Department of Natural Sciences, Novosibirsk State University, Pirogov Street 2, Novosibirsk 630090, Russian Federation
| | - Andrew G. Newman
- Institute of Cell and Neurobiology, Charité—Universitätsmedizin Berlin, Corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany
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32
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von Walden F, Fernandez-Gonzalo R, Pingel J, McCarthy J, Stål P, Pontén E. Epigenetic Marks at the Ribosomal DNA Promoter in Skeletal Muscle Are Negatively Associated With Degree of Impairment in Cerebral Palsy. Front Pediatr 2020; 8:236. [PMID: 32582584 PMCID: PMC7283884 DOI: 10.3389/fped.2020.00236] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Accepted: 04/20/2020] [Indexed: 12/12/2022] Open
Abstract
Introduction: Cerebral palsy (CP) is the most common motor impairment in children. Skeletal muscles in individuals with CP are typically weak, thin, and stiff. Whether epigenetic changes at the ribosomal DNA (rDNA) promoter are involved in this dysregulation remains unknown. Methods: Skeletal muscle samples were collected from 19 children with CP and 10 typically developed (TD) control children. Methylation of the rDNA promoter was analyzed using the Agena Epityper Mass array and gene expression by qRT-PCR. Results: Biceps brachii muscle ribosome biogenesis was suppressed in CP as compared to TD. Average methylation of the rDNA promoter was not different between CP and TD but negatively correlated to elbow flexor contracture in the CP group. Discussions: We observed a negative correlation between rDNA promoter methylation and degree of muscle contracture in the CP group. Children with CP with more severe motor impairment had less methylation of the rDNA promoter compared to less affected children. This finding suggests the importance of neural input and voluntary muscle movements for promoter methylation to occur in the biceps muscle.
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Affiliation(s)
- Ferdinand von Walden
- Division of Pediatric Neurology/Orthopedics/Rheumatology, Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden.,Department of Physiology, University of Kentucky, Lexington, KY, United States.,Center for Muscle Biology, University of Kentucky, Lexington, KY, United States
| | - Rodrigo Fernandez-Gonzalo
- Division of Clinical Physiology, Department of Laboratory Medicine, Karolinska Institutet, and Unit of Clinical Physiology, Karolinska University Hospital, Stockholm, Sweden
| | - Jessica Pingel
- Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark
| | - John McCarthy
- Department of Physiology, University of Kentucky, Lexington, KY, United States.,Center for Muscle Biology, University of Kentucky, Lexington, KY, United States
| | - Per Stål
- Laboratory of Muscle Biology, Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
| | - Eva Pontén
- Division of Pediatric Neurology/Orthopedics/Rheumatology, Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden
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33
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Robinson JF, Kapidzic M, Hamilton EG, Chen H, Puckett KW, Zhou Y, Ona K, Parry E, Wang Y, Park JS, Costello JF, Fisher SJ. Genomic Profiling of BDE-47 Effects on Human Placental Cytotrophoblasts. Toxicol Sci 2019; 167:211-226. [PMID: 30202865 DOI: 10.1093/toxsci/kfy230] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Despite gradual legislative efforts to phase out flame retardants (FRs) from the marketplace, polybrominated diphenyl ethers (PBDEs) are still widely detected in human maternal and fetal tissues, eg, placenta, due to their continued global application in consumer goods and inherent biological persistence. Recent studies in rodents and human placental cell lines suggest that PBDEs directly cause placental toxicity. During pregnancy, trophoblasts play key roles in uterine invasion, vascular remodeling, and anchoring of the placenta-fetal unit to the mother. Thus, to study the potential consequences of PBDE exposures on human placental development, we used an in vitro model: primary villous cytotrophoblasts (CTBs). Following exposures, the endpoints that were evaluated included cytotoxicity, function (migration, invasion), the transcriptome, and the methylome. In a concentration-dependent manner, common PBDE congeners, BDE-47 and -99, significantly reduced cell viability and increased death. Upon exposures to sub-cytotoxic concentrations (≤ 5 µM), we observed BDE-47 accumulation in CTBs with limited evidence of metabolism. At a functional level, BDE-47 hindered the ability of CTBs to migrate and invade. Transcriptomic analyses of BDE-47 effects suggested concentration-dependent changes in gene expression, involving stress pathways, eg, inflammation and lipid/cholesterol metabolism as well as processes underlying trophoblast fate, eg, differentiation, migration, and vascular morphogenesis. In parallel assessments, BDE-47 induced low-level global increases in methylation of CpG islands, including a subset that were proximal to genes with roles in cell adhesion/migration. Thus, using a primary human CTB model, we showed that PBDEs induced alterations at cellular and molecular levels, which could adversely impact placental development.
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Affiliation(s)
- Joshua F Robinson
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143.,Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143
| | - Mirhan Kapidzic
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143.,Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143
| | - Emily G Hamilton
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143.,Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143
| | - Hao Chen
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143.,Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143
| | - Kenisha W Puckett
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143.,Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143
| | - Yan Zhou
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143.,Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143
| | - Katherine Ona
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143.,Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143
| | - Emily Parry
- Environmental Chemistry Laboratory, Department of Toxic Substances Control, California Environmental Protection Agency, Berkeley, California 94710
| | - Yunzhu Wang
- Environmental Chemistry Laboratory, Department of Toxic Substances Control, California Environmental Protection Agency, Berkeley, California 94710
| | - June-Soo Park
- Environmental Chemistry Laboratory, Department of Toxic Substances Control, California Environmental Protection Agency, Berkeley, California 94710
| | - Joseph F Costello
- Department of Neurological Surgery, University of California, San Francisco (UCSF), San Francisco, California 94158
| | - Susan J Fisher
- Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143.,Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, California 94143
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34
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Peng Y, Wang Z, Wang Z, Yu F, Li J, Wong J. SUMOylation down-regulates rDNA transcription by repressing expression of upstream-binding factor and proto-oncogene c-Myc. J Biol Chem 2019; 294:19155-19166. [PMID: 31694914 DOI: 10.1074/jbc.ra119.010624] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2019] [Revised: 10/29/2019] [Indexed: 11/06/2022] Open
Abstract
Ribosome biogenesis is critical for proliferating cells and requires the coordinated activities of three eukaryotic RNA polymerases. We recently showed that the small ubiquitin-like modifier (SUMO) system controls the global level of RNA polymerase II (Pol II)-controlled transcription in mammalian cells by regulating cyclin-dependent kinase 9 activity. Here, we present evidence that the SUMO system also plays a critical role in the control of Pol I transcription. Using an siRNA-based knockdown approach, we found that multiple SUMO E3 ligases of the PIAS (protein inhibitor of activated STAT) family are involved in SUMO-mediated repression of ribosomal DNA (rDNA) gene transcription. We demonstrate that endogenous SUMO represses rDNA transcription primarily by repressing upstream-binding factor and proto-oncogene c-Myc expression and that ectopic overexpression of SUMO-associated enzymes additionally represses rDNA transcription via c-Myc SUMOylation and its subsequent degradation. The results of our study reveal a critical role of SUMOylation in the control of rDNA transcription, uncover the underlying mechanisms involved, and indicate that the SUMO system coordinates Pol I- and Pol II-mediated transcription in mammalian cells.
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Affiliation(s)
- Yu Peng
- Shanghai Key Laboratory of Regulatory Biology, Fengxian District Central Hospital-East China Normal University Joint Center of Translational Medicine, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Zhenxing Wang
- Shanghai Key Laboratory of Regulatory Biology, Fengxian District Central Hospital-East China Normal University Joint Center of Translational Medicine, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Zhiqiang Wang
- Shanghai Key Laboratory of Regulatory Biology, Fengxian District Central Hospital-East China Normal University Joint Center of Translational Medicine, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Fang Yu
- Shanghai Key Laboratory of Regulatory Biology, Fengxian District Central Hospital-East China Normal University Joint Center of Translational Medicine, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.,Department of Medicine, University of Florida, Gainesville, Florida 32610
| | - Jiwen Li
- Shanghai Key Laboratory of Regulatory Biology, Fengxian District Central Hospital-East China Normal University Joint Center of Translational Medicine, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Jiemin Wong
- Shanghai Key Laboratory of Regulatory Biology, Fengxian District Central Hospital-East China Normal University Joint Center of Translational Medicine, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China .,State Key Laboratory of Oncogene and Related Genes, Shanghai Cancer Institute and Shanghai Jiao Tong University, Shanghai 200240, China
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35
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Correll CC, Bartek J, Dundr M. The Nucleolus: A Multiphase Condensate Balancing Ribosome Synthesis and Translational Capacity in Health, Aging and Ribosomopathies. Cells 2019; 8:cells8080869. [PMID: 31405125 PMCID: PMC6721831 DOI: 10.3390/cells8080869] [Citation(s) in RCA: 84] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2019] [Revised: 07/31/2019] [Accepted: 08/06/2019] [Indexed: 12/21/2022] Open
Abstract
The nucleolus is the largest membrane-less structure in the eukaryotic nucleus. It is involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and is the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the intricate pathophysiological relationship between the nucleolus and protein synthesis has only recently begun to emerge. Here, we provide perspective on new principles governing nucleolar formation and the resulting multiphase organization driven by liquid-liquid phase separation. With recent advances in the structural analysis of ribosome formation, we highlight the current understanding of the step-wise assembly of pre-ribosomal subunits and the quality control required for proper function. Finally, we address how aging affects ribosome genesis and how genetic defects in ribosome formation cause ribosomopathies, complex diseases with a predisposition to cancer.
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Affiliation(s)
- Carl C Correll
- Center for Proteomics and Molecular Therapeutics, Rosalind Franklin University of Medicine & Science, North Chicago, IL 60064, USA.
| | - Jiri Bartek
- Danish Cancer Society Research Center, Genome Integrity Unit, DK-2100 Copenhagen, Denmark
- Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Karolinska Institute, SE-171 77 Stockholm, Sweden
| | - Miroslav Dundr
- Center for Cancer Cell Biology Immunology and Infection, Rosalind Franklin University of Medicine & Science, North Chicago, IL 60064, USA.
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36
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Genome Organization in and around the Nucleolus. Cells 2019; 8:cells8060579. [PMID: 31212844 PMCID: PMC6628108 DOI: 10.3390/cells8060579] [Citation(s) in RCA: 81] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2019] [Revised: 06/09/2019] [Accepted: 06/11/2019] [Indexed: 12/17/2022] Open
Abstract
The nucleolus is the largest substructure in the nucleus, where ribosome biogenesis takes place, and forms around the nucleolar organizer regions (NORs) that comprise ribosomal RNA (rRNA) genes. Each cell contains hundreds of rRNA genes, which are organized in three distinct chromatin and transcriptional states—silent, inactive and active. Increasing evidence indicates that the role of the nucleolus and rRNA genes goes beyond the control of ribosome biogenesis. Recent results highlighted the nucleolus as a compartment for the location and regulation of repressive genomic domains and, together with the nuclear lamina, represents the hub for the organization of the inactive heterochromatin. In this review, we aim to describe the crosstalk between the nucleolus and the rest of the genome and how distinct rRNA gene chromatin states affect nucleolus structure and are implicated in genome stability, genome architecture, and cell fate decision.
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37
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Chen H, Seifikar H, Larocque N, Kim Y, Khatib I, Fernandez CJ, Abello N, Robinson JF. Using a Multi-Stage hESC Model to Characterize BDE-47 Toxicity during Neurogenesis. Toxicol Sci 2019; 171:221-234. [PMID: 31173147 PMCID: PMC6736394 DOI: 10.1093/toxsci/kfz136] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2019] [Revised: 04/20/2019] [Accepted: 05/22/2019] [Indexed: 12/20/2022] Open
Abstract
While the ramifications associated with polybrominated diphenyl ethers (PBDE) exposures during human pregnancy have yet to be determined, increasing evidence in humans and animal models suggests that these compounds cause neurodevelopmental toxicity. Human embryonic stem cell models (hESCs) can be used to study the effects of environmental chemicals throughout the successive stages of neuronal development. Here, using a hESC differentiation model, we investigated the effects of common PBDE congeners (BDE-47 or -99) on the successive stages of early neuronal development. First, we determined the points of vulnerability to PBDEs across four stages of in vitro neural development by using assays to assess for cytotoxicity. Differentiated neural progenitors were identified to be more sensitive to PBDEs than their less differentiated counterparts. In follow-up investigations, we observed BDE-47 to inhibit functional processes critical for neurogenesis (e.g., proliferation, expansion) in hESC-derived neural precursor cells (NPCs) at sub-lethal concentrations. Finally, to determine the mechanism(s) underlying PBDE-toxicity, we conducted global transcriptomic and methylomic analyses of BDE-47. We identified 589 genes to be differentially expressed (DE) due to BDE-47 exposure, including molecules involved in oxidative stress mediation, cell cycle, hormone signaling, steroid metabolism, and neurodevelopmental pathways. In parallel analyses, we identified a broad significant increase in CpG methylation. In summary our results suggest, on a cellular level, PBDEs induce human neurodevelopmental toxicity in a concentration-dependent manner and sensitivity to these compounds is dependent on the developmental stage of exposure. Proposed mRNA and methylomic perturbations may underlie toxicity in early embryonic neuronal populations.
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Affiliation(s)
- Hao Chen
- Center for Reproductive Sciences and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, CA, USA
| | - Helia Seifikar
- Center for Reproductive Sciences and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, CA, USA
| | - Nicholas Larocque
- Center for Reproductive Sciences and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, CA, USA
| | - Yvonne Kim
- Center for Reproductive Sciences and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, CA, USA
| | - Ibrahim Khatib
- Center for Reproductive Sciences and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, CA, USA
| | - Charles J Fernandez
- Center for Reproductive Sciences and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, CA, USA
| | - Nicomedes Abello
- Center for Reproductive Sciences and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, CA, USA
| | - Joshua F Robinson
- Center for Reproductive Sciences and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco (UCSF), San Francisco, CA, USA
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38
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The chromatin landscape of the ribosomal RNA genes in mouse and human. Chromosome Res 2019; 27:31-40. [PMID: 30617621 DOI: 10.1007/s10577-018-09603-9] [Citation(s) in RCA: 43] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2018] [Revised: 12/21/2018] [Accepted: 12/26/2018] [Indexed: 12/21/2022]
Abstract
The rRNA genes of mouse and human encode the three major RNAs of the ribosome and as such are essential for growth and development. These genes are present in high copy numbers and arranged as direct repeats at the Nucleolar Organizer Regions on multiple chromosomes. Not all the rRNA genes are transcriptionally active, but the molecular mechanisms that determine activity are complex and still poorly understood. Recent studies applying a novel Deconvolution Chromatin Immunoprecipitation (DChIP-Seq) technique in conjunction with conditional gene inactivation provide new insights into the structure of the active rRNA genes and question previous assumptions on the role of chromatin and histone modifications. We suggest an alternative model for the active rRNA gene chromatin and discuss how this structure is determined and maintained.
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Lyu G, Zong L, Zhang C, Huang X, Xie W, Fang J, Guan Y, Zhang L, Ni T, Gu J, Tao W. Metastasis-related methyltransferase 1 (Merm1) represses the methyltransferase activity of Dnmt3a and facilitates RNA polymerase I transcriptional elongation. J Mol Cell Biol 2019; 11:78-90. [PMID: 30535232 DOI: 10.1093/jmcb/mjy023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2017] [Accepted: 03/20/2018] [Indexed: 11/13/2022] Open
Abstract
Stimulatory regulators for DNA methyltransferase activity, such as Dnmt3L and some Dnmt3b isoforms, affect DNA methylation patterns, thereby maintaining gene body methylation and maternal methylation imprinting, as well as the methylation landscape of pluripotent cells. Here we show that metastasis-related methyltransferase 1 (Merm1), a protein deleted in individuals with Williams-Beuren syndrome, acts as a repressive regulator of Dnmt3a. Merm1 interacts with Dnmt3a and represses its methyltransferase activity with the requirement of the binding motif for S-adenosyl-L-methionine. Functional analysis of gene regulation revealed that Merm1 is capable of maintaining hypomethylated rRNA gene bodies and co-localizes with RNA polymerase I in the nucleolus. Dnmt3a recruits Merm1, and in return, Merm1 ensures the binding of Dnmt3a to hypomethylated gene bodies. Such interplay between Dnmt3a and Merm1 facilitates transcriptional elongation by RNA polymerase I. Our findings reveal a repressive factor for Dnmt3a and uncover a molecular mechanism underlying transcriptional elongation of rRNA genes.
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Affiliation(s)
- Guoliang Lyu
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Le Zong
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Chao Zhang
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Xiaoke Huang
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Wenbing Xie
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Junnan Fang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Yiting Guan
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Lijun Zhang
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Ting Ni
- State Key Laboratory of Genetics Engineering & Ministry of Education (MOE) Key Laboratory of Contemporary Anthropology, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai0, China
| | - Jun Gu
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
| | - Wei Tao
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China
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40
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Olson CO, Pejhan S, Kroft D, Sheikholeslami K, Fuss D, Buist M, Ali Sher A, Del Bigio MR, Sztainberg Y, Siu VM, Ang LC, Sabourin-Felix M, Moss T, Rastegar M. MECP2 Mutation Interrupts Nucleolin-mTOR-P70S6K Signaling in Rett Syndrome Patients. Front Genet 2018; 9:635. [PMID: 30619462 PMCID: PMC6305968 DOI: 10.3389/fgene.2018.00635] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2018] [Accepted: 11/27/2018] [Indexed: 01/26/2023] Open
Abstract
Rett syndrome (RTT) is a severe and rare neurological disorder that is caused by mutations in the X-linked MECP2 (methyl CpG-binding protein 2) gene. MeCP2 protein is an important epigenetic factor in the brain and in neurons. In Mecp2-deficient neurons, nucleoli structures are compromised. Nucleoli are sites of active ribosomal RNA (rRNA) transcription and maturation, a process mainly controlled by nucleolin and mechanistic target of rapamycin (mTOR)-P70S6K signaling. Currently, it is unclear how nucleolin-rRNA-mTOR-P70S6K signaling from RTT cellular model systems translates into human RTT brain. Here, we studied the components of nucleolin-rRNA-mTOR-P70S6K signaling in the brain of RTT patients with common T158M and R255X mutations. Immunohistochemical examination of T158M brain showed disturbed nucleolin subcellular localization, which was absent in Mecp2-deficient homozygous male or heterozygote female mice, compared to wild type (WT). We confirmed by Western blot analysis that nucleolin protein levels are altered in RTT brain, but not in Mecp2-deficient mice. Further, we studied the expression of rRNA transcripts in Mecp2-deficient mice and RTT patients, as downstream molecules that are controlled by nucleolin. By data mining of published ChIP-seq studies, we showed MeCP2-binding at the multi-copy rRNA genes in the mouse brain, suggesting that rRNA might be a direct MeCP2 target gene. Additionally, we observed compromised mTOR-P70S6K signaling in the human RTT brain, a molecular pathway that is upstream of rRNA-nucleolin molecular conduits. RTT patients showed significantly higher phosphorylation of active mTORC1 or mTORC2 complexes compared to age- and sex-matched controls. Correlational analysis of mTORC1/2-P70S6K signaling pathway identified multiple points of deviation from the control tissues that may result in abnormal ribosome biogenesis in RTT brain. To our knowledge, this is the first report of deregulated nucleolin-rRNA-mTOR-P70S6K signaling in the human RTT brain. Our results provide important insight toward understanding the molecular properties of human RTT brain.
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Affiliation(s)
- Carl O Olson
- Regenerative Medicine Program, and Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
| | - Shervin Pejhan
- Regenerative Medicine Program, and Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
| | - Daniel Kroft
- Regenerative Medicine Program, and Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
| | - Kimia Sheikholeslami
- Regenerative Medicine Program, and Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada.,Faculty of Medicine, University of Toronto, Toronto, ON, Canada
| | - David Fuss
- Regenerative Medicine Program, and Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
| | - Marjorie Buist
- Regenerative Medicine Program, and Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
| | - Annan Ali Sher
- Regenerative Medicine Program, and Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
| | - Marc R Del Bigio
- Department of Pathology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
| | - Yehezkel Sztainberg
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States
| | - Victoria Mok Siu
- Division of Medical Genetics, Department of Paediatrics, Schulich School of Medicine, Western University, London, ON, Canada
| | - Lee Cyn Ang
- Department of Pathology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | - Marianne Sabourin-Felix
- Cancer Division of the Quebec University Hospital Research Centre, Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Quebec City, QC, Canada
| | - Tom Moss
- Cancer Division of the Quebec University Hospital Research Centre, Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Quebec City, QC, Canada
| | - Mojgan Rastegar
- Regenerative Medicine Program, and Department of Biochemistry and Medical Genetics, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
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41
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Karole AM, Chodisetty S, Ali A, Kumari N, Tyagi S. Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein. Cell Cycle 2018; 17:2684-2696. [PMID: 30489191 DOI: 10.1080/15384101.2018.1553338] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
Mixed-lineage leukemia (MLL) protein is the best-characterized member of SET family of histone 3 lysine 4 methyltransferase, known for its transcriptional-activation role during development. mll gene rearrangements cause multiple kinds of aggressive leukemia in both children and adults. An important 'first' step in understanding the role of MLL in leukemogenesis would be to identify its localization throughout the cell cycle. In order to fully understand the breath of MLL functions in proliferating cells, we have analyzed its sub-cellular localization during the cell cycle. Our results show that MLL localizes to nucleolus and centrosome in interphase. During mitosis, it localizes to centrosomes and midbody in addition to previously reported spindle apparatus. Our results show that MLLN is required to translocate MLLC to the nucleolus. These finding suggest functional roles for MLL in nucleolus and mitosis. We also show how MLL-fusion proteins (MLL-FPs) localize to the same sub-cellular organelles like endogenous MLL. Our results indicate that MLL-fusion proteins may not only disturb the cell homeostasis by gain-of-function of the chimeric protein, but also by interfering with the functions of endogenous MLL.
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Affiliation(s)
- Amit Mahendra Karole
- a Laboratory of Cell Cycle Regulation , Centre for DNA Fingerprinting and Diagnostics (CDFD) , Uppal , Hyderabad 500039 , India.,b Graduate Studies , Manipal Academy of Higher Education , Manipal , India
| | - Swathi Chodisetty
- a Laboratory of Cell Cycle Regulation , Centre for DNA Fingerprinting and Diagnostics (CDFD) , Uppal , Hyderabad 500039 , India.,b Graduate Studies , Manipal Academy of Higher Education , Manipal , India
| | - Aamir Ali
- a Laboratory of Cell Cycle Regulation , Centre for DNA Fingerprinting and Diagnostics (CDFD) , Uppal , Hyderabad 500039 , India.,b Graduate Studies , Manipal Academy of Higher Education , Manipal , India
| | - Nidhi Kumari
- a Laboratory of Cell Cycle Regulation , Centre for DNA Fingerprinting and Diagnostics (CDFD) , Uppal , Hyderabad 500039 , India
| | - Shweta Tyagi
- a Laboratory of Cell Cycle Regulation , Centre for DNA Fingerprinting and Diagnostics (CDFD) , Uppal , Hyderabad 500039 , India
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Abstract
The nucleolus as site of ribosome biogenesis holds a pivotal role in cell metabolism. It is composed of ribosomal DNA (rDNA), which is present as tandem arrays located in nucleolus organizer regions (NORs). In interphase cells, rDNA can be found inside and adjacent to nucleoli and the location is indicative for transcriptional activity of ribosomal genes-inactive rDNA (outside) versus active one (inside). Moreover, the nucleolus itself acts as a spatial organizer of non-nucleolar chromatin. Microscopy-based approaches offer the possibility to explore the spatially distinct localization of the different DNA populations in relation to the nucleolar structure. Recent technical developments in microscopy and preparatory methods may further our understanding of the functional architecture of nucleoli. This review will attempt to summarize the current understanding of mammalian nucleolar chromatin organization as seen from a microscopist's perspective.
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Affiliation(s)
- Christian Schöfer
- Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Schwarzspanierstr. 17, 1090, Vienna, Austria.
| | - Klara Weipoltshammer
- Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Schwarzspanierstr. 17, 1090, Vienna, Austria
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43
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Malinovskaya EM, Ershova ES, Golimbet VE, Porokhovnik LN, Lyapunova NA, Kutsev SI, Veiko NN, Kostyuk SV. Copy Number of Human Ribosomal Genes With Aging: Unchanged Mean, but Narrowed Range and Decreased Variance in Elderly Group. Front Genet 2018; 9:306. [PMID: 30131826 PMCID: PMC6090032 DOI: 10.3389/fgene.2018.00306] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Accepted: 07/19/2018] [Indexed: 01/24/2023] Open
Abstract
Introduction: The multi-copied genes coding for the human 18, 5.8, and 28S ribosomal RNA (rRNA) are located in five pairs of acrocentric chromosomes forming so-called rDNA. Human genome contains unmethylated, slightly methylated, and hypermethylated copies of rDNA. The major research question: What is the rDNA copy number (rDNA CN) and the content of hypermethylated rDNA as a function of age? Materials and Methods: We determined the rDNA CN in the blood leukocyte genomes of 651 subjects aged 17 to 91 years. The subjects were divided into two subgroups: “elderly” group (E-group, N = 126) – individuals over 72 years of age (the age of the population’s mean lifetime for Russia) and “non-elderly” group (NE-group, N = 525). The hypermethylated rDNA content was determined in the 40 DNA samples from the each group. The change in rDNA during replicative cell senescence was studied for the cultured skin fibroblast lines of five subjects from NE-group. Non-radioactive quantitative dot- and blot-hybridization techniques (NQH) were applied. Results: In the subjects from the E-group the mean rDNA CN was the same, but the range of variation was narrower compared to the NE-group: a range of 272 to 541 copies in E-group vs. 200 to 711 copies in NE-group. Unlike NE-group, the E-group genomes contained almost no hypermethylated rDNA copies. A case study of cultured skin fibroblasts from five subjects has shown that during the replicative senescence the genome lost hypermethylated rDNA copies only. Conclusion: In the elderly group, the mean rDNA CN is the same, but the range of variation is narrower compared with the younger subjects. During replicative senescence, the human fibroblast genome loses hypermethylated copies of rDNA. Two hypotheses were put forward: (1) individuals with either very low or very high rDNA content in their genomes do not survive till the age of the population’s mean lifetime; and/or (2) during the aging, the human genome eliminates hypermethylated copies of rDNA.
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44
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Kim JH, Dilthey AT, Nagaraja R, Lee HS, Koren S, Dudekula D, Wood Iii WH, Piao Y, Ogurtsov AY, Utani K, Noskov VN, Shabalina SA, Schlessinger D, Phillippy AM, Larionov V. Variation in human chromosome 21 ribosomal RNA genes characterized by TAR cloning and long-read sequencing. Nucleic Acids Res 2018; 46:6712-6725. [PMID: 29788454 PMCID: PMC6061828 DOI: 10.1093/nar/gky442] [Citation(s) in RCA: 59] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2018] [Accepted: 05/08/2018] [Indexed: 12/31/2022] Open
Abstract
Despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. We recovered ribosomal DNA segments from a single human chromosome 21 using transformation-associated recombination (TAR) cloning in yeast. Accurate long-read sequencing of 13 isolates covering ∼0.82 Mb of the chromosome 21 rDNA complement revealed substantial variation among tandem repeat rDNA copies, several palindromic structures and potential errors in the previous reference sequence. These clones revealed 101 variant positions in the 45S transcription unit and 235 in the intergenic spacer sequence. Approximately 60% of the 45S variants were confirmed in independent whole-genome or RNA-seq data, with 47 of these further observed in mature 18S/28S rRNA sequences. TAR cloning and long-read sequencing enabled the accurate reconstruction of multiple rDNA units and a new, high-quality 44 838 bp rDNA reference sequence, which we have annotated with variants detected from chromosome 21 of a single individual. The large number of variants observed reveal heterogeneity in human rDNA, opening up the possibility of corresponding variations in ribosome dynamics.
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MESH Headings
- Animals
- Cell Line
- Chromosomes, Human, Pair 21
- Cloning, Molecular
- DNA, Ribosomal/chemistry
- DNA, Ribosomal/isolation & purification
- DNA, Ribosomal Spacer/chemistry
- Genes, rRNA
- Genetic Variation
- Humans
- Mice
- Nucleic Acid Conformation
- Nucleolus Organizer Region/chemistry
- RNA, Ribosomal/chemistry
- RNA, Ribosomal/metabolism
- Sequence Analysis, DNA
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Affiliation(s)
- Jung-Hyun Kim
- National Cancer Institute, Developmental Therapeutics Branch, Bethesda, MD 20892, USA
| | - Alexander T Dilthey
- National Human Genome Research Institute, Computational and Statistical Genomics Branch, Bethesda, MD 20892, USA
| | - Ramaiah Nagaraja
- National Institute on Aging, Laboratory of Genetics and Genomics, Baltimore, MD 21224, USA
| | - Hee-Sheung Lee
- National Cancer Institute, Developmental Therapeutics Branch, Bethesda, MD 20892, USA
| | - Sergey Koren
- National Human Genome Research Institute, Computational and Statistical Genomics Branch, Bethesda, MD 20892, USA
| | - Dawood Dudekula
- National Institute on Aging, Laboratory of Genetics and Genomics, Baltimore, MD 21224, USA
| | - William H Wood Iii
- National Institute on Aging, Laboratory of Genetics and Genomics, Baltimore, MD 21224, USA
| | - Yulan Piao
- National Institute on Aging, Laboratory of Genetics and Genomics, Baltimore, MD 21224, USA
| | - Aleksey Y Ogurtsov
- National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD 20892, USA
| | - Koichi Utani
- National Cancer Institute, Developmental Therapeutics Branch, Bethesda, MD 20892, USA
| | - Vladimir N Noskov
- National Cancer Institute, Developmental Therapeutics Branch, Bethesda, MD 20892, USA
| | - Svetlana A Shabalina
- National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD 20892, USA
| | - David Schlessinger
- National Institute on Aging, Laboratory of Genetics and Genomics, Baltimore, MD 21224, USA
| | - Adam M Phillippy
- National Human Genome Research Institute, Computational and Statistical Genomics Branch, Bethesda, MD 20892, USA
| | - Vladimir Larionov
- National Cancer Institute, Developmental Therapeutics Branch, Bethesda, MD 20892, USA
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45
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Danson AF, Marzi SJ, Lowe R, Holland ML, Rakyan VK. Early life diet conditions the molecular response to post-weaning protein restriction in the mouse. BMC Biol 2018; 16:51. [PMID: 29720174 PMCID: PMC5930764 DOI: 10.1186/s12915-018-0516-5] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2018] [Accepted: 04/09/2018] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Environmental influences fluctuate throughout the life course of an organism. It is therefore important to understand how the timing of exposure impacts molecular responses. Herein, we examine the responses of two key molecular markers of dietary stress, namely variant-specific methylation at ribosomal DNA (rDNA) and small RNA distribution, including tRNA fragments, in a mouse model of protein restriction (PR) with exposure at pre- and/or post-weaning. RESULTS We first confirm that pre-weaning PR exposure modulates the methylation state of rDNA in a genotype-dependent manner, whereas post-weaning PR exposure has no such effect. Conversely, post-weaning PR induces a shift in small RNA distribution, but there is no effect in the pre-weaning PR model. Intriguingly, mice exposed to PR throughout their lives show neither of these two dietary stress markers, similar to controls. CONCLUSIONS The results show that the timing of the insult affects the nature of the molecular response but also, critically, that 'matching' diet exposure either side of weaning eliminates the stress response at the level of rDNA methylation and small RNA in sperm.
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Affiliation(s)
- Amy F Danson
- The Blizard Institute, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK
| | - Sarah J Marzi
- The Blizard Institute, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK
| | - Robert Lowe
- The Blizard Institute, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK
| | - Michelle L Holland
- Department of Medical and Molecular Genetics, King's College London, Guys Hospital, London, SE1 9RT, UK.
| | - Vardhman K Rakyan
- The Blizard Institute, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK.
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46
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Abstract
RNA metabolic labeling is a method of choice in the study of dynamic changes in the rate of gene transcription and RNA processing. It is particularly applicable to transcription of the ribosomal RNA genes and their processing products due to the very high levels of ribosomal RNA synthesis. Metabolic labeling can detect changes in ribosomal RNA transcription that occur within a few minutes as opposed to the still widely used RT-PCR or Northern blot procedures that measure RNA pool sizes and at best are able to detect changes occurring over several hours or several days. Here, we describe a metabolic labeling technique applicable to the measurement of ribosomal RNA synthesis and processing rates, as well as to the determination of RNA Polymerase I transcription elongation rates.
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47
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Herdman C, Mars JC, Stefanovsky VY, Tremblay MG, Sabourin-Felix M, Lindsay H, Robinson MD, Moss T. A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription. PLoS Genet 2017; 13:e1006899. [PMID: 28715449 PMCID: PMC5536353 DOI: 10.1371/journal.pgen.1006899] [Citation(s) in RCA: 61] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2017] [Revised: 07/31/2017] [Accepted: 06/27/2017] [Indexed: 11/19/2022] Open
Abstract
Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.
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Affiliation(s)
- Chelsea Herdman
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Canada
| | - Jean-Clement Mars
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Canada
| | - Victor Y. Stefanovsky
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
| | - Michel G. Tremblay
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
| | - Marianne Sabourin-Felix
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Canada
| | - Helen Lindsay
- Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland
- SIB Swiss Institute of Bioinformatics, University of Zürich, Zürich, Switzerland
| | - Mark D. Robinson
- Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland
- SIB Swiss Institute of Bioinformatics, University of Zürich, Zürich, Switzerland
| | - Tom Moss
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Canada
- * E-mail:
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48
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Tsekrekou M, Stratigi K, Chatzinikolaou G. The Nucleolus: In Genome Maintenance and Repair. Int J Mol Sci 2017; 18:ijms18071411. [PMID: 28671574 PMCID: PMC5535903 DOI: 10.3390/ijms18071411] [Citation(s) in RCA: 62] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2017] [Revised: 06/22/2017] [Accepted: 06/27/2017] [Indexed: 11/16/2022] Open
Abstract
The nucleolus is the subnuclear membrane-less organelle where rRNA is transcribed and processed and ribosomal assembly occurs. During the last 20 years, however, the nucleolus has emerged as a multifunctional organelle, regulating processes that go well beyond its traditional role. Moreover, the unique organization of rDNA in tandem arrays and its unusually high transcription rates make it prone to unscheduled DNA recombination events and frequent RNA:DNA hybrids leading to DNA double strand breaks (DSBs). If not properly repaired, rDNA damage may contribute to premature disease onset and aging. Deregulation of ribosomal synthesis at any level from transcription and processing to ribosomal subunit assembly elicits a stress response and is also associated with disease onset. Here, we discuss how genome integrity is maintained within nucleoli and how such structures are functionally linked to nuclear DNA damage response and repair giving an emphasis on the newly emerging roles of the nucleolus in mammalian physiology and disease.
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Affiliation(s)
- Maria Tsekrekou
- Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Nikolaou Plastira 100, 70013 Heraklion, Crete, Greece.
- Department of Biology, University of Crete, Vassilika Vouton, 71409 Heraklion, Crete, Greece.
| | - Kalliopi Stratigi
- Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Nikolaou Plastira 100, 70013 Heraklion, Crete, Greece.
- Department of Biology, University of Crete, Vassilika Vouton, 71409 Heraklion, Crete, Greece.
| | - Georgia Chatzinikolaou
- Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Nikolaou Plastira 100, 70013 Heraklion, Crete, Greece.
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49
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Heterochromatin and the molecular mechanisms of ‘parent-of-origin’ effects in animals. J Biosci 2016; 41:759-786. [DOI: 10.1007/s12038-016-9650-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
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50
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Overexpression of Ribosomal RNA in the Development of Human Cervical Cancer Is Associated with rDNA Promoter Hypomethylation. PLoS One 2016; 11:e0163340. [PMID: 27695092 PMCID: PMC5047480 DOI: 10.1371/journal.pone.0163340] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2016] [Accepted: 09/07/2016] [Indexed: 12/30/2022] Open
Abstract
The ribosomal RNA (rRNA) gene encodes rRNA for protein synthesis. Aberrant expression of the rRNA gene has been generally observed in tumor cells and levels of its promoter methylation as an epigenetic regulator affect rRNA gene transcription. The possible relationship between expression and promoter methylation of rDNA has not been examined in human clinical cervical cancer. Here we investigate rRNA gene expression by quantitative real time PCR, and promoter methylation levels by HpaII/MspI digestion and sodium bisulfite sequencing in the development of human cervical cancer. We find that indeed rRNA levels are elevated in most of cervical intraepithelial neoplasia (CIN) specimens as compared with non-cancer tissues. The rDNA promoter region in cervical intraepithelial neoplasia (CIN) tissues reveals significant hypomethylation at cytosines in the context of CpG dinucleotides, accompanied with rDNA chromatin decondensation. Furthermore treatment of HeLa cells with the methylation inhibitor drug 5-aza-2’-deoxycytidine (DAC) demonstrates the negative correlation between the expression of 45S rDNA and the methylation level in the rDNA promoter region. These data suggest that a decrease in rDNA promoter methylation levels can result in an increase of rRNA synthesis in the development of human cervical cancer.
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