|
1
|
West K, Nguyen TN, Tengler K, Kreiling N, Raney K, Ghosal G, Leung J. Autophosphorylation of the Tousled-like kinases TLK1 and TLK2 regulates recruitment to damaged chromatin via PCNA interaction. Nucleic Acids Res 2025; 53:gkae1279. [PMID: 39727191 PMCID: PMC11879137 DOI: 10.1093/nar/gkae1279] [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: 02/28/2024] [Revised: 12/10/2024] [Accepted: 12/13/2024] [Indexed: 12/28/2024] Open
Abstract
Tousled-like kinases 1 and 2 (TLK1 and 2) are cell cycle-regulated serine/threonine kinases that are involved in multiple biological processes. Mutation of TLK1 and 2 confer neurodegenerative diseases. Recent studies demonstrate that TLK1 and 2 are involved in DNA repair. However, there is no direct evidence that TLK1 and 2 function at DNA damage sites. Here, we show that both TLK1 and TLK2 are hyper-autophosphorylated at their N-termini, at least in part, mediated by their homo- or hetero- dimerization. We found that TLK1 and 2 hyper-autophosphorylation suppresses their recruitment to damaged chromatin. Furthermore, both TLK1 and 2 associate with PCNA specifically through their evolutionarily conserved non-canonical PCNA-interacting protein (PIP) box at the N-terminus, and mutation of the PIP-box abolishes their recruitment to DNA damage sites. Mechanistically, the TLK1 and 2 hyper-autophosphorylation masks the PIP-box and negatively regulates their recruitment to the DNA damage site. Overall, our study dissects the detailed genetic regulation of TLK1 and 2 at damaged chromatin, which provides important insights into their emerging roles in DNA repair.
Collapse
Affiliation(s)
- Kirk L West
- Department of Radiation Oncology, University of Arkansas for Medical Sciences, 4301 Markham St, Little Rock, AR 72205, USA
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 Markham St, Little Rock, AR 72205, USA
| | - Tram T N Nguyen
- Department of Radiation Oncology, University of Texas Health and Science Center, 7703 Floyd Curl Dr, San Antonio, TX 78229, USA
| | - Kyle A Tengler
- Department of Radiation Oncology, University of Texas Health and Science Center, 7703 Floyd Curl Dr, San Antonio, TX 78229, USA
| | - Natasha Kreiling
- Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, S 42nd &, Emile St, Omaha, NE 68198, USA
| | - Kevin D Raney
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 Markham St, Little Rock, AR 72205, USA
| | - Gargi Ghosal
- Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, S 42nd &, Emile St, Omaha, NE 68198, USA
| | - Justin W Leung
- Department of Radiation Oncology, University of Texas Health and Science Center, 7703 Floyd Curl Dr, San Antonio, TX 78229, USA
| |
Collapse
|
|
2
|
Bhoir S, De Benedetti A. Beyond the Horizon: Rethinking Prostate Cancer Treatment Through Innovation and Alternative Strategies. Cancers (Basel) 2024; 17:75. [PMID: 39796704 PMCID: PMC11718964 DOI: 10.3390/cancers17010075] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2024] [Revised: 12/10/2024] [Accepted: 12/27/2024] [Indexed: 01/13/2025] Open
Abstract
For nearly a century, fundamental observations that prostate cancer (PCa) cells nearly always require AR stimulation for sustained proliferation have led to a unidirectional quest to abrogate such a pathway. Similarly focused have been efforts to understand AR-driven processes in the context of elevated expression of its target genes, and much less so on products that become overexpressed when AR signaling is suppressed. Treatment with ARSI results in an increased expression of the TLK1B splice variant via a 'translational' derepression driven by the compensatory mTOR activation and consequent activation of the TLK1 > NEK1 > ATR > Chk1 and NEK1 > YAP axes. In due course, this results first in a pro-survival quiescence and then adaptation to ADT and CRPC progression. This constitutes a novel liability for PCa that we have targeted for several years and novel approaches.
Collapse
Affiliation(s)
- Siddhant Bhoir
- Department of Biochemistry and Molecular Biology, LSU Health Shreveport, Shreveport, LA 71103, USA;
- Department of Therapeutic Radiology, School of Medicine, Yale University, 15 York Street, New Haven, CT 06510, USA
| | - Arrigo De Benedetti
- Department of Biochemistry and Molecular Biology, LSU Health Shreveport, Shreveport, LA 71103, USA;
| |
Collapse
|
|
3
|
Shrestha B, Nieminen AI, Matilainen O. Loss of the histone chaperone UNC-85/ASF1 inhibits the epigenome-mediated longevity and modulates the activity of one-carbon metabolism. Cell Stress Chaperones 2024; 29:392-403. [PMID: 38608859 PMCID: PMC11039323 DOI: 10.1016/j.cstres.2024.04.003] [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] [Received: 01/22/2024] [Revised: 04/05/2024] [Accepted: 04/08/2024] [Indexed: 04/14/2024] Open
Abstract
Histone H3/H4 chaperone anti-silencing function 1 (ASF1) is a conserved factor mediating nucleosomal assembly and disassembly, playing crucial roles in processes such as replication, transcription, and DNA repair. Nevertheless, its involvement in aging has remained unclear. Here, we utilized the model organism Caenorhabditis elegans to demonstrate that the loss of UNC-85, the homolog of ASF1, leads to a shortened lifespan in a multicellular organism. Furthermore, we show that UNC-85 is required for epigenome-mediated longevity, as knockdown of the histone H3 lysine K4 methyltransferase ash-2 does not extend the lifespan of unc-85 mutants. In this context, we found that the longevity-promoting ash-2 RNA interference enhances UNC-85 activity by increasing its nuclear localization. Finally, our data indicate that the loss of UNC-85 increases the activity of one-carbon metabolism, and that downregulation of the one-carbon metabolism component dao-3/MTHFD2 partially rescues the short lifespan of unc-85 mutants. Together, these findings reveal UNC-85/ASF1 as a modulator of the central metabolic pathway and a factor regulating a pro-longevity response, thus shedding light on a mechanism of how nucleosomal maintenance associates with aging.
Collapse
Affiliation(s)
- Bideep Shrestha
- The Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Anni I Nieminen
- FIMM Metabolomics Unit, Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland
| | - Olli Matilainen
- The Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland.
| |
Collapse
|
|
4
|
West KL, Kreiling N, Raney KD, Ghosal G, Leung JW. Autophosphorylation of the Tousled-like kinases TLK1 and TLK2 regulates recruitment to damaged chromatin via PCNA interaction. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.04.22.590659. [PMID: 38712247 PMCID: PMC11071368 DOI: 10.1101/2024.04.22.590659] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/08/2024]
Abstract
Tousled-like kinases 1 and 2 (TLK1 and 2) are cell cycle-regulated serine/threonine kinases that are involved in multiple biological processes. Mutation of TLK1 and 2 confer neurodegenerative diseases. Recent studies demonstrate that TLK1 and 2 are involved in DNA repair. However, there is no direct evidence that TLK1 and 2 function at DNA damage sites. Here, we show that both TLK1 and TLK2 are hyper-autophosphorylated at their N-termini, at least in part, mediated by their homo- or hetero-dimerization. We found that TLK1 and 2 hyper-autophosphorylation suppresses their recruitment to damaged chromatin. Furthermore, both TLK1 and 2 associate with PCNA specifically through their evolutionarily conserved non-canonical PCNA-interacting protein (PIP) box at the N-terminus, and mutation of the PIP-box abolishes their recruitment to DNA damage sites. Mechanistically, the TLK1 and 2 hyper-autophosphorylation masks the PIP-box and negatively regulates their recruitment to the DNA damage site. Overall, our study dissects the detailed genetic regulation of TLK1 and 2 at damaged chromatin, which provides important insights into their emerging roles in DNA repair.
Collapse
Affiliation(s)
- Kirk L. West
- Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA
| | - Natasha Kreiling
- Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Kevin D. Raney
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA
| | - Gargi Ghosal
- Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Justin W Leung
- Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA
- Department of Radiation Oncology, University of Texas Health and Science Center, San Antonio, TX, 78229, USA
| |
Collapse
|
|
5
|
Sun RC, Li J, Li YX, Wang HZ, Dal E, Wang ML, Li YX. Tousled-like kinase 1 promotes gastric cancer progression by regulating the tumor growth factor-beta signaling pathway. World J Gastroenterol 2023; 29:5919-5934. [PMID: 38111505 PMCID: PMC10725561 DOI: 10.3748/wjg.v29.i44.5919] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/20/2023] [Revised: 10/23/2023] [Accepted: 11/14/2023] [Indexed: 11/27/2023] Open
Abstract
BACKGROUND The role of Tousled-like kinase 1 (TLK1) in in gastric cancer (GC) remains unclear. AIM To investigate the expression, biological function, and underlying mechanisms of TLK1 in GC. METHODS We measured TLK1 protein expression levels and localized TLK1 in GC cells and tissues by western blot and immunofluorescence, respectively. We transfected various GC cells with lentiviruses to create TLK1 overexpression and knockdown lines and established the functional roles of TLK1 through in vitro colony formation, 5-ethynyl-2`-deoxyuridine, and Transwell assays as well as flow cytometry. We applied bioinformatics to elucidate the signaling pathways associated with TLK1. We performed in vivo validation of TLK1 functions by inducing subcutaneous xenograft tumors in nude mice. RESULTS TLK1 was significantly upregulated in GC cells and tissues compared to their normal counterparts and was localized mainly to the nucleus. TLK1 knockdown significantly decreased colony formation, proliferation, invasion, and migration but increased apoptosis in GC cells. TLK1 overexpression had the opposite effects. Bioinformatics revealed, and subsequent experiments verified, that the tumor growth factor-beta signaling pathway was implicated in TLK1-mediated GC progression. The in vivo assays confirmed that TLK1 promotes tumorigenesis in GC. CONCLUSION The findings of the present study indicated that TLK1 plays a crucial role in GC progression and is, therefore, promising as a therapeutic target against this disease.
Collapse
Affiliation(s)
- Ruo-Chuan Sun
- Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China
| | - Jing Li
- Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China
| | - Ya-Xian Li
- Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China
| | - Hui-Zhen Wang
- Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China
| | - Emre Dal
- Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, United States
| | - Ming-Liang Wang
- Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China
| | - Yong-Xiang Li
- Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China
| |
Collapse
|
|
6
|
Bhoir S, De Benedetti A. Targeting Prostate Cancer, the 'Tousled Way'. Int J Mol Sci 2023; 24:11100. [PMID: 37446279 DOI: 10.3390/ijms241311100] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2023] [Revised: 07/01/2023] [Accepted: 07/03/2023] [Indexed: 07/15/2023] Open
Abstract
Androgen deprivation therapy (ADT) has been the mainstay of prostate cancer (PCa) treatment, with success in developing more effective inhibitors of androgen synthesis and antiandrogens in clinical practice. However, hormone deprivation and AR ablation have caused an increase in ADT-insensitive PCas associated with a poor prognosis. Resistance to ADT arises through various mechanisms, and most castration-resistant PCas still rely on the androgen axis, while others become truly androgen receptor (AR)-independent. Our research identified the human tousled-like kinase 1 (TLK1) as a crucial early mediator of PCa cell adaptation to ADT, promoting androgen-independent growth, inhibiting apoptosis, and facilitating cell motility and metastasis. Although explicit, the growing role of TLK1 biology in PCa has remained underrepresented and elusive. In this review, we aim to highlight the diverse functions of TLK1 in PCa, shed light on the molecular mechanisms underlying the transition from androgen-sensitive (AS) to an androgen-insensitive (AI) disease mediated by TLK1, and explore potential strategies to counteract this process. Targeting TLK1 and its associated signaling could prevent PCa progression to the incurable metastatic castration-resistant PCa (mCRPC) stage and provide a promising approach to treating PCa.
Collapse
Affiliation(s)
- Siddhant Bhoir
- Department of Biochemistry and Molecular Biology, LSU Health Shreveport, Shreveport, LA 71103, USA
| | - Arrigo De Benedetti
- Department of Biochemistry and Molecular Biology, LSU Health Shreveport, Shreveport, LA 71103, USA
| |
Collapse
|
|
7
|
Lee SB, Chang TY, Lee NZ, Yu ZY, Liu CY, Lee HY. Design, synthesis and biological evaluation of bisindole derivatives as anticancer agents against Tousled-like kinases. Eur J Med Chem 2022; 227:113904. [PMID: 34662748 DOI: 10.1016/j.ejmech.2021.113904] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2021] [Revised: 10/02/2021] [Accepted: 10/03/2021] [Indexed: 11/03/2022]
Abstract
This study presents the design, synthesis, and characterization of bisindole molecules as anti-cancer agents against Tousled-like kinases (TLKs). We show that compound 2 composed of an indirubin-3'-oxime group linked with a (N-methylpiperidin-2-yl)ethyl moiety possessed inhibitory activity toward both TLK1 and TLK2 in vitro and diminished the phosphorylation level of the downstream substrate anti-silencing function 1 (ASF1) in replicating cells. The treatment of compound 2 impaired DNA replication, slowed S-phase progression, and triggered DNA damage response in replicating cells. Structure optimization further discovered six derivatives exhibiting potent TLK inhibitory activity and revealed the importance of the tertiary amine-containing moiety of the side chain. Moreover, the derivatives 6, 17, 19, and 20 strongly suppressed the growth of triple-negative breast cancer MDA-MB-231 cells, non-small cell lung cancer A549 cells, and colorectal cancer HCT-116 cells, while normal lung fibroblast MRC5 and IMR90 cells showed a lower response to these compounds. Taken together, this study identifies tertiary amine-linked indirubin-3'-oximes as potent anticancer agents that inhibit TLK activity.
Collapse
Affiliation(s)
- Sung-Bau Lee
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan; Master Program in Clinical Genomics and Proteomics, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Ting-Yu Chang
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Nian-Zhe Lee
- Master Program in Clinical Genomics and Proteomics, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Zih-Yao Yu
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Chi-Yuan Liu
- School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Hsueh-Yun Lee
- Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan; Master Program in Clinical Genomics and Proteomics, College of Pharmacy, Taipei Medical University, Taipei, Taiwan; School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.
| |
Collapse
|
|
8
|
Interaction of TLK1 and AKTIP as a Potential Regulator of AKT Activation in Castration-Resistant Prostate Cancer Progression. PATHOPHYSIOLOGY 2021; 28:339-354. [PMID: 35366279 PMCID: PMC8830441 DOI: 10.3390/pathophysiology28030023] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Revised: 06/14/2021] [Accepted: 07/17/2021] [Indexed: 01/13/2023] Open
Abstract
Prostate cancer (PCa) progression is characterized by the emergence of resistance to androgen deprivation therapy (ADT). AKT/PKB has been directly implicated in PCa progression, often due to the loss of PTEN and activation of PI3K>PDK1>AKT signaling. However, the regulatory network of AKT remains incompletely defined. Here, we describe the functional significance of AKTIP in PCa cell growth. AKTIP, identified in an interactome analysis as a substrate of TLK1B (that itself is elevated following ADT), enhances the association of AKT with PDK1 and its phosphorylation at T308 and S473. The interaction between TLK1 and AKTIP led to AKTIP phosphorylation at T22 and S237. The inactivation of TLK1 led to reduced AKT phosphorylation, which was potentiated with AKTIP knockdown. The TLK1 inhibitor J54 inhibited the growth of the LNCaP cells attributed to reduced AKT activation. However, LNCaP cells that expressed constitutively active, membrane-enriched Myr-AKT (which is expected to be active, even in the absence of AKTIP) were also growth-inhibited with J54. This suggested that other pathways (like TLK1>NEK1>YAP) regulating proliferation are also suppressed and can mediate growth inhibition, despite compensation by Myr-AKT. Nonetheless, further investigation of the potential role of TLK1>AKTIP>AKT in suppressing apoptosis, and conversely its reversal with J54, is warranted.
Collapse
|
|
9
|
Segura-Bayona S, Villamor-Payà M, Attolini CSO, Koenig LM, Sanchiz-Calvo M, Boulton SJ, Stracker TH. Tousled-Like Kinases Suppress Innate Immune Signaling Triggered by Alternative Lengthening of Telomeres. Cell Rep 2021; 32:107983. [PMID: 32755577 PMCID: PMC7408502 DOI: 10.1016/j.celrep.2020.107983] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Revised: 06/30/2020] [Accepted: 07/09/2020] [Indexed: 12/11/2022] Open
Abstract
The Tousled-like kinases 1 and 2 (TLK1/2) control histone deposition through the ASF1 histone chaperone and influence cell cycle progression and genome maintenance, yet the mechanisms underlying TLK-mediated genome stability remain uncertain. Here, we show that TLK loss results in severe chromatin decompaction and altered genome accessibility, particularly affecting heterochromatic regions. Failure to maintain heterochromatin increases spurious transcription of repetitive elements and induces features of alternative lengthening of telomeres (ALT). TLK depletion culminates in a cGAS-STING-TBK1-mediated innate immune response that is independent of replication-stress signaling and attenuated by the depletion of factors required to produce extra-telomeric DNA. Analysis of human cancers reveals that chromosomal instability correlates with high TLK2 and low STING levels in many cohorts. Based on these findings, we propose that high TLK levels contribute to immune evasion in chromosomally unstable and ALT+ cancers.
TLK-deficient cells have increased accessibility at heterochromatin regions TLK1/2 suppress spurious transcription and telomere hyper-recombination Extra-telomeric DNA generated upon TLK loss promotes innate immune signaling cGAS-STING-TBK1 signaling in TLK-deficient cells is independent of replication stress
Collapse
Affiliation(s)
- Sandra Segura-Bayona
- Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona 08028, Spain; The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
| | - Marina Villamor-Payà
- Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona 08028, Spain
| | - Camille Stephan-Otto Attolini
- Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona 08028, Spain
| | - Lars M Koenig
- Division of Clinical Pharmacology, University Hospital, LMU Munich, 80337 Munich, Germany
| | - Maria Sanchiz-Calvo
- Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona 08028, Spain
| | - Simon J Boulton
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Travis H Stracker
- Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona 08028, Spain.
| |
Collapse
|
|
10
|
Xu X, Su S, Cao Y, Zhao S, Li W, Qin Y. Variation analysis of tousled like kinase 1 gene in patients with sporadic premature ovarian insufficiency. Gynecol Endocrinol 2020; 36:33-35. [PMID: 31362519 DOI: 10.1080/09513590.2019.1630606] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/26/2022] Open
Abstract
Tousled like kinase 1 (TLK1), a member of DNA repair family, participates in the regulation of chromatin assembly and is associated with early menopause and premature ovarian insufficiency (POI) in European women. However, whether the sequence variant in the TLK1 gene was causative for POI is still elusive. Here we performed direct sequencing of the TLK1 gene in 192 patients with sporadic POI. All exons and exon-intron boundaries of TLK1 were amplified and sequenced. Six known single-nucleotide polymorphisms were identified in POI, including rs149844334, rs11553951, rs757600673, rs2277339, rs113416007 and rs17283147. No novel variant was identified, which indicates that sequence variants in the coding region of TLK1 might be uncommon in Chinese women with POI. The role of TLK1 in POI pathogenesis needs to be further explored in larger cohorts from Chinese and other ethnic populations.
Collapse
Affiliation(s)
- Xiaofei Xu
- Center for Reproductive Medicine, Shandong University National Research Center for Assisted Reproductive Technology and Reproductive Genetics, The Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China
- Key Laboratory of Assisted Reproduction, Ministry of Education, Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China
| | - Shizhen Su
- Center for Reproductive Medicine, Shandong University National Research Center for Assisted Reproductive Technology and Reproductive Genetics, The Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China
| | - Yongzhi Cao
- Center for Reproductive Medicine, Shandong University National Research Center for Assisted Reproductive Technology and Reproductive Genetics, The Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China
| | - Shidou Zhao
- Center for Reproductive Medicine, Shandong University National Research Center for Assisted Reproductive Technology and Reproductive Genetics, The Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China
| | - Weiping Li
- Ren Ji Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China
| | - Yingying Qin
- Center for Reproductive Medicine, Shandong University National Research Center for Assisted Reproductive Technology and Reproductive Genetics, The Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China
| |
Collapse
|
|
11
|
Segura-Bayona S, Stracker TH. The Tousled-like kinases regulate genome and epigenome stability: implications in development and disease. Cell Mol Life Sci 2019; 76:3827-3841. [PMID: 31302748 PMCID: PMC11105529 DOI: 10.1007/s00018-019-03208-z] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2019] [Revised: 06/05/2019] [Accepted: 06/24/2019] [Indexed: 02/06/2023]
Abstract
The Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine-threonine kinases that have been implicated in DNA replication, DNA repair, transcription, chromatin structure, viral latency, cell cycle checkpoint control and chromosomal stability in various organisms. The functions of the TLKs appear to depend largely on their ability to regulate the H3/H4 histone chaperone ASF1, although numerous TLK substrates have been proposed. Over the last few years, a clearer picture of TLK function has emerged through the identification of new partners, the definition of specific roles in development and the elucidation of their structural and biochemical properties. In addition, the TLKs have been clearly linked to human disease; both TLK1 and TLK2 are frequently amplified in human cancers and TLK2 mutations have been identified in patients with neurodevelopmental disorders characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. A better understanding of the substrates, regulation and diverse roles of the TLKs is needed to understand their functions in neurodevelopment and determine if they are viable targets for cancer therapy. In this review, we will summarize current knowledge of TLK biology and its potential implications in development and disease.
Collapse
Affiliation(s)
- Sandra Segura-Bayona
- Department of Oncology, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, C/Baldiri Reixac 10, 08028, Barcelona, Spain.
- The Francis Crick Institute, London, UK.
| | - Travis H Stracker
- Department of Oncology, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, C/Baldiri Reixac 10, 08028, Barcelona, Spain.
| |
Collapse
|
|
12
|
Inactive Tlk associating with Tak1 increases p38 MAPK activity to prolong the G2 phase. Sci Rep 2019; 9:1885. [PMID: 30760733 PMCID: PMC6374402 DOI: 10.1038/s41598-018-36137-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2018] [Accepted: 11/09/2018] [Indexed: 12/16/2022] Open
Abstract
To guard genome integrity, response mechanisms coordinately execute the G2/M checkpoint in responding to stress. p38 MAPK is activated to prolong the G2 phase for completion of damage repair. Tlk activity is required for DNA repair, chromosome segregation and G2 recovery. However, the involvement of Tlk in G2 recovery differs from previous findings that Tlk overexpression delays the G2/M transition. To clarify this difference, genetic interaction experiments were performed using the second mitotic wave as model system. The results indicate that Tlk overexpression prolongs the G2 phase through p38 MAPK activation, independent of Tlk kinase activity. The results of co-immunoprecipitation, database search and RNAi screening suggest that eEF1α1 and Hsc70-5 links Tlk to Tak1. Reduced gene activities of Tlk, Hsc70-5, eEF1α1 and/or Tak1 couldn’t prolong the G2 phase induced by heat shock, indicating that these proteins work together to elevate p38 MAPK activity. In contrast, a high level of wild type Tlk decreases phosphorylated p38 MAPK levels. Thus, the difference is explained by a dual function of Tlk. When under stress, inactive Tlk increases p38 MAPK activity to prolong the G2 phase, and then activated Tlk modulates activities of p38 MAPK and Asf1 to promote G2 recovery afterwards.
Collapse
|
|
13
|
Shibata Y, Seki Y, Nishiwaki K. Maintenance of cell fates and regulation of the histone variant H3.3 by TLK kinase in Caenorhabditis elegans. Biol Open 2019; 8:bio.038448. [PMID: 30635266 PMCID: PMC6361200 DOI: 10.1242/bio.038448] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Cell-fate maintenance is important to preserve the variety of cell types that are essential for the formation and function of tissues. We previously showed that the acetylated histone-binding protein BET-1 maintains cell fate by recruiting the histone variant H2A.z. Here, we report that Caenorhabditis elegans TLK-1 and the histone H3 chaperone CAF1 prevent the accumulation of histone variant H3.3. In addition, TLK-1 and CAF1 maintain cell fate by repressing ectopic expression of transcription factors that induce cell-fate specification. Genetic analyses suggested that TLK-1 and BET-1 act in parallel pathways. In tlk-1 mutants, the loss of SIN-3, which promotes histone acetylation, suppressed a defect in cell-fate maintenance in a manner dependent on MYST family histone acetyltransferase MYS-2 and BET-1. sin-3 mutation also suppressed abnormal H3.3 incorporation. Thus, we propose a hypothesis that the regulation and interaction of histone variants play crucial roles in cell-fate maintenance through the regulation of selector genes. Summary: Histone H3 chaperone CAF1 maintains cell fate by repressing ectopic expression of genes for cell fate-specifying transcription factors. Accumulation of histone variant H3.3 correlates with defects in cell-fate maintenance.
Collapse
Affiliation(s)
- Yukimasa Shibata
- School of Science and Technology, Department of Bioscience, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan
| | - Yoshiyuki Seki
- School of Science and Technology, Department of Bioscience, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan
| | - Kiyoji Nishiwaki
- School of Science and Technology, Department of Bioscience, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan
| |
Collapse
|
|
14
|
Molecular basis of Tousled-Like Kinase 2 activation. Nat Commun 2018; 9:2535. [PMID: 29955062 PMCID: PMC6023931 DOI: 10.1038/s41467-018-04941-y] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2017] [Accepted: 06/06/2018] [Indexed: 12/21/2022] Open
Abstract
Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs. The Tousled-like kinase (TLKs) family belongs to a distinct branch of Ser/Thr kinases that exhibit the highest levels of activity during DNA replication. Here the authors present the crystal structure of the kinase domain from human TLK2 and propose an activation model for TLK2 based on biochemical and phosphoproteomics experiments.
Collapse
|
|
15
|
Lee J, Kim MS, Park SH, Jang YK. Tousled-like kinase 1 is a negative regulator of core transcription factors in murine embryonic stem cells. Sci Rep 2018; 8:334. [PMID: 29321513 PMCID: PMC5762884 DOI: 10.1038/s41598-017-18628-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2017] [Accepted: 12/13/2017] [Indexed: 11/19/2022] Open
Abstract
Although the differentiation of pluripotent cells in embryonic stem cells (ESCs) is often associated with protein kinase-mediated signaling pathways and Tousled-like kinase 1 (Tlk1) is required for development in several species, the role of Tlk1 in ESC function remains unclear. Here, we used mouse ESCs to study the function of Tlk1 in pluripotent cells. The knockdown (KD)-based Tlk1-deficient cells showed that Tlk1 is not essential for ESC self-renewal in an undifferentiated state. However, Tlk1-KD cells formed irregularly shaped embryoid bodies and induced resistance to differentiation cues, indicating their failure to differentiate into an embryoid body. Consistent with their failure to differentiate, Tlk1-KD cells failed to downregulate the expression of undifferentiated cell markers including Oct4, Nanog, and Sox2 during differentiation, suggesting a negative role of Tlk1. Interestingly, Tlk1 overexpression sufficiently downregulated the expression of core pluripotency factors possibly irrespective of its kinase activity, thereby leading to a partial loss of self-renewal ability even in an undifferentiated state. Moreover, Tlk1 overexpression caused severe growth defects and G2/M phase arrest as well as apoptosis. Collectively, our data suggest that Tlk1 negatively regulates the expression of pluripotency factors, thereby contributing to the scheduled differentiation of mouse ESCs.
Collapse
Affiliation(s)
- Jina Lee
- Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea.,Initiative for Biological Function and Systems, Yonsei University, Seoul, 03722, Republic of Korea
| | - Min Seong Kim
- Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea.,Initiative for Biological Function and Systems, Yonsei University, Seoul, 03722, Republic of Korea
| | - Su Hyung Park
- Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea.,Initiative for Biological Function and Systems, Yonsei University, Seoul, 03722, Republic of Korea.,Center for Genomic Integrity, Institute for Basic Science, Ulsan National Institute of Science and Technology, UNIST-gil 50, Ulsan, 689-798, Republic of Korea
| | - Yeun Kyu Jang
- Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea. .,Initiative for Biological Function and Systems, Yonsei University, Seoul, 03722, Republic of Korea.
| |
Collapse
|
|
16
|
Differential requirements for Tousled-like kinases 1 and 2 in mammalian development. Cell Death Differ 2017; 24:1872-1885. [PMID: 28708136 DOI: 10.1038/cdd.2017.108] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2016] [Revised: 06/02/2017] [Accepted: 06/05/2017] [Indexed: 12/20/2022] Open
Abstract
The regulation of chromatin structure is critical for a wide range of essential cellular processes. The Tousled-like kinases, TLK1 and TLK2, regulate ASF1, a histone H3/H4 chaperone, and likely other substrates, and their activity has been implicated in transcription, DNA replication, DNA repair, RNA interference, cell cycle progression, viral latency, chromosome segregation and mitosis. However, little is known about the functions of TLK activity in vivo or the relative functions of the highly similar TLK1 and TLK2 in any cell type. To begin to address this, we have generated Tlk1- and Tlk2-deficient mice. We found that while TLK1 was dispensable for murine viability, TLK2 loss led to late embryonic lethality because of placental failure. TLK2 was required for normal trophoblast differentiation and the phosphorylation of ASF1 was reduced in placentas lacking TLK2. Conditional bypass of the placental phenotype allowed the generation of apparently healthy Tlk2-deficient mice, while only the depletion of both TLK1 and TLK2 led to extensive genomic instability, indicating that both activities contribute to genome maintenance. Our data identifies a specific role for TLK2 in placental function during mammalian development and suggests that TLK1 and TLK2 have largely redundant roles in genome maintenance.
Collapse
|
|
17
|
Singh V, Connelly ZM, Shen X, De Benedetti A. Identification of the proteome complement of humanTLK1 reveals it binds and phosphorylates NEK1 regulating its activity. Cell Cycle 2017; 16:915-926. [PMID: 28426283 DOI: 10.1080/15384101.2017.1314421] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022] Open
Abstract
The Tousled Like kinases (TLKs) are involved in numerous cellular functions, including the DNA Damage Response (DDR), but only a handful of substrates have been identified thus far. Through a novel proteomic screen, we have now identified 165 human proteins interacting with TLK1, and we have focused this work on NEK1 because of its known role in the DDR, upstream of ATR and Chk1. TLK1 and NEK1 were found to interact by coIP, and their binding is strengthened following exposure of cells to H2O2. Following incubation with doxorubicin, TLK1 and NEK1 relocalize with nuclear repair foci along with γH2AX. TLK1 phosphorylated NEK1 at T141, which lies in the kinase domain, and caused an increase in its activity. Following DNA damage, addition of the TLK1 inhibitor, THD, or overexpression of NEK1-T141A mutant impaired ATR and Chk1 activation, indicating the existence of a TLK1>NEK1>ATR>Chk1 pathway. Indeed, overexpression of the NEK1-T141A mutant resulted in an altered cell cycle response after exposure of cells to oxidative stress, including bypass of G1 arrest and implementation of an intra S-phase checkpoint.
Collapse
Affiliation(s)
- Vibha Singh
- a Department of Biochemistry and Molecular Biology , Louisiana State University Health Sciences Center , Shreveport , LA , USA
| | - Zachary M Connelly
- a Department of Biochemistry and Molecular Biology , Louisiana State University Health Sciences Center , Shreveport , LA , USA
| | - Xinggui Shen
- b Pathology and Translational Pathobiology , Louisiana State University Health Sciences Center , Shreveport , LA , USA
| | - Arrigo De Benedetti
- a Department of Biochemistry and Molecular Biology , Louisiana State University Health Sciences Center , Shreveport , LA , USA
| |
Collapse
|
|
18
|
Awate S, De Benedetti A. TLK1B mediated phosphorylation of Rad9 regulates its nuclear/cytoplasmic localization and cell cycle checkpoint. BMC Mol Biol 2016; 17:3. [PMID: 26860083 PMCID: PMC4746922 DOI: 10.1186/s12867-016-0056-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2015] [Accepted: 01/26/2016] [Indexed: 01/09/2023] Open
Abstract
Background The Tousled like kinase 1B (TLK1B) is critical for DNA repair and survival of cells. Upon DNA damage, Chk1 phosphorylates TLK1B at S457 leading to its transient inhibition. Once TLK1B regains its kinase activity it phosphorylates Rad9 at S328. In this work we investigated the significance of this mechanism by overexpressing mutant TLK1B in which the inhibitory phosphorylation site was eliminated. Results and discussion These cells expressing TLK1B resistant to DNA damage showed constitutive phosphorylation of Rad9 S328 that occurred even in the presence of hydroxyurea (HU), and this resulted in a delayed checkpoint recovery. One possible explanation was that premature phosphorylation of Rad9 caused its dissociation from 9-1-1 at stalled replication forks, resulting in their collapse and prolonged activation of the S-phase checkpoint. We found that phosphorylation of Rad9 at S328 results in its dissociation from chromatin and redistribution to the cytoplasm. This results in double stranded breaks formation with concomitant activation of ATM and phosphorylation of H2AX. Furthermore, a Rad9 (S328D) phosphomimic mutant was exclusively localized to the cytoplasm and not the chromatin. Another Rad9 phosphomimic mutant (T355D), which is also a site phosphorylated by TLK1, localized normally. In cells expressing the mutant TLK1B treated with HU, Rad9 association with Hus1 and WRN was greatly reduced, suggesting again that its phosphorylation causes its premature release from stalled forks. Conclusions We propose that normally, the inactivation of TLK1B following replication arrest and genotoxic stress functions to allow the retention of 9-1-1 at the sites of damage or stalled forks. Following reactivation of TLK1B, whose synthesis is concomitantly induced by genotoxins, Rad9 is hyperphosphorylated at S328, resulting in its dissociation and inactivation of the checkpoint that occurs once repair is complete. Electronic supplementary material The online version of this article (doi:10.1186/s12867-016-0056-x) contains supplementary material, which is available to authorized users.
Collapse
Affiliation(s)
- Sanket Awate
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA, 71130, USA.
| | - Arrigo De Benedetti
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA, 71130, USA.
| |
Collapse
|
|
19
|
Yeh TH, Huang SY, Lan WY, Liaw GJ, Yu JY. Modulation of cell morphogenesis by tousled-like kinase in the Drosophila follicle cell. Dev Dyn 2015; 244:852-65. [PMID: 25981356 DOI: 10.1002/dvdy.24292] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2014] [Revised: 04/30/2015] [Accepted: 05/07/2015] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Tousled-like kinase (Tlk) is a conserved serine/threonine kinase regulating DNA replication, chromatin assembly, and DNA repair. Previous studies have suggested that Tlk is involved in cell morphogenesis in vitro. In addition, tlk genetically interact with Rho1, which encodes a key regulator of the cytoskeleton. However, whether Tlk plays a physiological role in cell morphogenesis and cytoskeleton rearrangement remains unknown. RESULTS In tlk mutant follicle cells, area of the apical domain was reduced. The density of microtubules was increased in tlk mutant cells. The density of actin filaments was increased in the apical region and decreased in the basal region. Because area of the apical domain was reduced, we examined the levels of proteins located in the apical region by using immunofluorescence. The fluorescence intensities of two adherens junction proteins Armadillo (Arm) and DE-cadherin (DE-cad), atypical protein kinase C (aPKC), and Notch, were all increased in tlk mutant cells. The basolateral localized Discs large (Dlg) shifted apically in tlk mutant cells. CONCLUSIONS Increase of protein densities in the apical region might be resulted from disruption of the cytoskeleton and shrinkage of the apical domain. Together, these data suggest a novel role of Tlk in maintaining cell morphology, possibly through modulating the cytoskeleton.
Collapse
Affiliation(s)
- Tsung-Han Yeh
- Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan
| | - Shu-Yu Huang
- Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan
| | - Wan-Yu Lan
- Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan
| | - Gwo-Jen Liaw
- Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan
| | - Jenn-Yah Yu
- Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.,Brain Research Center, National Yang-Ming University, Taipei, Taiwan
| |
Collapse
|
|
20
|
Tousled-like kinase mediated a new type of cell death pathway in Drosophila. Cell Death Differ 2015; 23:146-57. [PMID: 26088162 DOI: 10.1038/cdd.2015.77] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2014] [Revised: 04/21/2015] [Accepted: 05/05/2015] [Indexed: 01/09/2023] Open
Abstract
Programmed cell death (PCD) has an important role in sculpting organisms during development. However, much remains to be learned about the molecular mechanism of PCD. We found that ectopic expression of tousled-like kinase (tlk) in Drosophila initiated a new type of cell death. Furthermore, the TLK-induced cell death is likely to be independent of the canonical caspase pathway and other known caspase-independent pathways. Genetically, atg2 RNAi could rescue the TLK-induced cell death, and this function of atg2 was likely distinct from its role in autophagy. In the developing retina, loss of tlk resulted in reduced PCD in the interommatidial cells (IOCs). Similarly, an increased number of IOCs was present in the atg2 deletion mutant clones. However, double knockdown of tlk and atg2 by RNAi did not have a synergistic effect. These results suggested that ATG2 may function downstream of TLK. In addition to a role in development, tlk and atg2 RNAi could rescue calcium overload-induced cell death. Together, our results suggest that TLK mediates a new type of cell death pathway that occurs in both development and calcium cytotoxicity.
Collapse
|
|
21
|
Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication. Nat Commun 2014; 5:3394. [PMID: 24598821 PMCID: PMC3977046 DOI: 10.1038/ncomms4394] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2013] [Accepted: 02/05/2014] [Indexed: 12/25/2022] Open
Abstract
During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-Like Kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and dissect how phosphorylation impacts on human Asf1 function. The divergent C-terminal tail of Asf1a is phosphorylated at several sites and this is required for timely progression through S phase. Consistent with this, biochemical analysis of wild-type and phosphomimetic Asf1a shows that phosphorylation enhances binding to histones and the downstream chaperones CAF-1 and HIRA. Moreover, we find that TLK phosphorylation of Asf1a is induced in cells experiencing deficiency of new histones and that TLK interaction with Asf1a involves its histone-binding pocket. We thus propose that TLK signaling promotes histone supply in S phase by targeting histone-free Asf1 and stimulating its ability to shuttle histones to sites of chromatin assembly.
Collapse
|
|
22
|
Ronald S, Awate S, Rath A, Carroll J, Galiano F, Dwyer D, Kleiner-Hancock H, Mathis JM, Vigod S, De Benedetti A. Phenothiazine Inhibitors of TLKs Affect Double-Strand Break Repair and DNA Damage Response Recovery and Potentiate Tumor Killing with Radiomimetic Therapy. Genes Cancer 2013; 4:39-53. [PMID: 23946870 DOI: 10.1177/1947601913479020] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2012] [Accepted: 01/25/2013] [Indexed: 11/16/2022] Open
Abstract
The Tousled-like kinases (TLKs) are involved in chromatin assembly, DNA repair, and transcription. Two TLK genes exist in humans, and their expression is often dysregulated in cancer. TLKs phosphorylate Asf1 and Rad9, regulating double-strand break (DSB) repair and the DNA damage response (DDR). TLKs maintain genomic stability and are important therapeutic intervention targets. We identified specific inhibitors of TLKs from several compound libraries, some of which belong to the family of phenothiazine antipsychotics. The inhibitors prevented the TLK-mediated phosphorylation of Rad9(S328) and impaired checkpoint recovery and DSB repair. The inhibitor thioridazine (THD) potentiated tumor killing with chemotherapy and also had activity alone. Staining for γ-H2AX revealed few positive cells in untreated tumors, but large numbers in mice treated with low doxorubicin or THD alone, possibly the result of the accumulation of DSBs that are not promptly repaired as they may occur in the harsh tumor growth environment.
Collapse
Affiliation(s)
- Sharon Ronald
- Department of Biochemistry and Molecular Biology and the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
23
|
Li L, Sase A, Patil S, Sunyer B, Höger H, Smalla KH, Stork O, Lubec G. Distinct set of kinases induced after retrieval of spatial memory discriminate memory modulation processes in the mouse hippocampus. Hippocampus 2013; 23:672-83. [PMID: 23536525 DOI: 10.1002/hipo.22127] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/20/2013] [Indexed: 12/15/2022]
Abstract
Protein phosphorylation and dephosphorylation events play a key role in memory formation and various protein kinases and phosphatases have been firmly associated with memory performance. Here, we determined expression changes of protein kinases and phosphatases following retrieval of spatial memory in CD1 mice in a Morris Water Maze task, using antibody microarrays and confirmatory Western blot. Comparing changes following single and consecutive retrieval, we identified stably and differentially expressed kinases, some of which have never been implicated before in memory functions. On the basis of these findings we define a small signaling network associated with spatial memory retrieval. Moreover, we describe differential regulation and correlation of expression levels with behavioral performance of polo-like kinase 1. Together with its recently observed genetic association to autism-spectrum disorders our data suggest a role of this kinase in balancing preservation and flexibility of learned behavior.
Collapse
Affiliation(s)
- Lin Li
- Department of Pediatrics, Medical University of Vienna, Vienna, Austria
| | | | | | | | | | | | | | | |
Collapse
|
|
24
|
De Benedetti A. The Tousled-Like Kinases as Guardians of Genome Integrity. ISRN MOLECULAR BIOLOGY 2012; 2012:627596. [PMID: 23869254 PMCID: PMC3712517 DOI: 10.5402/2012/627596] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The Tousled-like kinases (TLKs) function in processes of chromatin assembly, including replication, transcription, repair, and chromosome segregation. TLKs interact specifically (and phosphorylate) with the chromatin assembly factor Asf1, a histone H3-H4 chaperone, histone H3 itself at Ser10, and also Rad9, a key protein involved in DNA repair and cell cycle signaling following DNA damage. These interactions are believed to be responsible for the action of TLKs in double-stranded break repair and radioprotection and also in the propagation of the DNA damage response. Hence, I propose that TLKs play key roles in maintenance of genome integrity in many organisms of both kingdoms. In this paper, I highlight key issues of the known roles of these proteins, particularly in the context of DNA repair (IR and UV), their possible relevance to genome integrity and cancer development, and as possible targets for intervention in cancer management.
Collapse
Affiliation(s)
- Arrigo De Benedetti
- Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA
| |
Collapse
|
|
25
|
Caenorhabditis elegans dosage compensation regulates histone H4 chromatin state on X chromosomes. Mol Cell Biol 2012; 32:1710-9. [PMID: 22393255 DOI: 10.1128/mcb.06546-11] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Dosage compensation equalizes X-linked gene expression between the sexes. This process is achieved in Caenorhabditis elegans by hermaphrodite-specific, dosage compensation complex (DCC)-mediated, 2-fold X chromosome downregulation. How the DCC downregulates gene expression is not known. By analyzing the distribution of histone modifications in nuclei using quantitative fluorescence microscopy, we found that H4K16 acetylation (H4K16ac) is underrepresented and H4K20 monomethylation (H4K20me1) is enriched on hermaphrodite X chromosomes in a DCC-dependent manner. Depletion of H4K16ac also requires the conserved histone deacetylase SIR-2.1, while enrichment of H4K20me1 requires the activities of the histone methyltransferases SET-1 and SET-4. Our data suggest that the mechanism of dosage compensation in C. elegans involves redistribution of chromatin-modifying activities, leading to a depletion of H4K16ac and an enrichment of H4K20me1 on the X chromosomes. These results support conserved roles for histone H4 chromatin modification in worm dosage compensation analogous to those seen in flies, using similar elements and opposing strategies to achieve differential 2-fold changes in X-linked gene expression.
Collapse
|
|
26
|
Finding a balance: how diverse dosage compensation strategies modify histone h4 to regulate transcription. GENETICS RESEARCH INTERNATIONAL 2011; 2012:795069. [PMID: 22567401 PMCID: PMC3335593 DOI: 10.1155/2012/795069] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/15/2011] [Accepted: 08/08/2011] [Indexed: 01/21/2023]
Abstract
Dosage compensation balances gene expression levels between the sex chromosomes and autosomes and sex-chromosome-linked gene expression levels between the sexes. Different dosage compensation strategies evolved in different lineages, but all involve changes in chromatin. This paper discusses our current understanding of how modifications of the histone H4 tail, particularly changes in levels of H4 lysine 16 acetylation and H4 lysine 20 methylation, can be used in different contexts to either modulate gene expression levels twofold or to completely inhibit transcription.
Collapse
|
|
27
|
Ronald S, Sunavala-Dossabhoy G, Adams L, Williams B, De Benedetti A. The expression of Tousled kinases in CaP cell lines and its relation to radiation response and DSB repair. Prostate 2011; 71:1367-73. [PMID: 21647934 DOI: 10.1002/pros.21358] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/14/2010] [Accepted: 01/14/2011] [Indexed: 12/22/2022]
Abstract
BACKGROUND The Tousled-like kinases (TLKs) function in processes of chromatin assembly, including replication, transcription, repair, and chromosome segregation. TLK1/1B interacts specifically with the chromatin assembly factor Asf1, a histone H3-H4 chaperone, and with Rad9, a protein involved in DNA repair, and these interactions are believed to be responsible for the action of TLKs in double-strand break repair and radioprotection. METHODS Western blotting and RT-PCR were used to analyze the expression of TLK1, TLK1B, and TLK2 in a panel of prostate cancer (CaP) cell lines. The pattern of radiotolerance in the cell lines was analyzed in parallel. DU145 and PC-3 cells were also probed with assays utilizing transfected plasmids that could be cleaved in vivo with adeno-expressed HO nuclease to assess the potential contribution of TLK1/1B in DSB repair. RESULTS This is the first report of TLKs' expression in a panel of CaP cell lines and their relationship to radioresistance. Furthermore, expression of TLK1B in non-expressing PC-3 cells rendered them highly resistant to radiation, and conversely, knockdown to TLK1/1B in expressing DU145 reduced their radiotolerance. CONCLUSIONS TLKs appear to be intimately linked to the pattern of resistance to DNA damage, and specifically DSBs, a finding that was not reported before for any cell lines, and certainly not systematically for human prostate cell lines.
Collapse
Affiliation(s)
- Sharon Ronald
- Department of Biochemistry and Molecular Biology and the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA
| | | | | | | | | |
Collapse
|
|
28
|
Silencing of Tousled-like kinase 1 sensitizes cholangiocarcinoma cells to cisplatin-induced apoptosis. Cancer Lett 2010; 296:27-34. [DOI: 10.1016/j.canlet.2010.03.011] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2010] [Revised: 03/11/2010] [Accepted: 03/17/2010] [Indexed: 12/19/2022]
|
|
29
|
Furuhashi H, Takasaki T, Rechtsteiner A, Li T, Kimura H, Checchi PM, Strome S, Kelly WG. Trans-generational epigenetic regulation of C. elegans primordial germ cells. Epigenetics Chromatin 2010; 3:15. [PMID: 20704745 PMCID: PMC3146070 DOI: 10.1186/1756-8935-3-15] [Citation(s) in RCA: 92] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2010] [Accepted: 08/12/2010] [Indexed: 12/21/2022] Open
Abstract
Background The processes through which the germline maintains its continuity across generations has long been the focus of biological research. Recent studies have suggested that germline continuity can involve epigenetic regulation, including regulation of histone modifications. However, it is not clear how histone modifications generated in one generation can influence the transcription program and development of germ cells of the next. Results We show that the histone H3K36 methyltransferase maternal effect sterile (MES)-4 is an epigenetic modifier that prevents aberrant transcription activity in Caenorhabditis elegans primordial germ cells (PGCs). In mes-4 mutant PGCs, RNA Pol II activation is abnormally regulated and the PGCs degenerate. Genetic and genomewide analyses of MES-4-mediated H3K36 methylation suggest that MES-4 activity can operate independently of ongoing transcription, and may be predominantly responsible for maintenance methylation of H3K36 in germline-expressed loci. Conclusions Our data suggest a model in which MES-4 helps to maintain an 'epigenetic memory' of transcription that occurred in germ cells of previous generations, and that MES-4 and its epigenetic product are essential for normal germ cell development.
Collapse
|
|
30
|
Yeh CH, Yang HJ, Lee IJ, Wu YC. Caenorhabditis elegans TLK-1 controls cytokinesis by localizing AIR-2/Aurora B to midzone microtubules. Biochem Biophys Res Commun 2010; 400:187-93. [PMID: 20705056 DOI: 10.1016/j.bbrc.2010.07.146] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2010] [Accepted: 07/24/2010] [Indexed: 11/26/2022]
Abstract
Defects in chromosome condensation, segregation or cytokinesis during mitosis disrupt genome integrity and cause organismal death or tumorigenesis. The conserved kinase AIR-2/Aurora B is required for normal execution of all these important mitotic events in Caenorhabditis elegans. TLK-1 has been recently shown to be a substrate and activator of AIR-2 in the presence of another AIR-2 activator ICP-1/INCENP, and to cooperate with AIR-2 to ensure proper mitotic chromosome segregation. However, whether TLK-1 may contribute to chromosome condensation or cytokinesis is unclear. A time-lapse microscopy analysis showed that tlk-1 mutants are defective in chromosome condensation and cytokinesis, in addition to chromosome segregation, during mitosis. Our data indicate that TLK-1 contributes to chromosome condensation and segregation, at least in part, in a manner that is distinct from the ICP-1-mediated mechanism and does not involve loading AIR-2 or condensin proteins to mitotic chromosomes. Moreover, TLK-1 functions in cytokinesis by localizing AIR-2 to the midzone microtubules. The localization pattern of TLK-1 is different from those of ICP-1 and AIR-2, revealing differences in dynamic regulation and association of TLK-1 and ICP-1 towards AIR-2 in vivo. Interestingly, human TLK2 could functionally substitute for tlk-1, suggesting that the mitotic roles of TLK members might be evolutionarily conserved.
Collapse
Affiliation(s)
- Chan-Hsien Yeh
- Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan
| | | | | | | |
Collapse
|
|
31
|
Birge LM, Pitts ML, Richard BH, Wilkinson GS. Length polymorphism and head shape association among genes with polyglutamine repeats in the stalk-eyed fly, Teleopsis dalmanni. BMC Evol Biol 2010; 10:227. [PMID: 20663190 PMCID: PMC3055267 DOI: 10.1186/1471-2148-10-227] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2010] [Accepted: 07/27/2010] [Indexed: 12/03/2022] Open
Abstract
BACKGROUND Polymorphisms of single amino acid repeats (SARPs) are a potential source of genetic variation for rapidly evolving morphological traits. Here, we characterize variation in and test for an association between SARPs and head shape, a trait under strong sexual selection, in the stalk-eyed fly, Teleopsis dalmanni. Using an annotated expressed sequence tag database developed from eye-antennal imaginal disc tissues in T. dalmanni we identified 98 genes containing nine or more consecutive copies of a single amino acid. We then quantify variation in length and allelic diversity for 32 codon and 15 noncodon repeat regions in a large outbred population. We also assessed the frequency with which amino acid repeats are either gained or lost by identifying sequence similarities between T. dalmanni SARP loci and their orthologs in Drosophila melanogaster. Finally, to identify SARP containing genes that may influence head development we conducted a two-generation association study after assortatively mating for extreme relative eyespan. RESULTS We found that glutamine repeats occur more often than expected by amino acid abundance among 3,400 head development genes in T. dalmanni and D. melanogaster. Furthermore, glutamine repeats occur disproportionately in transcription factors. Loci with glutamine repeats exhibit heterozygosities and allelic diversities that do not differ from noncoding dinucleotide microsatellites, including greater variation among X-linked than autosomal regions. In the majority of cases, repeat tracts did not overlap between T. dalmanni and D. melanogaster indicating that large glutamine repeats are gained or lost frequently during Dipteran evolution. Analysis of covariance reveals a significant effect of parental genotype on mean progeny eyespan, with body length as a covariate, at six SARP loci [CG33692, ptip, band4.1 inhibitor LRP interactor, corto, 3531953:1, and ecdysone-induced protein 75B (Eip75B)]. Mixed model analysis of covariance using the eyespan of siblings segregating for repeat length variation confirms that significant genotype-phenotype associations exist for at least one sex at five of these loci and for one gene, CG33692, longer repeats were associated with longer relative eyespan in both sexes. CONCLUSION Among genes expressed during head development in stalk-eyed flies, long codon repeats typically contain glutamine, occur in transcription factors and exhibit high levels of heterozygosity. Furthermore, the presence of significant associations within families between repeat length and head shape indicates that six genes, or genes linked to them, contribute genetic variation to the development of this extremely sexually dimorphic trait.
Collapse
Affiliation(s)
- Leanna M Birge
- Department of Biology, University of Maryland, College Park, MD 20742 USA
- University College London, Research Department of Genetics, Evolution and Environment, Wolfson House, 4 Stephenson Way, London, NW1 2HE, UK
| | - Marie L Pitts
- Department of Biology, The College of William and Mary, Williamsburg, VA 23187 USA
| | - Baker H Richard
- Sackler Institute for Comparative Genomics, American Museum of Natural History, New York, NY, 10024 USA
| | - Gerald S Wilkinson
- Department of Biology, University of Maryland, College Park, MD 20742 USA
| |
Collapse
|
|
32
|
De Benedetti A. Tousled kinase TLK1B mediates chromatin assembly in conjunction with Asf1 regardless of its kinase activity. BMC Res Notes 2010; 3:68. [PMID: 20222959 PMCID: PMC2845150 DOI: 10.1186/1756-0500-3-68] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2010] [Accepted: 03/11/2010] [Indexed: 11/12/2022] Open
Abstract
Background The Tousled Like Kinases (TLKs) are involved in chromatin dynamics, including DNA replication and repair, transcription, and chromosome segregation. Indeed, the first two TLK1 substrates were identified as the histone H3 and Asf1 (a histone H3/H4 chaperone), which immediately suggested a function in chromatin remodeling. However, despite the straightforward assumption that TLK1 acts simply by phosphorylating its substrates and hence modifying their activity, TLK1 also acts as a chaperone. In fact, a kinase-dead (KD) mutant of TLK1B is functional in stimulating chromatin assembly in vitro. However, subtle effects of Asf1 phosphorylation are more difficult to probe in chromatin assembly assays. Not until very recently was the Asf1 site phosphorylated by TLK1 identified. This has allowed for probing directly the functionality of a site-directed mutant of Asf1 in chromatin assembly assays. Findings Addition of either wt or non-phosphorylatable mutant Asf1 to nuclear extract stimulates chromatin assembly on a plasmid. Similarly, TLK1B-KD stimulates chromatin assembly and it synergizes in reactions with supplemental Asf1 (wt or non-phosphorylatable mutant). Conclusions Although the actual function of TLKs as mediators of Asf1 activity cannot be easily studied in vivo, particularly since in mammalian cells there are two TLK genes and two Asf1 genes, we were able to study specifically the stimulation of chromatin assembly in vitro. In such assays, clearly the TLK1 kinase activity was not critical, as neither a non-phosphorylatable Asf1 nor use of the TLK1B-KD impaired the stimulation of nucleosome formation.
Collapse
Affiliation(s)
- Arrigo De Benedetti
- Department of Biochemistry and Molecular Biology and the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA.
| |
Collapse
|
|
33
|
Canfield C, Rains J, De Benedetti A. TLK1B promotes repair of DSBs via its interaction with Rad9 and Asf1. BMC Mol Biol 2009; 10:110. [PMID: 20021694 PMCID: PMC2803485 DOI: 10.1186/1471-2199-10-110] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2009] [Accepted: 12/20/2009] [Indexed: 11/10/2022] Open
Abstract
Background The Tousled-like kinases are involved in chromatin assembly, DNA repair, transcription, and chromosome segregation. Previous evidence indicated that TLK1B can promote repair of plasmids with cohesive ends in vitro, but it was inferred that the mechanism was indirect and via chromatin assembly, mediated by its interaction with the chromatin assembly factor Asf1. We recently identified Rad9 as a substrate of TLK1B, and we presented evidence that the TLK1B-Rad9 interaction plays some role in DSB repair. Hence the relative contribution of Asf1 and Rad9 to the protective effect of TLK1B in DSBs repair is not known. Using an adeno-HO-mediated cleavage system in MM3MG cells, we previously showed that overexpression of either TLK1B or a kinase-dead protein (KD) promoted repair and the assembly of Rad9 in proximity of the DSB at early time points post-infection. This established that it is a chaperone activity of TLK1B and not directly the kinase activity that promotes recruitment of 9-1-1 to the DSB. However, the phosphorylation of Rad9(S328) by TLK1B appeared important for mediating a cell cycle checkpoint, and thus, this phosphorylation of Rad9 may have other effects on 9-1-1 functionality. Results Here we present direct evidence that TLK1B can promote repair of linearized plasmids with incompatible ends that require processing prior to ligation. Immunodepletion of Rad9 indicated that Rad9 was important for processing the ends preceding ligation, suggesting that the interaction of TLK1B with Rad9 is a key mediator for this type of repair. Ligation of incompatible ends also required DNA-PK, as addition of wortmannin or immunodepletion of Ku70 abrogated ligation. Depletion of Ku70 prevented the ligation of the plasmid but did not affect stimulation of the fill-in of the ends by added TLK1B, which was attributed to Rad9. From experiments with the HO-cleavage system, we now show that Rad17, a subunit of the "clamp loader", associates normally with the DSB in KD-overexpressing cells. However, the subsequent release of Rad17 and Rad9 upon repair of the DSB was significantly slower in these cells compared to controls or cells expressing wt-TLK1B. Conclusions TLKs play important roles in DNA repair, not only by modulation of chromatin assembly via Asf1, but also by a more direct function in processing the ends of a DSB via interaction with Rad9. Inhibition of Rad9 phosphorylation in KD-overexpressing cells may have consequences in signaling completion of the repair and cell cycle re-entry, and could explain a loss of viability from DSBs in these cells.
Collapse
Affiliation(s)
- Caroline Canfield
- Department of Biochemistry and Molecular Biology and the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, 71130, USA.
| | | | | |
Collapse
|
|
34
|
De Benedetti A. Tousled kinase TLK1B counteracts the effect of Asf1 in inhibition of histone H3-H4 tetramer formation. BMC Res Notes 2009; 2:128. [PMID: 19586531 PMCID: PMC2713256 DOI: 10.1186/1756-0500-2-128] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2009] [Accepted: 07/08/2009] [Indexed: 11/15/2022] Open
Abstract
Background The Tousled-like kinases (TLKs) function in processes of chromatin assembly, including replication, transcription, repair, and chromosome segregation. TLK1 interacts specifically with the chromatin assembly factor Asf1, a histone H3–H4 chaperone, and with Rad9, a protein involved in DNA repair. Asf1 binds to the H3–H4 dimer at the same interface that is used for formation of the core tetramer, and hence Asf1 is implicated in disruption of the tetramer during transcription, although Asf1 also has a function in chromatin assembly during replication and repair. Findings We have used protein crosslinking with purified components to probe the interaction between H3, H4, Asf1, and TLK1B. We found that TLK1B, by virtue of its binding to Asf1, can restore formation of H3–H4 tetramers that is sterically prevented by adding Asf1. Conclusion We suggest that TLK1B binds to Asf1 in a manner that interferes with its binding to the H3–H4 dimer, thereby allowing for H3–H4 tetramerization. A description of the function of TLK1 and Asf1 in chromatin remodeling is presented.
Collapse
Affiliation(s)
- Arrigo De Benedetti
- Department of Biochemistry and Molecular Biology and the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA.
| |
Collapse
|
|
35
|
Li HH, Chiang CS, Huang HY, Liaw GJ. mars and tousled-like kinase act in parallel to ensure chromosome fidelity in Drosophila. J Biomed Sci 2009; 16:51. [PMID: 19486529 PMCID: PMC2705347 DOI: 10.1186/1423-0127-16-51] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2008] [Accepted: 06/01/2009] [Indexed: 01/08/2023] Open
Abstract
Background High levels of Hepatoma Up-Regulated Protein (HURP) and Tousled-Like Kinase (TLK) transcripts are found in hepatocellular carcinoma. HURP overexpression induces anchorage-independent growth of 293-T cells and enhances a rough-eye phenotype resulting from tlk overexpression in Drosophila. In addition, both HURP and Mars, a Drosophila HURP sequence homologue, promote polymerization of mitotic spindles. Thus, the genetic interaction of mars with tlk might be required for accurate chromosome segregation. Methods To reveal whether chromosome fidelity was decreased, the frequency of gynandromorphy, an individual with both male and female characteristics, and of non-disjunction were measured in the progeny from parents with reduced mars and/or tlk activities and analyzed by Student's t-test. To show that the genetic interaction between mars and tlk is epistatic or parallel, a cytological analysis of embryos with either reduced or increased activities of mars and/or tlk was used to reveal defects in mitotic-spindle morphology and chromosome segregation. Results A significant but small fraction of the progeny from parents with reduced mars activity showed gynandromorphy and non-disjunction. Results of cytological analysis revealed that the decrease in chromosome fidelity was a result of delayed polymerization of the mitotic spindle, which led to asynchronous chromosome segregation in embryos that had reduced mars activity. By removing one copy of tousled-like kinase (tlk) from flies with reduced mars activity, chromosome fidelity was further reduced. This was indicated by an increased in the non-disjunction rate and more severe asynchrony. However, the morphology of the mitotic spindles in the embryos at metaphase where both gene activities were reduced was similar to that in mars embryos. Furthermore, tlk overexpression did not affect the morphology of the mitotic spindles and the cellular localization of Mars protein. Conclusion Chromosome fidelity in progeny from parents with reduced mars and/or tlk activity was impaired. The results from cytological studies revealed that mars and tlk function in parallel and that a balance between mars activity and tlk activity is required for cells to progress through mitosis correctly, thus ensuring chromosome fidelity.
Collapse
Affiliation(s)
- Hsing-Hsi Li
- Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, 112 Taiwan, ROC
| | | | | | | |
Collapse
|
|
36
|
Sunavala-Dossabhoy G, De Benedetti A. Tousled homolog, TLK1, binds and phosphorylates Rad9; TLK1 acts as a molecular chaperone in DNA repair. DNA Repair (Amst) 2009; 8:87-102. [DOI: 10.1016/j.dnarep.2008.09.005] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2008] [Revised: 06/02/2008] [Accepted: 09/02/2008] [Indexed: 11/30/2022]
|
|
37
|
Heallen TR, Adams HP, Furuta T, Verbrugghe KJ, Schumacher JM. An Afg2/Spaf-related Cdc48-like AAA ATPase regulates the stability and activity of the C. elegans Aurora B kinase AIR-2. Dev Cell 2008; 15:603-16. [PMID: 18854144 DOI: 10.1016/j.devcel.2008.08.005] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2008] [Revised: 07/16/2008] [Accepted: 08/19/2008] [Indexed: 02/02/2023]
Abstract
The Aurora B kinase is the enzymatic core of the chromosomal passenger complex, which is a critical regulator of mitosis. To identify novel regulators of Aurora B, we performed a genome-wide screen for suppressors of a temperature-sensitive lethal allele of the C. elegans Aurora B kinase AIR-2. This screen uncovered a member of the Afg2/Spaf subfamily of Cdc48-like AAA ATPases as an essential inhibitor of AIR-2 stability and activity. Depletion of CDC-48.3 restores viability to air-2 mutant embryos and leads to abnormally high AIR-2 levels at the late telophase/G1 transition. Furthermore, CDC-48.3 binds directly to AIR-2 and inhibits its kinase activity from metaphase through telophase. While canonical p97/Cdc48 proteins have been assigned contradictory roles in the regulation of Aurora B, our results identify a member of the Afg2/Spaf AAA ATPases as a critical in vivo inhibitor of this kinase during embryonic development.
Collapse
Affiliation(s)
- Todd R Heallen
- Department of Genetics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA
| | | | | | | | | |
Collapse
|
|
38
|
Brès V, Yoh SM, Jones KA. The multi-tasking P-TEFb complex. Curr Opin Cell Biol 2008; 20:334-40. [PMID: 18513937 DOI: 10.1016/j.ceb.2008.04.008] [Citation(s) in RCA: 186] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2008] [Revised: 04/17/2008] [Accepted: 04/23/2008] [Indexed: 11/24/2022]
Abstract
P-TEFb (CycT1:Cdk9), the metazoan RNA polymerase II Ser2 C-terminal domain (CTD) kinase, regulates transcription elongation at many genes and integrates mRNA synthesis with histone modification, pre-mRNA processing, and mRNA export. Recruitment of P-TEFb to target genes requires deubiquitination of H2Bub, phosphorylation of H3S10, and the bromodomain protein, Brd4. Brd4 activates growth-related genes in the G1 phase of the cell cycle and can also tether P-TEFb to mitotic chromosomes, possibly to mark sites of active transcription throughout cell division. P-TEFb co-operates with c-Myc during transactivation and cell transformation, and also requires SKIP (c-Ski-interacting protein), an mRNA elongation and splicing factor. Some functions of the P-TEFb/Ser2P CTD are executed by the Spt6 transcription elongation factor, which binds directly to the phosphorylated CTD and recruits the Iws1 ('interacts with Spt6') protein. Iws1, in turn, interacts with the REF1/Aly nuclear export adaptor and stimulates the kinetics of mRNA export. Given the prominent role of Spt6 in regulating chromatin structure, the CTD-bound Spt6:Iws1 complex may also control histone modifications during elongation. Following transcription, P-TEFb accompanies the mature mRNA to the cytoplasm to promote translation elongation.
Collapse
Affiliation(s)
- Vanessa Brès
- Regulatory Biology Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037-1099, USA
| | | | | |
Collapse
|
|
39
|
Grigsby IF, Finger FP. UNC-85, a C. elegans homolog of the histone chaperone Asf1, functions in post-embryonic neuroblast replication. Dev Biol 2008; 319:100-9. [PMID: 18490010 DOI: 10.1016/j.ydbio.2008.04.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2007] [Revised: 04/08/2008] [Accepted: 04/08/2008] [Indexed: 11/28/2022]
Abstract
Normal animal development requires accurate cell divisions, not only in the early stages of rapid embryonic cleavages, but also in later developmental stages. The Caenorhabditis elegans unc-85 gene is implicated only in cell divisions that occur post-embryonically, primarily in terminal neuronal lineages. Variable post-embryonic cell division failures in ventral cord motoneuron precursors result in uncoordinated locomotion of unc-85 mutant larvae by the second larval stage. These neuroblast cell division failures often result in unequally sized daughter nuclei, and sometimes in nuclear fusions. Using a combination of conventional mapping techniques and microarray analysis, we cloned the unc-85 gene, and find that it encodes one of two C. elegans homologs of the yeast Anti-silencing function 1 (Asf1) histone chaperone. The unc-85 gene is expressed in replicating cells throughout development, and the protein is localized in nuclei. Examination of null mutants confirms that embryonic neuroblast cell divisions occur normally, but post-embryonic neuroblast cell divisions fail. Analysis of the DNA content of the mutant neurons indicates that defective replication in post-embryonic neuroblasts gives rise to ventral cord neurons with an average DNA content of approximately 2.5 n. We conclude that UNC-85 functions in post-embryonic DNA replication in ventral cord motor neuron precursors.
Collapse
Affiliation(s)
- Iwen F Grigsby
- Biology Department and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Biotech-BCHM-2, Troy, NY 12180, USA
| | | |
Collapse
|
|
40
|
Riefler GM, Dent SYR, Schumacher JM. Tousled-mediated activation of Aurora B kinase does not require Tousled kinase activity in vivo. J Biol Chem 2008; 283:12763-8. [PMID: 18334486 DOI: 10.1074/jbc.m709034200] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
The Aurora kinases comprise an evolutionarily conserved protein family that is required for a variety of cell division events, including spindle assembly, chromosome segregation, and cytokinesis. Emerging evidence suggests that once phosphorylated, a subset of Aurora substrates can enhance Aurora kinase activity. Our previous work revealed that the Caenorhabditis elegans Tousled-like kinase TLK-1 is a substrate and activator of the AIR-2 Aurora B kinase in vitro and that partial loss of TLK-1 enhances the mitotic defects of an air-2 mutant. However, given that these experiments were performed in vitro and with partial loss of function alleles in vivo, a necessary step forward in our understanding of the relationship between the Aurora B and Tousled kinases is to prove that TLK-1 expression is sufficient for Aurora B activation in vivo. Here, we report that heterologous expression of wild-type and kinase-inactive forms of TLK-1 suppresses the lethality of temperature-sensitive mutants of the yeast Aurora B kinase Ipl1. Moreover, kinase-dead TLK-1 associates with and augments the activity of Ipl1 in vivo. Together, these results provide critical and compelling evidence that Tousled has a bona fide kinase-independent role in the activation of Aurora B kinases in vivo.
Collapse
Affiliation(s)
- Gary M Riefler
- Department of Molecular Genetics, M.D Anderson Cancer Center, University of Texas, Houston, Texas 77030, USA
| | | | | |
Collapse
|
|
41
|
Bender LB, Suh J, Carroll CR, Fong Y, Fingerman IM, Briggs SD, Cao R, Zhang Y, Reinke V, Strome S. MES-4: an autosome-associated histone methyltransferase that participates in silencing the X chromosomes in the C. elegans germ line. Development 2006; 133:3907-17. [PMID: 16968818 PMCID: PMC2435371 DOI: 10.1242/dev.02584] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Germ cell development in C. elegans requires that the X chromosomes be globally silenced during mitosis and early meiosis. We previously found that the nuclear proteins MES-2, MES-3, MES-4 and MES-6 regulate the different chromatin states of autosomes versus X chromosomes and are required for germline viability. Strikingly, the SET-domain protein MES-4 is concentrated on autosomes and excluded from the X chromosomes. Here, we show that MES-4 has histone H3 methyltransferase (HMT) activity in vitro, and is required for histone H3K36 dimethylation in mitotic and early meiotic germline nuclei and early embryos. MES-4 appears unlinked to transcription elongation, thus distinguishing it from other known H3K36 HMTs. Based on microarray analysis, loss of MES-4 leads to derepression of X-linked genes in the germ line. We discuss how an autosomally associated HMT may participate in silencing genes on the X chromosome, in coordination with the direct silencing effects of the other MES proteins.
Collapse
Affiliation(s)
- Laurel B. Bender
- Department of Biology, Indiana University, Bloomington, IN 47405, USA
- Authors for correspondence (e-mail: ; )
| | - Jinkyo Suh
- Department of Biology, Indiana University, Bloomington, IN 47405, USA
| | - Coleen R. Carroll
- Department of Biology, Indiana University, Bloomington, IN 47405, USA
| | - Youyi Fong
- Department of Biology, Indiana University, Bloomington, IN 47405, USA
| | - Ian M. Fingerman
- Department of Biochemistry, Purdue Cancer Center, Purdue University, West Lafayette, IN 47907, USA
| | - Scott D. Briggs
- Department of Biochemistry, Purdue Cancer Center, Purdue University, West Lafayette, IN 47907, USA
| | - Ru Cao
- Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC 27599, USA
| | - Yi Zhang
- Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC 27599, USA
| | - Valerie Reinke
- Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Susan Strome
- Department of Biology, Indiana University, Bloomington, IN 47405, USA
- Authors for correspondence (e-mail: ; )
| |
Collapse
|
|
42
|
Sen SP, De Benedetti A. TLK1B promotes repair of UV-damaged DNA through chromatin remodeling by Asf1. BMC Mol Biol 2006; 7:37. [PMID: 17054786 PMCID: PMC1626478 DOI: 10.1186/1471-2199-7-37] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2006] [Accepted: 10/20/2006] [Indexed: 11/21/2022] Open
Abstract
Background The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila, or expression of a dominant negative mutant in mouse mammary cells causes loss of nuclear divisions and chromosome mis-segregation. TLK1B is a splice variant of TLK1 and it confers radioresistance in a normal mammary mouse cell line possibly due to increased chromatin remodeling capacity, but the mechanism of resistance remains to be fully elucidated. Results We now show that TLK1B also affords protection against UV radiation. We find that nuclear extracts isolated from TLK1B-containing mouse cells promote more efficient chromatin assembly than comparable extracts lacking TLK1B. TLK1B-containing extracts are also more efficient in repair of UV-damaged plasmid DNA assembled into nucleosomes. One of the two known substrates of TLK1 (or TLK1B) is the histone chaperone Asf1, and immuno-inactivation experiments suggest that TLK1B increases UV-repair through the action of Asf1 on chromatin assembly/disassembly. Conclusion Our studies provide evidence for TLK1B-mediated phosphorylation of Asf1 triggering DNA repair. We suggest that this occurs via Asf1-mediated chromatin assembly at the sites of UV damage.
Collapse
Affiliation(s)
- Siddhartha P Sen
- Department of Biochemistry and Molecular Biology and the Feist Weiller Cancer Center, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA
| | - Arrigo De Benedetti
- Department of Biochemistry and Molecular Biology and the Feist Weiller Cancer Center, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA
| |
Collapse
|
|
43
|
Franks DM, Izumikawa T, Kitagawa H, Sugahara K, Okkema PG. C. elegans pharyngeal morphogenesis requires both de novo synthesis of pyrimidines and synthesis of heparan sulfate proteoglycans. Dev Biol 2006; 296:409-20. [PMID: 16828468 DOI: 10.1016/j.ydbio.2006.06.008] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2006] [Revised: 05/18/2006] [Accepted: 06/02/2006] [Indexed: 11/21/2022]
Abstract
The C. elegans pharynx undergoes elongation and morphogenesis to its characteristic bi-lobed shape between the 2- and 3-fold stages of embryogenesis. During this period, the pharyngeal muscles and marginal cells forming the isthmus between the anterior and posterior pharyngeal bulbs elongate and narrow. We have identified the spontaneous mutant pyr-1(cu8) exhibiting defective pharyngeal isthmus elongation, cytoskeletal organization defects, and maternal effect lethality. pyr-1 encodes CAD, a trifunctional enzyme required for de novo pyrimidine synthesis, and pyr-1(cu8) mutants are rescued by supplying exogenous pyrimidines. Similar pharyngeal defects and maternal effect lethality were found in sqv-1, sqv-8, rib-1 and rib-2 mutants, which affect enzymes involved in heparan sulfate proteoglycan (HSPG) synthesis. rib-1 mutant lethality was enhanced in a pyr-1 mutant background, indicating that HSPG synthesis is very sensitive to decreased pyrimidine pools, and HS disaccharides are moderately decreased in both rib-1 and pyr-1 mutants. We hypothesize that HSPGs are necessary for pharyngeal isthmus elongation, and pyr-1 functions upstream of proteoglycan synthesizing enzymes by providing precursors of UDP-sugars essential for HSPG synthesis.
Collapse
Affiliation(s)
- Dawn M Franks
- Department of Biological Sciences and the Laboratory for Molecular Biology, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, IL 60607, USA
| | | | | | | | | |
Collapse
|
|
44
|
Dardick C, Ronald P. Plant and animal pathogen recognition receptors signal through non-RD kinases. PLoS Pathog 2006; 2:e2. [PMID: 16424920 PMCID: PMC1331981 DOI: 10.1371/journal.ppat.0020002] [Citation(s) in RCA: 170] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2005] [Accepted: 12/14/2005] [Indexed: 12/18/2022] Open
Abstract
Plants and animals mediate early steps of the innate immune response through pathogen recognition receptors (PRRs). PRRs commonly associate with or contain members of a monophyletic group of kinases called the interleukin-1 receptor-associated kinase (IRAK) family that include Drosophila Pelle, human IRAKs, rice XA21 and Arabidopsis FLS2. In mammals, PRRs can also associate with members of the receptor-interacting protein (RIP) kinase family, distant relatives to the IRAK family. Some IRAK and RIP family kinases fall into a small functional class of kinases termed non-RD, many of which do not autophosphorylate the activation loop. We surveyed the yeast, fly, worm, human, Arabidopsis, and rice kinomes (3,723 kinases) and found that despite the small number of non-RD kinases in these genomes (9%-29%), 12 of 15 kinases known or predicted to function in PRR signaling fall into the non-RD class. These data indicate that kinases associated with PRRs can largely be predicted by the lack of a single conserved residue and reveal new potential plant PRR subfamilies.
Collapse
Affiliation(s)
- Christopher Dardick
- United States Department of Agriculture, Agricultural Research Service, Appalachian Fruit Research Station, Kearneysville, West Virginia, United States of America
- * To whom correspondence should be addressed. E-mail: (CD); (PR)
| | - Pamela Ronald
- Department of Plant Pathology, University of California Davis, Davis, California, United States of America
- * To whom correspondence should be addressed. E-mail: (CD); (PR)
| |
Collapse
|
|
45
|
Adhvaryu KK, Morris SA, Strahl BD, Selker EU. Methylation of histone H3 lysine 36 is required for normal development in Neurospora crassa. EUKARYOTIC CELL 2005; 4:1455-64. [PMID: 16087750 PMCID: PMC1214527 DOI: 10.1128/ec.4.8.1455-1464.2005] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The SET domain is an evolutionarily conserved domain found predominantly in histone methyltransferases (HMTs). The Neurospora crassa genome includes nine SET domain genes (set-1 through set-9) in addition to dim-5, which encodes a histone H3 lysine 9 HMT required for DNA methylation. We demonstrate that Neurospora set-2 encodes a histone H3 lysine 36 (K36) methyltransferase and that it is essential for normal growth and development. We used repeat induced point mutation to make a set-2 mutant (set-2(RIP1)) with multiple nonsense mutations. Western analyses revealed that the mutant lacks SET-2 protein and K36 methylation. An amino-terminal fragment that includes the AWS, SET, and post-SET domains of SET-2 proved sufficient for K36 HMT activity in vitro. Nucleosomes were better substrates than free histones. The set-2(RIP1) mutant grows slowly, conidiates poorly, and is female sterile. Introducing the wild-type gene into the mutant complemented the defects, confirming that they resulted from loss of set-2 function. We replaced the wild-type histone H3 gene (hH3) with an allele producing a Lys to Leu substitution at position 36 and found that this hH3(K36L) mutant phenocopied the set-2(RIP1) mutant, confirming that the observed defects in growth and development result from inability to methylate K36 of H3. Finally, we used chromatin immunoprecipitation to demonstrate that actively transcribed genes in Neurospora crassa are enriched for H3 methylated at lysines 4 and 36. Taken together, our results suggest that methylation of K36 in Neurospora crassa is essential for normal growth and development.
Collapse
Affiliation(s)
- Keyur K Adhvaryu
- Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA
| | | | | | | |
Collapse
|
|
46
|
Han Z, Riefler GM, Saam JR, Mango SE, Schumacher JM. The C. elegans Tousled-like kinase contributes to chromosome segregation as a substrate and regulator of the Aurora B kinase. Curr Biol 2005; 15:894-904. [PMID: 15916946 PMCID: PMC2653428 DOI: 10.1016/j.cub.2005.04.019] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2005] [Revised: 03/30/2005] [Accepted: 04/01/2005] [Indexed: 11/18/2022]
Abstract
BACKGROUND The Aurora kinases control multiple aspects of mitosis, among them centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora activity is regulated in part by a subset of Aurora substrates that, once phosphorylated, can enhance Aurora kinase activity. Aurora A substrate activators include TPX2 and Ajuba, whereas the only known Aurora B substrate activator is the chromosomal passenger INCENP. RESULTS We report that the C. elegans Tousled kinase TLK-1 is a second substrate activator of the Aurora B kinase AIR-2. Tousled kinase (Tlk) expression and activity have been linked to ongoing DNA replication, and Tlk can phosphorylate the chromatin assembly factor Asf. Here, we show that TLK-1 is phosphorylated by AIR-2 during prophase/prometaphase and that phosphorylation increases TLK-1 kinase activity in vitro. Phosphorylated TLK-1 increases AIR-2 kinase activity in a manner that is independent of TLK-1 kinase activity but depends on the presence of ICP-1/INCENP. In vivo, TLK-1 and AIR-2 cooperate to ensure proper mitotic chromosome segregation. CONCLUSIONS The C. elegans Tousled kinase TLK-1 is a substrate and activator of the Aurora B kinase AIR-2. These results suggest that Tousled kinases have a previously unrecognized role in mitosis and that Aurora B associates with discrete regulatory complexes that may impart distinct substrate specificities and functions to the Aurora B kinase.
Collapse
Affiliation(s)
- Zhenbo Han
- Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
| | - Gary M. Riefler
- Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
- Genes and Development Program, Graduate School of Biomedical Sciences, The University of Texas-Houston, Houston, Texas 77030
| | - Jennifer R. Saam
- Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112
| | - Susan E. Mango
- Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112
| | - Jill M. Schumacher
- Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
- Genes and Development Program, Graduate School of Biomedical Sciences, The University of Texas-Houston, Houston, Texas 77030
- Correspondence:
| |
Collapse
|
|
47
|
Abstract
Aurora B kinases play important roles during mitosis in eukaryotic cells; new work in Caenorhabditis elegans has identified the Tousled kinase TLK-1 as a substrate activator of the model nematode's Aurora B kinase AIR-2 which acts to ensure proper chromosome segregation during cell division.
Collapse
Affiliation(s)
- Christopher T Richie
- Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA
| | | |
Collapse
|
|
48
|
Morris SA, Shibata Y, Noma KI, Tsukamoto Y, Warren E, Temple B, Grewal SIS, Strahl BD. Histone H3 K36 methylation is associated with transcription elongation in Schizosaccharomyces pombe. EUKARYOTIC CELL 2005; 4:1446-54. [PMID: 16087749 PMCID: PMC1214526 DOI: 10.1128/ec.4.8.1446-1454.2005] [Citation(s) in RCA: 92] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/23/2005] [Accepted: 05/29/2005] [Indexed: 11/20/2022]
Abstract
Set2 methylation of histone H3 at lysine 36 (K36) has recently been shown to be associated with RNA polymerase II (Pol II) elongation in Saccharomyces cerevisiae. However, whether this modification is conserved and associated with transcription elongation in other organisms is not known. Here we report the identification and characterization of the Set2 ortholog responsible for K36 methylation in the fission yeast Schizosaccharomyces pombe. We find that similar to the budding yeast enzyme, S. pombe Set2 is also a robust nucleosome-selective H3 methyltransferase that is specific for K36. Deletion of the S. pombe set2+ gene results in complete abolishment of K36 methylation as well as a slow-growth phenotype on plates containing synthetic medium. These results indicate that Set2 is the sole enzyme responsible for this modification in fission yeast and is important for cell growth under stressed conditions. Using the chromatin immunoprecipitation assay, we demonstrate that K36 methylation in S. pombe is associated with the transcribed regions of Pol II-regulated genes and is devoid in regions that are not transcribed by Pol II. Consistent with a role for Set2 in transcription elongation, we find that S. pombe Set2 associates with the hyperphosphorylated form of Pol II and can fully rescue K36 methylation and Pol II interaction in budding yeast cells deleted for Set2. These results, along with our finding that K36 methylation is highly conserved among eukaryotes, imply a conserved role for this modification in the transcription elongation process.
Collapse
Affiliation(s)
- Stephanie A Morris
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
49
|
Kodym R, Henöckl C, Fürweger C. Identification of the human DEAD-box protein p68 as a substrate of Tlk1. Biochem Biophys Res Commun 2005; 333:411-7. [PMID: 15950181 DOI: 10.1016/j.bbrc.2005.05.136] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2005] [Accepted: 05/18/2005] [Indexed: 12/20/2022]
Abstract
The activity of the human protein kinase Tlk1 is down-regulated within minutes after exposure of cells to ionizing radiation. In order to identify signaling pathways which might be relevant in the radiation response of mammalian cells we screened nuclear proteins for substrates of Tlk1. Among several proteins one could be identified as p68 RNA helicase. Furthermore, it could be shown that Tlk1 phosphorylates immunoprecipitated p68. The phosphorylation of the C-terminal fragment of p68 by rTlk1 reduced its affinity to single stranded RNA in a gel shift assay. In addition, it could be demonstrated that increasing the Tlk1 activity in HT1080 cells by forced Tlk1 overexpression leads to an increased phosphorylation of endogenous p68, arguing that p68 might be a physiological substrate of Tlk1. These findings open the possibility that Tlk1 might participate in diverse biologic functions like cell growth and differentiation, pre-mRNA splicing, and transcriptional coactivation.
Collapse
Affiliation(s)
- Reinhard Kodym
- Department of Radiobiology, Clinic for Radiotherapy and Radiobiology, Medical University of Vienna, Vienna, Austria.
| | | | | |
Collapse
|
|
50
|
Fourquin C, Vinauger-Douard M, Fogliani B, Dumas C, Scutt CP. Evidence that CRABS CLAW and TOUSLED have conserved their roles in carpel development since the ancestor of the extant angiosperms. Proc Natl Acad Sci U S A 2005; 102:4649-54. [PMID: 15767586 PMCID: PMC555504 DOI: 10.1073/pnas.0409577102] [Citation(s) in RCA: 77] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The carpel is the female reproductive organ specific to flowering plants. We aim to define the genes that controlled carpel development in the common ancestor of this group as a step toward determining the molecular events that were responsible for the evolution of the carpel. CRABS CLAW (CRC) and TOUSLED (TSL) control important aspects of carpel development in the model plant, Arabidopsis thaliana. The basal angiosperm species Amborella trichopoda and Cabomba aquatica very likely represent the two most early diverging groups of flowering plants. We have identified putative orthologues of CRC and TSL from A. trichopoda and C. aquatica, respectively. We demonstrate the expression patterns of these genes in carpels to be very highly conserved, both spatially and temporally, with those of their Arabidopsis orthologues. We argue that CRC and TSL in Arabidopsis are likely to have conserved their respective roles in carpel development since the common ancestor of the living flowering plants. We conclude that a divergent role shown for the CRC orthologue in rice, DROOPING LEAF, most probably arose specifically in the monocot lineage. We show that, in addition to its expression in carpels, the TSL orthologue of C. aquatica is expressed in tissues that contribute to buoyancy and argue that its role in these tissues may have arisen later than its role in carpel development.
Collapse
MESH Headings
- Arabidopsis/genetics
- Arabidopsis/metabolism
- Arabidopsis/physiology
- Arabidopsis Proteins/genetics
- Arabidopsis Proteins/physiology
- DNA, Plant/genetics
- Evolution, Molecular
- Flowers/growth & development
- Flowers/metabolism
- Gene Expression Regulation, Developmental
- Gene Expression Regulation, Plant
- Genes, Plant
- In Situ Hybridization
- Magnoliopsida/genetics
- Magnoliopsida/growth & development
- Magnoliopsida/metabolism
- Microscopy, Electron, Scanning
- Molecular Sequence Data
- Oryza/genetics
- Oryza/growth & development
- Oryza/metabolism
- Phylogeny
- Protein Serine-Threonine Kinases/genetics
- Protein Serine-Threonine Kinases/physiology
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- RNA, Plant/genetics
- RNA, Plant/metabolism
- Transcription Factors/genetics
- Transcription Factors/physiology
Collapse
Affiliation(s)
- Chloé Fourquin
- Laboratoire de Reproduction et Développement des Plantes, Unité Mixte de Recherche 5667, Université Claude Bernard-Lyon, 46 Allée d'Italie, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France
| | | | | | | | | |
Collapse
|