|
1
|
Yang XC, Wu XL, Li WH, Wu XJ, Shen QY, Li YX, Peng S, Hua JL. OCT6 inhibits differentiation of porcine-induced pluripotent stem cells through MAPK and PI3K signaling regulation. Zool Res 2022; 43:911-922. [PMID: 36052561 PMCID: PMC9700490 DOI: 10.24272/j.issn.2095-8137.2022.220] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Accepted: 09/01/2022] [Indexed: 08/18/2023] Open
Abstract
As a transcription factor of the Pit-Oct-Unc (POU) domain family, octamer-binding transcription factor 6 ( OCT6) participates in various aspects of stem cell development and differentiation. At present, however, its role in porcine-induced pluripotent stem cells (piPSCs) remains unclear. Here, we explored the function of OCT6 in piPSCs. We found that piPSCs overexpressing OCT6 maintained colony morphology and pluripotency under differentiation conditions, with a similar gene expression pattern to that of non-differentiated piPSCs. Functional analysis revealed that OCT6 attenuated the adverse effects of extracellular signal-regulated kinase (ERK) signaling pathway inhibition on piPSC pluripotency by activating phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling activity. Our research sheds new light on the mechanism by which OCT6 promotes PSC maintenance.
Collapse
Affiliation(s)
- Xin-Chun Yang
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Xiao-Long Wu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Wen-Hao Li
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Xiao-Jie Wu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Qiao-Yan Shen
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Yun-Xiang Li
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Sha Peng
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China
| | - Jin-Lian Hua
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi 712100, China. E-mail:
| |
Collapse
|
|
2
|
The Divergent and Conserved Expression Profile of Turtle Nanog Gene Comparing with Fish and Mammals. BIOLOGY 2022; 11:biology11091342. [PMID: 36138820 PMCID: PMC9495436 DOI: 10.3390/biology11091342] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/27/2022] [Revised: 08/19/2022] [Accepted: 09/05/2022] [Indexed: 11/17/2022]
Abstract
Nanog is a homeodomain-containing transcription factor, and it plays a vital role in maintaining the pluripotency of embryonic stem cells. Nanog’s function has been well studied in many species. However, there is lack of reporting on the Nanog gene in reptile. Here, we identified a 1032 bp cDNA sequence of a Nanog gene in Pelidiscus sinensis, known as PsNanog. PsNanog has a highly conserved HD domain and shares a high identity with that of Chelonia mydas and the lowest identity with Oryzias latipes. Similarly, PsNanog presented a tight cluster with C. mydas Nanog, but was far from those of teleosts. Additionally, we cloned a length of 1870 bp PsNanog promoter. Dual luciferase assay showed that the DNA fragment of −1560 to +1 exhibited a high promoter activity. The RT-PCR and RT-qPCR results showed that PsNanog was predominantly expressed in ovary, and then in testis. The in situ hybridization and immunohistochemical analysis showed that PsNanog was expressed in the early primary oocytes and the cytoplasm of the cortical region of stage VIII oocytes in ovary, and distributed in most stages of germ cells in testis. Collectively, the results imply that PsNanog probably has the conserved function in regulating germ cell development across phyla and is also a pluripotent cell gene and expressed in germ cells, which is similar to that in teleosts and mammals.
Collapse
|
|
3
|
Gisler S, Gonçalves JP, Akhtar W, de Jong J, Pindyurin AV, Wessels LFA, van Lohuizen M. Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency. Nat Commun 2019; 10:1598. [PMID: 30962441 PMCID: PMC6453899 DOI: 10.1038/s41467-019-09551-w] [Citation(s) in RCA: 43] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2018] [Accepted: 03/14/2019] [Indexed: 12/16/2022] Open
Abstract
Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR-Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. Here we leverage our TRIP technology to integrate ~ 1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5' overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency.
Collapse
Affiliation(s)
- Santiago Gisler
- Division of Molecular Genetics, Oncode and The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
| | - Joana P Gonçalves
- Department of Intelligent Systems, Delft University of Technology, Van Mourik Broekmanweg 6, Delft, 2628 XE, The Netherlands
- Division of Molecular Carcinogenesis, Oncode and The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
| | - Waseem Akhtar
- Division of Molecular Genetics, Oncode and The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
| | - Johann de Jong
- Division of Molecular Carcinogenesis, Oncode and The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
- Data & Translational Sciences Group, UCB Biosciences GmbH, Alfred-Nobel-Straße 10, Monheim am Rhein, 40789, Germany
| | - Alexey V Pindyurin
- Institute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, Acad. Lavrentiev Ave. 8, Novosibirsk, 630090, Russia
- Division of Gene Regulation, Oncode and The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands
| | - Lodewyk F A Wessels
- Department of Intelligent Systems, Delft University of Technology, Van Mourik Broekmanweg 6, Delft, 2628 XE, The Netherlands.
- Division of Molecular Carcinogenesis, Oncode and The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands.
| | - Maarten van Lohuizen
- Division of Molecular Genetics, Oncode and The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands.
| |
Collapse
|
|
4
|
Patra SK, Vemulawada C, Soren MM, Sundaray JK, Panda MK, Barman HK. Molecular characterization and expression patterns of Nanog gene validating its involvement in the embryonic development and maintenance of spermatogonial stem cells of farmed carp, Labeo rohita. J Anim Sci Biotechnol 2018; 9:45. [PMID: 29992021 PMCID: PMC5994655 DOI: 10.1186/s40104-018-0260-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2017] [Accepted: 04/25/2018] [Indexed: 12/24/2022] Open
Abstract
Background The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells (SSCs) via interacting with transcription factors such as Oct4 and Sox2 in mammals. However, knowledge of its exact mechanistic pathways remains unexploited. Very little is known about teleost Nanog. Information on the Nanog gene of farmed rohu carp (Labeo rohita) is lacking. We cloned and characterized the Nanog gene of rohu carp to understand the expression pattern in early developmental stages and also deduced the genomic organization including promoter elements. Results Rohu Nanog (LrNanog) cDNA comprised an open reading frame of 1,161 nucleotides bearing a structural homeodomain; whereas, the genomic structure contained four exons and three introns suggesting that it is homologous to mammalian counterparts. Phylogenetically, it was closely related to freshwater counterparts. Protein sequence (386 AA of 42.65 kDa) comparison revealed its low similarity with other vertebrate counterparts except that of the conserved homeodomain. Tissue distribution analysis revealed the existence of LrNanog transcripts only in adult gonads. The heightened abundances in the ovary and proliferating spermatogonia suggested its participations in maternal inheritance and male germ cell development. The potentiating abundances from fertilized egg onwards peaking at blastula stage vis- à-vis decreasing levels from gastrula stage onwards demonstrated its role in embryonic stem cell development. We also provided evidence of its presence in SSCs by western blotting analysis. Further, the promoter region was characterized, predicting a basal core promoter and other consensus elements. Conclusion The molecular characterization of LrNanog and its documented expression profiling at transcript and protein levels are indicative of its functional linkage with embryonic/spermatogonial stem cell maintenance. This is the first report of LrNanog genomic organization including its promoter sequence information with predicted regulatory elements of a large-bodied carp species. This will be useful for elucidating its mechanism expression in future. Nanog could be used as a potential biomarker for proliferating carp SSCs.
Collapse
Affiliation(s)
- Swagat K Patra
- 1Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, Odisha 751002 India
| | - Chakrpani Vemulawada
- 1Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, Odisha 751002 India
| | - Meenati M Soren
- 1Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, Odisha 751002 India
| | - Jitendra K Sundaray
- 1Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, Odisha 751002 India
| | - Manoj K Panda
- 2Center of Biotechnology, Siksha 'O' Anusandhan University, Bhubaneswar, India
| | - Hirak K Barman
- 1Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, Odisha 751002 India
| |
Collapse
|
|
5
|
Zhou Y, Song N, Li X, Han Y, Ren Z, Xu JX, Han YC, Li F, Jia X. Changes in the methylation status of the Oct3/4, Nanog, and Sox2 promoters in stem cells during regeneration of rat tracheal epithelium after injury. Oncotarget 2018; 8:2984-2994. [PMID: 27935870 PMCID: PMC5356857 DOI: 10.18632/oncotarget.13818] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2016] [Accepted: 11/21/2016] [Indexed: 11/25/2022] Open
Abstract
We investigated the relationship between promoter methylation and tracheal stem cell activation. We developed a model of rat tracheal epithelium regeneration after 5-fluorouracil (5-FU)-induced injury. Using immunohistochemistry and Western blotting, the expression levels of the stem cell pluripotency regulator Oct3/4 and differentiation marker CK14 were measured after 5-FU treatment. The methylation status of the Oct3/4, Nanog, and Sox2 promoters was investigated using methylation-specific PCR. Additionally, the effects of 5-azacytidine (5-azaC), a demethylating agent, on Oct3/4, Nanog, and Sox2 mRNA and protein expression were evaluated. Finally, we measured the activity of the maintenance and de novo DNA methyltransferases DNMT1, DNMT3a, and DNMT3b. Our data indicate that Oct3/4, Sox2, and Nanog are transiently expressed in response to 5-FU-induced injury, and then they are gradually silenced as the cells differentiate. DNA methylation can result in silencing of gene expression, and it can determine whether tracheal stem cells are in an active or dormant state. Treatment with 5-FU reversed the methylation of the Oct3/4, Nanog, and Sox2 promoters, which corresponded to increases in Oct3/4, Nanog, and Sox2 mRNA and protein. Thus, both maintenance and de novo methyltransferases are involved in regulating tracheal stem cell dormancy and activation.
Collapse
Affiliation(s)
- Ying Zhou
- Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China.,Department of Pathology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China.,Department of Emergency, First Affiliated Hospital of China Medical University, Shenyang, 110001, China
| | - Nan Song
- Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China.,Department of Pathology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China
| | - Xin Li
- Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China.,Department of Pathology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China.,Department of Physiology, College of Life Science and Biopharmaceutics of Shenyang Pharmaceutical University, Shenyang, 110016, China
| | - Ying Han
- Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China.,Department of Pathology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China.,Department of Pathology, Shenyang Medical College, Shenyang, 110001, China
| | - Zihan Ren
- Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China.,Department of Pathology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China
| | - Jing-Xian Xu
- Department of Ophthalmology, The 4th Affiliated Hospital, Eye Institute, China Medical University, The Key Laboratory of Lens Research, Shenyang 110005, China
| | - Yu-Chen Han
- Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China.,Department of Pathology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China
| | - Fang Li
- Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China.,Department of Pathology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China.,IVF Michigan, Bloomfield Hills, MI, 48304, USA
| | - Xinshan Jia
- Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China.,Department of Pathology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China
| |
Collapse
|
|
6
|
Kelly GM, Gatie MI. Mechanisms Regulating Stemness and Differentiation in Embryonal Carcinoma Cells. Stem Cells Int 2017; 2017:3684178. [PMID: 28373885 PMCID: PMC5360977 DOI: 10.1155/2017/3684178] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2016] [Revised: 01/10/2017] [Accepted: 02/08/2017] [Indexed: 02/06/2023] Open
Abstract
Just over ten years have passed since the seminal Takahashi-Yamanaka paper, and while most attention nowadays is on induced, embryonic, and cancer stem cells, much of the pioneering work arose from studies with embryonal carcinoma cells (ECCs) derived from teratocarcinomas. This original work was broad in scope, but eventually led the way for us to focus on the components involved in the gene regulation of stemness and differentiation. As the name implies, ECCs are malignant in nature, yet maintain the ability to differentiate into the 3 germ layers and extraembryonic tissues, as well as behave normally when reintroduced into a healthy blastocyst. Retinoic acid signaling has been thoroughly interrogated in ECCs, especially in the F9 and P19 murine cell models, and while we have touched on this aspect, this review purposely highlights how some key transcription factors regulate pluripotency and cell stemness prior to this signaling. Another major focus is on the epigenetic regulation of ECCs and stem cells, and, towards that end, this review closes on what we see as a new frontier in combating aging and human disease, namely, how cellular metabolism shapes the epigenetic landscape and hence the pluripotency of all stem cells.
Collapse
Affiliation(s)
- Gregory M. Kelly
- Department of Biology, Molecular Genetics Unit, Western University, London, ON, Canada
- Collaborative Program in Developmental Biology, Western University, London, ON, Canada
- Department of Paediatrics and Department of Physiology and Pharmacology, Western University, London, ON, Canada
- Child Health Research Institute, London, ON, Canada
- Ontario Institute for Regenerative Medicine, Toronto, ON, Canada
- The Hospital for Sick Children, Toronto, ON, Canada
| | - Mohamed I. Gatie
- Department of Biology, Molecular Genetics Unit, Western University, London, ON, Canada
- Collaborative Program in Developmental Biology, Western University, London, ON, Canada
| |
Collapse
|
|
7
|
Choi DH, Kim DJ, Song KD, Park HH, Ko TH, Pyao Y, Chung KM, Cha SH, Sin YS, Kim NH, Lee WK. Characterization of the Nanog 5'-flanking Region in Bovine. ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 2016; 29:1383-91. [PMID: 27165025 PMCID: PMC5003962 DOI: 10.5713/ajas.16.0032] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/12/2016] [Revised: 03/29/2016] [Accepted: 04/07/2016] [Indexed: 11/27/2022]
Abstract
Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5′-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5′-flanking sequences revealed that the −134 to −19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.
Collapse
Affiliation(s)
- Don-Ho Choi
- The Institute of Hankook Life Science, Seoul 110-812, Korea
| | - Duk-Jung Kim
- The Institute of Hankook Life Science, Seoul 110-812, Korea.,Department of Animal Science, Chungbuk National University, Cheongju, 361-763, Korea
| | - Ki-Duk Song
- Department of Animal Biotechnology, Chonbuk National University, Jeonju 561-756, Korea
| | - Hwan-Hee Park
- Department of Biomedical Sciences, School of Medicine, Inha University, Incheon 400-712, Korea
| | - Tae Hyun Ko
- Department of Biomedical Sciences, School of Medicine, Inha University, Incheon 400-712, Korea
| | - Yuliya Pyao
- Department of Biomedical Sciences, School of Medicine, Inha University, Incheon 400-712, Korea
| | - Ku-Min Chung
- The Institute of Hankook Life Science, Seoul 110-812, Korea
| | - Seok Ho Cha
- Department of Parasitology and Tropical Medicine, School of Medicine, Inha University, Incheon 400-712, Korea
| | - Young-Su Sin
- Department of Animal Science, Singu University, Seongnam, 462-743, Korea
| | - Nam-Hyung Kim
- Department of Animal Science, Chungbuk National University, Cheongju, 361-763, Korea
| | - Woon-Kyu Lee
- Department of Biomedical Sciences, School of Medicine, Inha University, Incheon 400-712, Korea
| |
Collapse
|
|
8
|
Carrett-Dias M, Almeida LK, Pereira JL, Almeida DV, Filgueira DMVB, Marins LF, Votto APDS, Trindade GS. Cell differentiation and the multiple drug resistance phenotype in human erythroleukemic cells. Leuk Res 2016; 42:13-20. [DOI: 10.1016/j.leukres.2016.01.008] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2015] [Revised: 01/14/2016] [Accepted: 01/18/2016] [Indexed: 12/16/2022]
|
|
9
|
Wang X, Wang Y, Zuo Q, Li D, Zhang W, Lian C, Tang B, Xiao T, Wang M, Wang K, Li B, Zhang Y. The synergistic effect of 5Azadc and TSA on maintenance of pluripotency of chicken ESCs by overexpression of NANOG gene. In Vitro Cell Dev Biol Anim 2016; 52:488-96. [PMID: 26822431 DOI: 10.1007/s11626-015-9993-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2015] [Accepted: 12/20/2015] [Indexed: 11/30/2022]
Abstract
NANOG is a transcription factor that functions in embryonic stem cells (ESCs) and a key factor in maintaining pluripotency. Here, we cloned the NANOG gene promoter from the Rugao yellow chicken and constructed a dual luciferase reporter vector to detect its transcriptional activity and analyze the effects of 5-aza-2'-deoxycytidine (5-Azadc) and trichostatin A (TSA) on NANOG promoter activity and ESC pluripotency maintenance in vitro. NANOG transcriptional activity was enhanced when 5-Azadc and TSA were used alone or together, suggesting the possibility of elevated methylation of the CpG island in the NANOG regulatory region. When ESCs were cultured in basic medium with 5-Azadc and TSA in vitro, significantly more cell colonies were maintained in the 5-Azadc + TSA group than in the control group, which had many differentiated cells and few cell colonies after 6 d of induction. On the tenth day of induction, the cells in the control group fully differentiated and no cell colonies remained, but many cell colonies were present in the 5-Azadc + TSA group. The expression of NANOG in the cell colonies was confirmed by indirect immunofluorescence. Furthermore, ESCs could be passaged to the 12th generation under 5-Azadc and TSA treatment and maintained their pluripotency. Thus, we showed that 5-Azadc and TSA can effectively maintain chicken ESC pluripotency in vitro by increasing NANOG gene expression.
Collapse
Affiliation(s)
- Xiaoyan Wang
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Yingjie Wang
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Qisheng Zuo
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Dong Li
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Wenhui Zhang
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Chao Lian
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Beibei Tang
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Tianrong Xiao
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Man Wang
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China.,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Kehua Wang
- Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou, 225125, Jiangsu Province, People's Republic of China
| | - Bichun Li
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China. .,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
| | - Yani Zhang
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu Province, People's Republic of China. .,Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
| |
Collapse
|
|
10
|
Yang F, Zhang J, Liu Y, Cheng D, Wang H. Structure and functional evaluation of porcine NANOG that is a single-exon gene and has two pseudogenes. Int J Biochem Cell Biol 2015; 59:142-52. [DOI: 10.1016/j.biocel.2014.12.009] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2014] [Revised: 12/11/2014] [Accepted: 12/15/2014] [Indexed: 12/25/2022]
|
|
11
|
Sun D, Qin L, Xu Y, Liu JX, Tian LP, Qian HX. Influence of adriamycin on changes in Nanog, Oct-4, Sox2, ARID1 and Wnt5b expression in liver cancer stem cells. World J Gastroenterol 2014; 20:6974-6980. [PMID: 24944491 PMCID: PMC4051940 DOI: 10.3748/wjg.v20.i22.6974] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/08/2014] [Revised: 04/28/2014] [Accepted: 05/19/2014] [Indexed: 02/07/2023] Open
Abstract
AIM: To determine the influence of Adriamycin (ADM) on the changes in Nanog, Oct4, Sox2, as well as, in ARID1 and Wnt5b expression in liver cancer stem cells.
METHODS: The MHCC97-L and HCCLM3 liver cancer cell lines were selected as the cell models in this study, and were routinely cultured. The 50% lethal dose (LD50) in the cell lines was detected by the MTT assay. Expression changes in liver cancer stem cell related genes (Nanog, Oct-4, Sox2, ARID1, and Wnt5b) were detected by western blot following treatment with ADM (LD50).
RESULTS: The LD50 of ADM in MHCC97-L cells was lower than that in HCCLM3 cells (0.4123 ± 0.0236 μmol/L vs 0.5259 ± 0.0125 μmol/L, P < 0.05). Wnt5b and Nanog were expressed in both MHCC97-L and HCCLM3 cells, while only Sox2 was expressed in HCCLM3 cells. However, neither ARID1A nor Oct4 was detected in these two cell lines. Genes, related to the stem cells, showed different expression in liver cancer cells with different metastatic potential following treatment with ADM (LD50). Wnt5b protein increased gradually within 4 h of ADM (LD50) treatment, while Nanog decreased (P < 0.05). After 12 h, Wnt5b decreased gradually, while Nanog increased steadily (P < 0.05). In addition, only Sox2 was expressed in HCCLM3 cells with high metastatic potential following ADM (LD50) treatment. The expression of Sox2 increased gradually with ADM (LD50) in HCCLM3 cells (P < 0.05).
CONCLUSION: ADM increased the death rate of MHCC97-L and HCCLM3 cells, while the growth suppressive effect of ADM was higher in MHCC97-L cells than in HCCLM3 cells.
Collapse
|
|
12
|
Guo Y, Lei L, Ma X, Wang H. Characterization of the proximal region of the goat NANOG promoter that is used for monitoring cell reprogramming and early embryo development. Vet J 2014; 199:80-7. [DOI: 10.1016/j.tvjl.2013.10.004] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2013] [Revised: 09/22/2013] [Accepted: 10/01/2013] [Indexed: 12/20/2022]
|
|
13
|
Cloning, expression promoter analysis of vasa gene in Japanese flounder (Paralichthys olivaceus). Comp Biochem Physiol B Biochem Mol Biol 2014; 167:41-50. [DOI: 10.1016/j.cbpb.2013.06.004] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2013] [Revised: 06/11/2013] [Accepted: 06/17/2013] [Indexed: 11/23/2022]
|
|
14
|
Ibrahim EE, Babaei-Jadidi R, Nateri AS. The streptavidin/biotinylated DNA/protein bound complex protocol for determining the association of c-JUN protein with NANOG promoter. ACTA ACUST UNITED AC 2013; Chapter 1:Unit 1B.10. [PMID: 23661244 DOI: 10.1002/9780470151808.sc01b10s25] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Chromatin immunoprecipitation (ChIP) is a widely used and pre-eminent technique for detecting the association of an individual protein or a particular protein complex with its specific DNA sequence(s) in vivo. Herein we introduce a novel and simple biotinylated-oligonucleotide-mediated ChIP method for testing specific binding of the c-JUN protein to the M1-DNA-regulatory element in the NANOG promoter. We prepared a 260-bp DNA PCR amplicon containing -300 bp to -59 bp, relative to the transcriptional start site of the human NANOG gene, which was transfected into mouse embryonic fibroblasts (MEF) containing wild-type (c-jun(+/+)) or knockout c-jun (c-jun(-/-)) alleles. Whole cells that were cross-linked using formaldehyde and protein-DNA interactions were immunoprecipitated using streptavidin-coupled Dynabeads. Protein-DNA cross-links were reversed during incubation at 95°C, and protein samples were visualized using SDS-PAGE electrophoresis and western blotting. This streptavidin/biotinylated DNA/protein-bound complex protocol can be used for detecting the interactions between multiple transcription factors and their DNA binding sites.
Collapse
Affiliation(s)
- Elsayed E Ibrahim
- Cancer Genetics and Stem Cell Group, Division of Pre-Clinical Oncology, University of Nottingham, Nottingham, United Kingdom
| | | | | |
Collapse
|
|
15
|
Identification and characterization of a nanog homolog in Japanese flounder (Paralichthys olivaceus). Gene 2013; 531:411-21. [PMID: 24013085 DOI: 10.1016/j.gene.2013.08.030] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2013] [Revised: 07/28/2013] [Accepted: 08/10/2013] [Indexed: 02/06/2023]
Abstract
The homeodomain-containing transcription factor nanog plays a key role in maintaining the pluripotency and self-renewal of embryonic stem cells in mammals. Stem cells offered as a significant and effective tool for generation of transgenic animals and preservation of genetic resources. The molecular genetic organization and expression of nanog gene in marine fish have not been reported yet. In this study, we isolated and characterized the flounder nanog gene as a first step towards understanding the mechanism of the plurpotency of fish stem cells and develop a potential molecular marker to identify the stem cells in vivo and in vitro. Phylogenetic, gene structure and chromosome synteny analysis provided the evidence that Po-nanog is homologous to the mammalian nanog gene. Protein sequence comparison showed that flounder Nanog shared low similarity with other vertebrate orthologs except for a conserved homeodomain. Quantitative RT-PCR analysis showed that flounder nanog was maternally expressed, and the transcripts were present from the one-cell stage to the neurula stage with the peaking at blastula stage. Whole mount in situ hybridization analyses demonstrated that the transcripts were present in all blastomeres of the early embryo. Tissue distribution analysis indicated that nanog was detectable only in gonads. Further, the expression was significantly high in ovary than in testis. In situ hybridization revealed that the transcripts were located in the cytoplasm of the oogonia and oocytes in ovary, only in the spermatogonia but no spermatocytes or spermatids in testis. The promoter region was also analyzed to have several basal core promoter elements and transcription factor binding sites. All these results suggest that Po-Nanog may have a conservative function between teleosts and mammals.
Collapse
|
|
16
|
Galvin-Burgess KE, Travis ED, Pierson KE, Vivian JL. TGF-β-superfamily signaling regulates embryonic stem cell heterogeneity: self-renewal as a dynamic and regulated equilibrium. Stem Cells 2013; 31:48-58. [PMID: 23081664 DOI: 10.1002/stem.1252] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2012] [Accepted: 09/12/2012] [Indexed: 01/06/2023]
Abstract
Embryonic stem cells dynamically fluctuate between phenotypic states, as defined by expression levels of genes such as Nanog, while remaining pluripotent. The dynamic phenotype of stem cells is in part determined by gene expression control and dictated by various signaling pathways and transcriptional regulators. We sought to define the activities of two TGF-β-related signaling pathways, bone morphogenetic protein (BMP) and Nodal signaling, in modulating mouse embryonic stem (ES) cell heterogeneity in undifferentiated culture conditions. Both BMP and Nodal signaling pathways were seen to be active in distinct Nanog subpopulations, with subtle quantitative differences in activity. Pharmacological and genetic modulation of BMP or Nodal signaling strongly influenced the heterogeneous state of undifferentiated ES cells, as assessed by dynamic expression of Nanog reporters. Inhibition of Nodal signaling enhanced BMP activity, which through the downstream target Id factors, enhanced the capacity of ES cells to remain in the Nanog-high epigenetic state. The combined inhibition of Nodal and BMP signaling resulted in the accumulation of Nanog-negative cells, even in the presence of LIF, uncovering a shared role for BMP and Nodal signaling in maintaining Nanog expression and repression of differentiation. These results demonstrate a complex requirement for both arms of TGF-β-related signaling to influence the dynamic cellular phenotype of undifferentiated ES cells in serum-based media, and that differing subpopulations of ES cells in heterogeneous culture have distinct responses to these signaling pathways. Several pathways, including BMP, Nodal, and FGF signaling, have important regulatory function in defining the steady-state distribution of heterogeneity of stem cells.
Collapse
Affiliation(s)
- Katherine E Galvin-Burgess
- Department of Pathology and Laboratory, Institute for Reproductive Health and Regenerative Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160, USA
| | | | | | | |
Collapse
|
|
17
|
Ibrahim EE, Babaei-Jadidi R, Saadeddin A, Spencer-Dene B, Hossaini S, Abuzinadah M, Li N, Fadhil W, Ilyas M, Bonnet D, Nateri AS. Embryonic NANOG activity defines colorectal cancer stem cells and modulates through AP1- and TCF-dependent mechanisms. Stem Cells 2013; 30:2076-87. [PMID: 22851508 DOI: 10.1002/stem.1182] [Citation(s) in RCA: 110] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Embryonic NANOG (NANOG1) is considered as an important regulator of pluripotency while NANOGP8 (NANOG-pseudogene) plays a role in tumorigenesis. Herein, we show NANOG is expressed from both NANOG1 and NANOGP8 in human colorectal cancers (CRC). Enforced NANOG1-expression increases clonogenic potential and tumor formation in xenograft models, although it is expressed only in a small subpopulation of tumor cells and is colocalized with endogenous nuclear β-catenin(High) . Moreover, single NANOG1-CRCs form spherical aggregates, similar to the embryoid body of embryonic stem cells (ESCs), and express higher levels of stem-like Wnt-associated target genes. Furthermore, we show that NANOG1-expression is positively regulated by c-JUN and β-catenin/TCF4. Ectopic expression of c-Jun in murine Apc(Min/+) -ESCs results in the development of larger xenograft tumors with higher cell density compared to controls. Chromatin immunoprecipitation assays demonstrate that c-JUN binds to the NANOG1-promoter via the octamer M1 DNA element. Collectively, our data suggest that β-Catenin/TCF4 and c-JUN together drive a subpopulation of CRC tumor cells that adopt a stem-like phenotype via the NANOG1-promoter.
Collapse
Affiliation(s)
- Elsayed E Ibrahim
- Cancer Genetics and Stem Cell Group, Division of Pre-Clinical Oncology, University of Nottingham, Nottingham, UK
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
18
|
Lei L, Li L, Du F, Chen CH, Wang H, Keefer CL. Monitoring bovine fetal fibroblast reprogramming utilizing a bovine NANOG promoter-driven EGFP reporter system. Mol Reprod Dev 2013; 80:193-203. [PMID: 23280629 DOI: 10.1002/mrd.22147] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2012] [Accepted: 12/17/2012] [Indexed: 01/23/2023]
Abstract
NANOG is an essential transcription factor involved in the proliferation and maintenance of embryonic stem cells (ESC) and reprogramming of somatic cells to a pluripotent state. Oct4 and Nanog promoter-driven enhanced green fluorescent protein (EGFP) reporters have been employed for establishing lines of induced pluripotent stem cells (iPSC) from mouse, human, and pig. In ruminants, including cattle, in which no fully validated ESC lines have been established, iPSC generated by reprogramming somatic cells to an ESC-like state may prove useful in the production of genetically modified livestock. In this study, utility of the bovine NANOG reporter was tested for use with cattle. Seven proximal bovine NANOG promoter fragments of different size were fused to the LUC gene, and were tested in mouse ESC lines using a dual-luciferase assay. Three of the bovine NANOG promoters, 315 bp (-134/+181), 446 bp (-265/+181), and 1,100 bp (-919/+181), were fused to a nuclear localized signal EGFP reporter gene. The fidelity of these constructs was analyzed by transfection into mouse ESC and bovine fetal fibroblasts (bFFs), and subsequent reprogramming of the bFF. Fusion of the transgenic bFF with human teratocarcinoma (NTERA2) cells induced nuclear expression of the EGFP reporter. Similarly, bFF-derived somatic cell nuclear transfer (SCNT) embryos expressed EGFP in a stage- and location-appropriate manner. Following reprogramming of transgenic bFFs for 10 days with an Oct4-Sox2-Klf4-cMyc vector, iPSC expressed EGFP and alkaline phosphatase. These results indicate that NANOG reporters can be used to monitor nuclear reprogramming of bFFs and to distinguish cell allocation in SCNT-derived embryos.
Collapse
Affiliation(s)
- Lei Lei
- College of Veterinary Medicine, Shaanxi Center for Stem Cell Engineering and Technology, Northwest A&F University, Yangling, P.R. China
| | | | | | | | | | | |
Collapse
|
|
19
|
Jung SM, Ju JH. Application of Induced Pluripotent Stem Cells in Rheumatology. JOURNAL OF RHEUMATIC DISEASES 2013. [DOI: 10.4078/jrd.2013.20.5.286] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Affiliation(s)
- Seung Min Jung
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Korea
| | - Ji Hyeon Ju
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Korea
| |
Collapse
|
|
20
|
Singh N, Sharma R, George A, Singla SK, Palta P, Manik R, Chauhan MS, Singh D. Cloning and characterization of buffalo NANOG gene: alternative transcription start sites, splicing, and polyadenylation in embryonic stem cell-like cells. DNA Cell Biol 2012; 31:721-31. [PMID: 22011250 PMCID: PMC3358104 DOI: 10.1089/dna.2011.1410] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2011] [Revised: 09/02/2011] [Accepted: 09/02/2011] [Indexed: 01/06/2023] Open
Abstract
NANOG is a critical homeodomain transcription factor responsible for maintaining embryonic stem cell (ESC) self-renewal and pluripotency. In the present study, we isolated, sequenced, and characterized the NANOG gene in buffalo ESC-like cells. Here, we demonstrated that NANOG mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and five different polyadenylation sites. The TSSs identified by 5'-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5'-RACE) were positioned at 182, 95, 35, and 17 nucleotides upstream relative to the translation initiation codon. 3'-RACE experiment revealed the presence of tandem polyadenylation signals, which leads to the expression of at least five different 3'-untranslated regions (269, 314, 560, 566, and 829 nucleotides). Expression analysis showed that these alternatively polyadenylated transcripts expressed differentially. Sequence analysis showed that the open reading frame of buffalo NANOG codes for a 300-amino-acid-long protein. Further, results showed that alternative splicing leads to the expression of two types of transcript variants encoded by four and five exons. In silico analysis of cloned 5'-flanking region (3366 nucleotides upstream of translation start codon) identified several putative transcription factors binding sites in addition to a TATA box and CAAT box at -30 and -139 bp (upstream to the distal most TSS), respectively, in the buffalo NANOG promoter.
Collapse
Affiliation(s)
- Natwar Singh
- Molecular Endocrinology Laboratory, Animal Biochemistry Division, National Dairy Research Institute (NDRI), Karnal, Haryana, India
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, Haryana, India
| | - Ruchi Sharma
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, Haryana, India
| | - Aman George
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, Haryana, India
| | - Suresh K. Singla
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, Haryana, India
| | - Prabhat Palta
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, Haryana, India
| | - Radhaysham Manik
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, Haryana, India
| | - Manmohan S. Chauhan
- Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, Haryana, India
| | - Dheer Singh
- Molecular Endocrinology Laboratory, Animal Biochemistry Division, National Dairy Research Institute (NDRI), Karnal, Haryana, India
| |
Collapse
|
|
21
|
Hackett CH, Greve L, Novakofski KD, Fortier LA. Comparison of gene-specific DNA methylation patterns in equine induced pluripotent stem cell lines with cells derived from equine adult and fetal tissues. Stem Cells Dev 2011; 21:1803-11. [PMID: 21988203 DOI: 10.1089/scd.2011.0055] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Cellular pluripotency is associated with expression of the homeobox transcription factor genes NANOG, SOX2, and POU5F1 (OCT3/4 protein). Some reports suggest that mesenchymal progenitor cells (MPCs) may express increased quantities of these genes, creating the possibility that MPCs are more "pluripotent" than other adult cell types. The objective of this study was to determine whether equine bone marrow-derived MPCs had gene expression or DNA methylation patterns that differed from either early fetal-derived or terminally differentiated adult cells. Specifically, this study compared DNA methylation of the NANOG and SOX2 promoter regions and concurrent gene expression of NANOG, SOX2, and POU5F1 in equine induced pluripotent stem (iPS) cells, fetal fibroblasts, fetal brain cells, adult chondrocytes, and MPCs. Results indicate that NANOG and POU5F1 were not detectable in appreciable quantities in tissues other than the equine iPS cell lines. Equine iPS cells expressed large quantities of all three genes examined. Significantly increased quantities of SOX2 were noted in iPS cells and both fetal-derived cell types compared with adult cells. MPCs and adult chondrocytes expressed equivalent, low quantities of SOX2. Further, NANOG and SOX2 expression inversely correlated with the DNA methylation pattern in the promoter region, such that as gene expression increased, DNA methylation decreased. The equine iPS cell lines examined demonstrated DNA methylation and gene expression patterns that were consistent with pluripotency features described in other species. Results do not support previous reports that NANOG, SOX2, and POU5F1 are poised for increased activity in MPCs compared with other adult cells.
Collapse
Affiliation(s)
- Catherine H Hackett
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
| | | | | | | |
Collapse
|
|
22
|
Marandel L, Labbe C, Bobe J, Le Bail PY. nanog 5'-upstream sequence, DNA methylation, and expression in gametes and early embryo reveal striking differences between teleosts and mammals. Gene 2011; 492:130-7. [PMID: 22037485 DOI: 10.1016/j.gene.2011.10.037] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2011] [Revised: 10/11/2011] [Accepted: 10/12/2011] [Indexed: 01/14/2023]
Abstract
The nanog gene plays a major role in vertebrate development and was only recently discovered in teleosts. In order to gain new insight into its regulation in gametes and early embryo in teleost fish, the present study aimed at characterizing nanog upstream sequence features and DNA methylation, as well as early embryonic expression pattern in a Cyprinid fish, the goldfish. Using an in silico approach, we were able to demonstrate that despite the existence of conserved regulatory motifs in the promoter region of the nanog gene, specific features known to play a major role in the regulation of Nanog in mammals were missing in teleosts. The analysis of CpG sites in the upstream region of the nanog genes in goldfish revealed a significant DNA methylation state in oocytes while a hypomethylated state was observed in sperm. Using both quantitative PCR and whole mount in situ hybridization, we were able to clearly demonstrate the maternal inheritance of the nanog transcript in goldfish. Corresponding mRNA levels subsequently decreased during early gastrulation. Together, our results reveal striking differences in expression and DNA methylation patterns in gametes and during early development and in upstream region features between teleosts and mammals that are consistent with the hypothesis of a rapid evolution of the Nanog gene in vertebrates, at least in some lineages.
Collapse
Affiliation(s)
- Lucie Marandel
- INRA, UR1037 SCRIBE, IFR140, Biogenouest, Campus de Beaulieu, F-35042 Rennes, France.
| | | | | | | |
Collapse
|
|
23
|
Fuellen G, Struckmann S. Evolution of gene regulation of pluripotency--the case for wiki tracks at genome browsers. Biol Direct 2010; 5:67. [PMID: 21190561 PMCID: PMC3024949 DOI: 10.1186/1745-6150-5-67] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2010] [Accepted: 12/29/2010] [Indexed: 12/23/2022] Open
Abstract
Background Experimentally validated data on gene regulation are hard to obtain. In particular, information about transcription factor binding sites in regulatory regions are scattered around in the literature. This impedes their systematic in-context analysis, e.g. the inference of their conservation in evolutionary history. Results We demonstrate the power of integrative bioinformatics by including curated transcription factor binding site information into the UCSC genome browser, using wiki and custom tracks, which enable easy publication of annotation data. Data integration allows to investigate the evolution of gene regulation of the pluripotency-associated genes Oct4, Sox2 and Nanog. For the first time, experimentally validated transcription factor binding sites in the regulatory regions of all three genes were assembled together based on manual curation of data from 39 publications. Using the UCSC genome browser, these data were then visualized in the context of multi-species conservation based on genomic alignment. We confirm previous hypotheses regarding the evolutionary age of specific regulatory patterns, establishing their "deep homology". We also confirm some other principles of Carroll's "Genetic theory of Morphological Evolution", such as "mosaic pleiotropy", exemplified by the dual role of Sox2 reflected in its regulatory region. Conclusions We were able to elucidate some aspects of the evolution of gene regulation for three genes associated with pluripotency. Based on the expected return on investment for the community, we encourage other scientists to contribute experimental data on gene regulation (original work as well as data collected for reviews) to the UCSC system, to enable studies of the evolution of gene regulation on a large scale, and to report their findings. Reviewers This article was reviewed by Dr. Gustavo Glusman and Dr. Juan Caballero, Institute for Systems Biology, Seattle, USA (nominated by Dr. Doron Lancet, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel), Dr. Niels Grabe, TIGA Center (BIOQUANT) and Medical Systems Biology Group, Institute of Medical Biometry and Informatics, University Hospital Heidelberg, Germany (nominated by Dr. Mikhail Gelfand, Department of Bioinformatics, Institute of Information Transfer Problems, Russian Academy of Science, Moscow, Russian Federation) and Dr. Franz-Josef Müller, Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA, USA and University Hospital for Psychiatry and Psychotherapy (part of ZIP gGmbH), University of Kiel, Germany (nominated by Dr. Trey Ideker, University of California, San Diego, La Jolla CA, United States).
Collapse
Affiliation(s)
- Georg Fuellen
- Institute for Biostatistics and Informatics in Medicine and Ageing Research - IBIMA, University of Rostock, Medical Faculty, Ernst-Heydemann-Str. 8, 18057 Rostock, Germany.
| | | |
Collapse
|
|
24
|
Kim JS, Kim BS, Kim J, Park CS, Chung IY. The phosphoinositide-3-kinase/Akt pathway mediates the transient increase in Nanog expression during differentiation of F9 cells. Arch Pharm Res 2010; 33:1117-25. [PMID: 20661723 DOI: 10.1007/s12272-010-0719-y] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2010] [Revised: 05/11/2010] [Accepted: 05/19/2010] [Indexed: 11/28/2022]
Abstract
Nanog is a key determinant that maintains self-renewal and pluripotency of embryonic stem cells and represses their differentiation to endoderm. In this study, we examined the regulation of Nanog expression by phosphoinositide-3-kinase (PI3K)/Akt pathway during retinoic acid (RA)-induced differentiation of F9 embryonic carcinoma cells. Nanog protein expression was transiently upregulated up to 6 h after RA treatment and then declined. In agreement, a murine Nanog promoter reporter assay revealed that promoter activity increased during early stage of differentiation, but decreased when F9 cells became fully differentiated. RA treatment of F9 cells also led to a transient and parallel increase in both Akt and glycogen synthase kinase 3beta phosphorylations. Nanog expression was diminished in the early stage by LY294002, a PI3K inhibitor, but was not affected in the late stage despite considerable inhibition of Akt phosphorylation and endoderm marker expression by the inhibitor. These data suggest that RA-induced PI3K/Akt activation in the early stage of differentiation is required for Nanog expression, which becomes independent of PI3K/Akt signaling once the differentiation is established. Thus, Nanog expression appears to be differently regulated by the PI3K/Akt pathway depending on differentiation stage.
Collapse
|
|
25
|
Kang J, Shakya A, Tantin D. Stem cells, stress, metabolism and cancer: a drama in two Octs. Trends Biochem Sci 2009; 34:491-9. [PMID: 19733480 DOI: 10.1016/j.tibs.2009.06.003] [Citation(s) in RCA: 81] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2009] [Revised: 05/30/2009] [Accepted: 06/03/2009] [Indexed: 01/16/2023]
Abstract
It is a classic story of two related transcription factors. Oct4 is a potent regulator of pluripotency during early mammalian embryonic development, and is notable for its ability to convert adult somatic cells to pluripotency. The widely expressed Oct1 protein shares significant homology with Oct4, binds to the same sequences, regulates common target genes, and shares common modes of upstream regulation, including the ability to respond to cellular stress. Both proteins are also associated with malignancy, yet Oct1 cannot substitute for Oct4 in the generation of pluripotency. The molecular underpinnings of these phenomena are emerging, as are the consequences for adult stem cells and cancer, and thereby hangs a tale.
Collapse
Affiliation(s)
- Jinsuk Kang
- Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84112, USA
| | | | | |
Collapse
|
|
26
|
Kalmar T, Lim C, Hayward P, Muñoz-Descalzo S, Nichols J, Garcia-Ojalvo J, Martinez Arias A. Regulated fluctuations in nanog expression mediate cell fate decisions in embryonic stem cells. PLoS Biol 2009; 7:e1000149. [PMID: 19582141 PMCID: PMC2700273 DOI: 10.1371/journal.pbio.1000149] [Citation(s) in RCA: 417] [Impact Index Per Article: 26.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2008] [Accepted: 05/28/2009] [Indexed: 01/06/2023] Open
Abstract
The notion that the differentiated state of a cell population is determined simply by expression of specific marker genes is changing. In this work, the authors reveal that a pluripotent cell population comprises cells with temporal fluctuations in the expression of Nanog. There is evidence that pluripotency of mouse embryonic stem (ES) cells is associated with the activity of a network of transcription factors with Sox2, Oct4, and Nanog at the core. Using fluorescent reporters for the expression of Nanog, we observed that a population of ES cells is best described by a dynamic distribution of Nanog expression characterized by two peaks defined by high (HN) and low (LN) Nanog expression. Typically, the LN state is 5%–20% of the total population, depending on the culture conditions. Modelling of the activity of Nanog reveals that a simple network of Oct4/Sox2 and Nanog activity can account for the observed distribution and its properties as long as the transcriptional activity is tuned by transcriptional noise. The model also predicts that the LN state is unstable, something that is born out experimentally. While in this state, cells can differentiate. We suggest that transcriptional fluctuations in Nanog expression are an essential element of the pluripotent state and that the function of Sox2, Oct4, and Nanog is to act as a network that promotes and maintains transcriptional noise to interfere with the differentiation signals. Embryonic stem (ES) cells are a pluripotent cell population derived from early mammalian embryos. An intrinsic feature of ES cells is their phenotypic heterogeneity: they display promiscuous activation of lineage-specific genes and exhibit a fluctuating flow of differentiating cells. A gene regulatory network (GRN) centred around the transcription factors Sox2, Oct4, and Nanog is essential for the establishment and the maintenance of the pluripotent state. Previous studies had suggested that ES cells can reversibly change their state of Nanog expression without losing pluripotency. Here, we extend these studies by quantifying and monitoring the expression of Nanog in a Nanog-GFP reporter cell line. We show that Nanog levels undergo slow, random fluctuations in ES cells, giving rise to heterogeneous cell populations. We identify two states, one stable, characterized by high levels of expression (HN), and another with low levels of Nanog expression (LN), which is highly unstable. While in the LN state, cells are more likely to differentiate depending on the culture medium. Mathematical modelling shows that a simple excitable system driven by transcriptional noise can account for the observed distributions and behaviours in gene expression. Our study suggests that rather than a discrete state dependent on the fixed expression of a small set of genes, pluripotency is best represented by a state of dynamic heterogeneity of a population driven by transcriptional noise, and that the function of the gene regulatory network centred around Nanog might be to generate dynamic heterogeneities at the population level.
Collapse
Affiliation(s)
- Tibor Kalmar
- Department of Genetics, University of Cambridge, Cambridge, United Kingdom
| | - Chea Lim
- Department of Genetics, University of Cambridge, Cambridge, United Kingdom
| | - Penelope Hayward
- Department of Genetics, University of Cambridge, Cambridge, United Kingdom
| | | | - Jennifer Nichols
- Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, United Kingdom
| | - Jordi Garcia-Ojalvo
- Departament de Fisica i Enginyeria Nuclear, Universitat Politecnica de Catalunya, Colom 11, Terrassa, Spain
| | | |
Collapse
|
|
27
|
Zhong X, Jin Y. Critical roles of coactivator p300 in mouse embryonic stem cell differentiation and Nanog expression. J Biol Chem 2009; 284:9168-75. [PMID: 19150979 DOI: 10.1074/jbc.m805562200] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
p300 is a well known histone acetyltransferase and coactivator that plays pivotal roles in many physiological processes. Despite extensive research for the functions of p300 in embryogenesis and transcription regulation, its roles in regulating embryonic stem (ES) cell pluripotency are poorly understood. To address this issue, we investigated the self-renewal ability and early differentiation process in both wild-type mouse ES cells and ES cells derived from p300 knock-out (p300(-/-)) mice. We found that p300 ablation did not affect self-renewal capacity overtly when ES cells were maintained under undifferentiated conditions. However, the absence of p300 caused a significantly abnormal expression pattern of germ layer markers when differentiation was induced by embryoid body (EB) formation. Interestingly, the expression level of pluripotency marker Nanog but not Oct4 was markedly lower in EBs from p300(-/-) ES cells compared with that in EBs from wild-type ES cells. Exogenous expression of Nanog rescued abnormal expression of extra-embryonic endoderm marker partially but not mesoderm and ectoderm markers. Furthermore, we demonstrate that p300 was directly involved in modulating Nanog expression. Importantly, epigenetic modification of histone acetylation at the distal regulatory region of Nanog was found to be dependent on the presence of p300, which could contribute to the mechanism of regulating Nanog expression by p300. Collectively, our results show that p300 plays an important role in the differentiation process of ES cells and provide the first evidence for the involvement of p300 in regulating Nanog expression during differentiation, probably through epigenetic modification of histone on Nanog.
Collapse
Affiliation(s)
- Xiaomin Zhong
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai Jiao Tong University School of Medicine, China
| | | |
Collapse
|
|
28
|
Milewski WM, Temple KA, Wesselschmidt RL, Hara M. Generation of embryonic stem cells from mouse insulin I promoter-green fluorescent protein transgenic mice and characterization in a teratoma model. In Vitro Cell Dev Biol Anim 2008; 45:1-5. [PMID: 18855079 DOI: 10.1007/s11626-008-9142-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2008] [Accepted: 08/25/2008] [Indexed: 11/29/2022]
Abstract
Insulin-secreting pancreatic beta cells play a key role in the pathogenesis of diabetes mellitus. Potential new treatments for this disease include cell-replacement therapies using embryonic stem cells (ESCs). We have generated ESCs from a transgenic mouse model, mouse insulin 1 promoter (MIP) green fluorescent protein (GFP) mice, in which embryonic and adult beta cells are genetically tagged with GFP. The aim of the present study is to examine the differentiation potential of MIP-GFP ESCs in the microenvironment of the kidney capsule. The ESCs grew rapidly and formed a teratoma with GFP-expressing beta-like cells present in clusters that formed a cord-like structure similar to what is seen in the embryonic pancreas. These structures also included glucagon-expressing alpha cells and amylase-expressing acinar cells. Electron microscopic analysis showed insulin-like granules in columnar epithelium with microvilli adjacent to exocrine-like granule-containing cells. The MIP-GFP ESCs should be a useful research tool to study the differentiation capacity of ESCs toward pancreatic lineages.
Collapse
Affiliation(s)
- Wieslawa M Milewski
- Department of Medicine, The University of Chicago, 5841 South Maryland Avenue, MC1027, Chicago, IL 60637, USA
| | | | | | | |
Collapse
|
|
29
|
Yamanaka S. Pluripotency and nuclear reprogramming. Philos Trans R Soc Lond B Biol Sci 2008; 363:2079-87. [PMID: 18375377 DOI: 10.1098/rstb.2008.2261] [Citation(s) in RCA: 95] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Embryonic stem cells are promising donor cell sources for cell transplantation therapy, which may in the future be used to treat various diseases and injuries. However, as is the case for organ transplantation, immune rejection after transplantation is a potential problem with this type of therapy. Moreover, the use of human embryos presents serious ethical difficulties. These issues may be overcome if pluripotent stem cells are generated from patients' somatic cells. Here, we review the molecular mechanisms underlying pluripotency and the currently known methods of inducing pluripotency in somatic cells.
Collapse
Affiliation(s)
- Shinya Yamanaka
- Center for iPS Cell Research & Application, Kyoto University, Kyoto 606-8507, Japan.
| |
Collapse
|
|
30
|
Minematsu T, Harumi T, Naito M. Germ cell-specific expression of GFP gene induced by chickenvasahomologue (Cvh) promoter in early chicken embryos. Mol Reprod Dev 2008; 75:1515-22. [DOI: 10.1002/mrd.20894] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
|
|
31
|
Estrogen-related receptor beta interacts with Oct4 to positively regulate Nanog gene expression. Mol Cell Biol 2008; 28:5986-95. [PMID: 18662995 DOI: 10.1128/mcb.00301-08] [Citation(s) in RCA: 134] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Embryonic stem (ES) cell self-renewal is regulated by transcription factors, including Oct4, Sox2, and Nanog. A number of additional transcriptional regulators of ES cell self-renewal have recently been identified, including the orphan nuclear receptor estrogen-related receptor beta (Esrrb). However, the mode of action of Esrrb in ES cells is unknown. Here, using an Oct4 affinity screen, we identify Esrrb as an Oct4 partner protein. Esrrb can interact with Oct4 independently of DNA. Esrrb is recruited near the Oct-Sox element in the Nanog proximal promoter, where it positively regulates Nanog expression. Esrrb recruitment to the Nanog promoter requires both the presence of Oct4 and a degenerate estrogen-related receptor DNA element. Consistent with its role in Nanog regulation, expression of the Esrrb protein within the Oct4-positive ES cell population is mosaic and correlates with the mosaic expression of the Nanog protein. Together with previous reports that Nanog may regulate Esrrb gene expression, our results suggest that Esrrb and Nanog act as part of a feedback regulatory circuit that modulates the fluctuating self-renewal capacity of ES cell populations.
Collapse
|
|
32
|
Yi F, Merrill BJ. Stem cells and TCF proteins: a role for beta-catenin--independent functions. ACTA ACUST UNITED AC 2007; 3:39-48. [PMID: 17873380 DOI: 10.1007/s12015-007-0003-9] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/1999] [Revised: 11/30/1999] [Accepted: 11/30/1999] [Indexed: 12/18/2022]
Abstract
The Wnt signal transduction pathway has been shown to stimulate stem cell self renewal and has been shown to cause cancer in humans. One interesting aspect of Wnt signaling is that it utilizes downstream DNA-binding transcription factors, called Tcf proteins, which can activate transcription of target genes in the presence of a Wnt signal and repress the expression of target genes in the absence of a Wnt signal. Since Tcf proteins are present in Wnt-stimulated and unstimulated stem cells, understanding how Tcf proteins regulate target gene expression in each state offers the potential to understand how stem cells regulate their self-renewal, differentiation, and proliferation. In this article, we will review recent work elucidating the roles Tcf-protein interactions in the context of stem cells and cancer.
Collapse
Affiliation(s)
- Fei Yi
- Molecular Biology Research Building, Department of Biochemistry and Molecular Genetics, University of Illinois, 900 S. Ashland Ave., Chicago, IL 60607, USA
| | | |
Collapse
|
|
33
|
Pereira L, Yi F, Merrill BJ. Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal. Mol Cell Biol 2006; 26:7479-91. [PMID: 16894029 PMCID: PMC1636872 DOI: 10.1128/mcb.00368-06] [Citation(s) in RCA: 235] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
The dual function of stem cells requires them not only to form new stem cells through self-renewal but also to form lineage-committed cells through differentiation. Embryonic stem cells (ESC), which are derived from the blastocyst inner cell mass, retain properties of self-renewal and the potential for lineage commitment. To balance self-renewal and differentiation, ESC must carefully control the levels of several transcription factors, including Nanog, Sox2, and Oct4. While molecular mechanisms promoting transcription of these genes have been described, mechanisms preventing excessive levels in self-renewing ESC remain unknown. By examining the function of the TCF family of transcription factors in ESC, we have found that Tcf3 is necessary to limit the steady-state levels of Nanog mRNA, protein, and promoter activity in self-renewing ESC. Chromatin immunoprecipitation and promoter reporter assays showed that Tcf3 bound to a promoter regulatory region of the Nanog gene and repressed its transcriptional activity in ESC through a Groucho interaction domain-dependent process. The absence of Tcf3 caused delayed differentiation of ESC in vitro as elevated Nanog levels persisted through 5 days of embryoid body formation. These new data support a model wherein Tcf3-mediated control of Nanog levels allows stem cells to balance the creation of lineage-committed and undifferentiated cells.
Collapse
Affiliation(s)
- Laura Pereira
- Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland Ave., Molecular Biology Research Building, Room 1268, Mail Code 669, Chicago, IL 60607, USA
| | | | | |
Collapse
|
|
34
|
Imamura M, Miura K, Iwabuchi K, Ichisaka T, Nakagawa M, Lee J, Kanatsu-Shinohara M, Shinohara T, Yamanaka S. Transcriptional repression and DNA hypermethylation of a small set of ES cell marker genes in male germline stem cells. BMC DEVELOPMENTAL BIOLOGY 2006; 6:34. [PMID: 16859545 PMCID: PMC1564388 DOI: 10.1186/1471-213x-6-34] [Citation(s) in RCA: 105] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/12/2006] [Accepted: 07/21/2006] [Indexed: 01/06/2023]
Abstract
BACKGROUND We previously identified a set of genes called ECATs (ES cell-associated transcripts) that are expressed at high levels in mouse ES cells. Here, we examine the expression and DNA methylation of ECATs in somatic cells and germ cells. RESULTS In all ECATs examined, the promoter region had low methylation levels in ES cells, but higher levels in somatic cells. In contrast, in spite of their lack of pluripotency, male germline stem (GS) cells expressed most ECATs and exhibited hypomethylation of ECAT promoter regions. We observed a similar hypomethylation of ECAT loci in adult testis and isolated sperm. Some ECATs were even less methylated in male germ cells than in ES cells. However, a few ECATs were not expressed in GS cells, and most of them targets of Oct3/4 and Sox2. The Octamer/Sox regulatory elements were hypermethylated in these genes. In addition, we found that GS cells express little Sox2 protein and low Oct3/4 protein despite abundant expression of their transcripts. CONCLUSION Our results suggest that DNA hypermethylation and transcriptional repression of a small set of ECATs, together with post-transcriptional repression of Oct3/4 and Sox2, contribute to the loss of pluripotency in male germ cells.
Collapse
Affiliation(s)
- Masanori Imamura
- Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | - Kyoko Miura
- Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | - Kumiko Iwabuchi
- Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | - Tomoko Ichisaka
- Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | - Masato Nakagawa
- Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | - Jiyoung Lee
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
| | - Mito Kanatsu-Shinohara
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
- Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
| | - Takashi Shinohara
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
| | - Shinya Yamanaka
- Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
- CREST, Japan Science and Technology Agency, Saitama, Japan
| |
Collapse
|
|
35
|
Wu DY, Yao Z. Functional analysis of two Sp1/Sp3 binding sites in murine Nanog gene promoter. Cell Res 2006; 16:319-22. [PMID: 16541131 DOI: 10.1038/sj.cr.7310040] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5' flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements within the Nanog promoter, namely Oct-4 and Sox-2 binding sites, have been identified to regulate the transcriptional activity of Nanog gene. In this report, we identified the role of two putative Sp1 binding sites located in the Nanog gene 5'-flanking region in regulation of murine Nanog gene transcription. Mutation studies showed that the two sites were essential for the Nanog promoter activity. Gel shift and supershift analysis showed that both sites specifically bind Sp1 and Sp3. Furthermore, overexpression of dominant-negative Sp1 or Sp3 mutants significantly inhibits Nanog promoter activity. These results suggest that the transcription factor Sp1 and Sp3 are important for Murine Nanog gene expression.
Collapse
Affiliation(s)
- Da Yong Wu
- Laboratory of Molecular Cell Biology, Laboratory of Stem Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
| | | |
Collapse
|
|
36
|
Player A, Wang Y, Bhattacharya B, Rao M, Puri RK, Kawasaki ES. Comparisons between Transcriptional Regulation and RNA Expression in Human Embryonic Stem Cell Lines. Stem Cells Dev 2006; 15:315-23. [PMID: 16846370 DOI: 10.1089/scd.2006.15.315] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Recent studies have focused on transcriptional regulation and gene expression profiling of human embryonic stem cells (hESCs). However, little information is available regarding the relationship between RNA expression and transcriptional regulation, which is critical in the complete understanding of pluripotency and differentiation of hESCs. In the current study, we determined RNA expression of three different hESC lines compared to Human universal reference RNA expression (HuU-RNA) using a full genome expression microarray, and compared our results to target genes previously identified using ChIP-on-chip analysis. The objective was to identify genes common between the two methods, and generate a more reliable list of embryonic signature genes. Even though hESCs were obtained from different sources and maintained under different conditions, a considerable number of genes could be identified as common between RNA expression and transcriptional regulation analyses. As an example, results from ChIP-on-chip studies show that OCT4, SOX2, and NANOG co-occupy SOX2, OCT4, TDGF1, GJA1, SET, and DPPA4 genes. The results are consistent with RNA expression analyses that demonstrate these genes as differently expressed in our hESC lines, further substantiating their role across cell types and confirming their importance as embryonic signatures. In addition, we report the differential expression of growth arrest-specific (GAS) family of genes in hESC. GAS2L1 and GAS3 members of this family appear to be transcriptionally regulated by OCT4, SOX2, or NANOG, whereas GAS5 and GAS6 are not; all of the genes are differentially expressed, as determined by microarray and validated via quantitative (Q)- PCR. Collectively, these data provide insight into the relationship between gene expression and transcriptional regulation, resulting in a reliable list of genes associated with hESCs.
Collapse
Affiliation(s)
- Audrey Player
- Advanced Technology Center, Microarray Facility, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
| | | | | | | | | | | |
Collapse
|
|
37
|
Yamazaki Y, Fujita TC, Low EW, Alarcón VB, Yanagimachi R, Marikawa Y. Gradual DNA demethylation of the Oct4 promoter in cloned mouse embryos. Mol Reprod Dev 2006; 73:180-8. [PMID: 16245355 DOI: 10.1002/mrd.20411] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
During differentiation, somatic cell nuclei acquire unique patterns of epigenetic modifications, such as DNA methylation, which affect the transcriptional activity of specific genes. Upon transfer into oocytes, however, the somatic nucleus undergoes reprogramming of these epigenetic modifications to achieve pluripotency. Oct4 is one of the critical pluripotency regulators, and is expressed in the germ line, including the pluripotent early embryonic cells. Previous studies showed that the upstream regulatory sequences of the Oct4 gene are distinctly methylated in somatic cells, and the DNA methylation of the regulatory sequences suppresses the transcriptional activity. Thus, successful reprogramming of the somatic cell nucleus to gain pluripotency must be accompanied by the demethylation of the Oct4 regulatory sequences. Here, we investigated the methylation pattern of the Oct4 promoter during early development of cloned mouse embryos. We found that the Oct4 promoter was only gradually demethylated during the early cleavage stages and that the ineffective demethylation of the promoter was associated with developmental retardation. We also found that the upstream sequences of the other pluripotency regulators, namely Nanog, Sox2, and Foxd3, were considerably under-methylated in cumulus cells. These results suggest that the Oct4 gene, as compared to the other pluripotency regulators, needs to undergo extensive demethylation during nuclear reprogramming, and that the failure of such demethylation is associated with inefficient development of cloned somatic cell embryos.
Collapse
Affiliation(s)
- Yukiko Yamazaki
- Institute for Biogenesis Research, Department of Anatomy and Reproductive Biology, University of Hawaii John A. Burns School of Medicine, Honolulu, HI 96822, USA
| | | | | | | | | | | |
Collapse
|
|
38
|
The homeodomain protein Nanog and pluripotency in mouse embryonic stem cells. Biochem Soc Trans 2005; 33:1518-21. [PMID: 16246159 DOI: 10.1042/bst0331518] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Intrinsic regulators of the pluripotency of mouse ES (embryonic stem) cells include the homeodomain proteins Oct4 and the recently identified Nanog. When overexpressed, Nanog displays the unique attribute of robustly sustaining ES cell self-renewal in the absence of the otherwise requisite extracellular stimulation by LIF (leukaemia inhibitory factor) and BMP (bone morphogenetic protein). Here, we review our current understanding of the function of Nanog in pluripotent stem cells both in vitro and in vivo.
Collapse
|
|
39
|
Zou GM, Chen JJ, Ni J. LIGHT induces differentiation of mouse embryonic stem cells associated with activation of ERK5. Oncogene 2005; 25:463-9. [PMID: 16247486 DOI: 10.1038/sj.onc.1208987] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) is a recently cloned type II transmembrane protein belonging to the TNF family that was originally identified as a weak inducer of apoptosis. This cytokine has been extensively defined in its role on T-cell regulation and dendritic cell maturation. However, whether this cytokine regulates stem cell proliferation and/or differentiation remains unknown. In this study, we transduced exogenous LIGHT into embryonic stem cells (ES cells) and found it induced their differentiation. The expression of phospho-STAT3, Nanog and Oct-4 was reduced in LIGHT-transduced ES cells compared with wild-type ES cells. LIGHT-transduced ES cells exhibit a low level of SSEA-1 surface antigen and alkaline phosphatase staining compared with wild-type cells. Introduction of LIGHT into ES cells results in the dephosphorylation of MKP-3 and activation of extracellular signal-regulated kinase (ERK)5. When ERK5 was inhibited by the specific inhibitor PD184352 or knocked down by ERK5 siRNA, reduction of Oct-4 and SSEA-1 expression was rescued. We conclude that LIGHT overrides Leukemia inhibitory factor to induce ES cell differentiation associated with activation of ERK5.
Collapse
Affiliation(s)
- G-M Zou
- Department of Internal Medicine, University of Iowa School of Medicine, Iowa City, IA, USA.
| | | | | |
Collapse
|