scholarly journals NCOR1 and OCT4 Facilitate Early Reprogramming by Co-Suppressing Fibroblast Gene Expression

2019 ◽  
Author(s):  
Georgina Peñalosa-Ruiz ◽  
Klaas W. Mulder ◽  
Gert Jan C. Veenstra
Keyword(s):  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Embryonic Fibroblasts ◽  
Chromatin Regulators ◽  
Highly Correlated ◽  
Induced Pluripotent ◽  
Transcriptomic Changes ◽  
Shed Light ◽  
Pluripotency Genes

ABSTRACTReprogramming somatic cells to induced pluripotent stem cells (iPSC) succeeds only in a small fraction of cells within the population. Reprogramming occurs in distinctive stages, each facing its own bottlenecks. It initiates with overexpression of transcription factors OCT4, SOX2, KLF4 and c-MYC (OSKM) in somatic cells such as mouse embryonic fibroblasts (MEFs). OSKM bind chromatin, silencing the somatic identity and starting the stepwise reactivation of the pluripotency program. However, inefficient suppression of the somatic lineage leads to unwanted epigenetic memory from the tissue of origin, even in successfully generated iPSCs. Thus, it is essential to shed more light on chromatin regulators and processes involved in dissolving the somatic identity. Recent work characterized the role of transcriptional co-repressors NCOR1 and NCOR2 (also known as NCoR and SMRT), showing that they cooperate with c-MYC to silence pluripotency genes during late reprogramming stages. NCOR1/NCOR2 were also proposed to be involved in silencing fibroblast identity, however it is unclear how this happens. Here, we shed light on the role of NCOR1 in early reprogramming. We show that siRNA-mediated ablation of NCOR1 and OCT4 results in very similar phenotypes, including transcriptomic changes and highly correlated high content colony phenotypes.. Both NCOR1 and OCT4 bind to promoters co-occupied by c-MYC in MEFs. During early reprogramming, downregulation of one group of somatic MEF-expressed genes requires both NCOR1 and OCT4, whereas another group of MEF-expressed genes is downregulated by NCOR1 but not OCT4. Our data suggest that NCOR1, assisted by OCT4 and c-MYC, facilitates transcriptional inactivation of genes with high expression in MEFs, which need to be suppressed to bypass an early reprogramming block. This way, NCOR1 facilitates early reprogramming progression.

scholarly journals The corepressor NCOR1 and OCT4 facilitate early reprogramming by suppressing fibroblast gene expression

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8952 ◽  
Author(s):  
Georgina Peñalosa-Ruiz ◽  
Klaas W. Mulder ◽  
Gert Jan C. Veenstra
Keyword(s):  
Transcriptional Repression ◽  
Somatic Cells ◽  
Chromatin Regulators ◽  
Highly Correlated ◽  
Induced Pluripotent ◽  
Transcriptional Corepressors ◽  
Transcriptomic Changes ◽  
Shed Light ◽  
Pluripotency Genes

Reprogramming somatic cells to induced pluripotent stem cells (iPSC) succeeds only in a small fraction of cells within the population. Reprogramming occurs in distinctive stages, each facing its own bottlenecks. It initiates with overexpression of transcription factors OCT4, SOX2, KLF4 and c-MYC (OSKM) in somatic cells such as mouse embryonic fibroblasts (MEFs). OSKM bind chromatin, silencing the somatic identity and starting the stepwise reactivation of the pluripotency programme. However, inefficient suppression of the somatic lineage leads to unwanted epigenetic memory from the tissue of origin, even in successfully generated iPSCs. Thus, it is essential to shed more light on chromatin regulators and processes involved in dissolving the somatic identity. Recent work characterised the role of transcriptional corepressors NCOR1 and NCOR2 (also known as NCoR and SMRT), showing that they cooperate with c-MYC to silence pluripotency genes during late reprogramming stages. NCOR1/NCOR2 were also proposed to be involved in silencing fibroblast identity, however it is unclear how this happens. Here, we shed light on the role of NCOR1 in early reprogramming. We show that siRNA-mediated ablation of NCOR1 and OCT4 results in very similar phenotypes, including transcriptomic changes and highly correlated high-content colony phenotypes. Both NCOR1 and OCT4 bind to promoters co-occupied by c-MYC in MEFs. During early reprogramming, downregulation of one group of somatic MEF-expressed genes requires both NCOR1 and OCT4, whereas another group of MEF-expressed genes is downregulated by NCOR1 but not OCT4. Our data suggest that NCOR1, assisted by OCT4 and c-MYC, facilitates transcriptional repression of genes with high expression in MEFs, which is necessary to bypass an early reprogramming block; this way, NCOR1 facilitates early reprogramming progression.

223 ISOLATION OF RABBIT PLURIPOTENCY GENES TO GENERATE RABBIT INDUCED PLURIPOTENT STEM CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 223 ◽  
Author(s):  
Z. Tancos ◽  
O. Ujhelly ◽  
M. K. Pirity ◽  
A. Dinnyes
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Promising Tool ◽  
Human Genes ◽  
Pluripotency Gene ◽  
Induced Pluripotent ◽  
Pluripotency Genes

Induced pluripotent stem cells (iPSC) technology, which allows direct reprogramming of somatic cells to a pluripotent state, is a promising tool for gene-function studies disease modelling, drug screening, toxicology tests and to generate knockout animal models. The goal of the current work was to close the gap in knowledge with regard to the molecular biological background for rabbit iPS work by isolating the putative pluripotency genes from the rabbit, based on the sequences published for other species. The sequence of known pluripotency genes (Oct4, Sox2, Klf4, c-Myc, Nanog) were analysed and primers designed based on the similarity of sequences. Sequences of each individual rabbit pluripotency gene was compared to other vertebrates (e.g. human, mouse, bovine) phylogenetically. Rabbit ESCs and blastocyst stage embryos were collected from superovulated rabbits to isolate total RNA. Genes of interest were amplified using RT-PCR and electrophoretically separated for cDNA fragment isolation. Isolated and subcloned cDNA fragments were sequenced and analysed. Our results showed that after restriction digestion the size of amplified and cloned rabbit Oct4, Sox2, Klf4, c-Myc and Nanog gene fragments correspond to the expected amplicon size. Furthermore, sequence confirmation by DNA sequencing has been completed in the case of Oct4, c-Myc, Klf4 and Nanog. The homology of these genes to that of their mouse and human orthologs were as follows: Oct4: at Na level 79% homologue to mouse, 85% homologue to human, at Aa level 81% homologue to mouse, 90% homologue to human; Klf4: at Na level 98% homologue to mouse, 85% homologue to human, at Aa level 95% homologue to mouse, 84% homologue to human; c-myc: at Na level 88% homologue to mouse, 92% homologue to human, at Aa level 91% homologue to mouse and 94% homologue to human; Nanog: at Na level 71% homologue to mouse, 78% homologue to human, at Aa level 55% homologue to mouse, 66% homologue to human. In conclusion, we have revealed differences at both Na and Aa level in all four major rabbit pluripotency gene sequences in comparison to their mammalian orthologs which might partially explain difficulties in generation of rabbit iPSC capable of germline transmission. Our further goal is to apply rabbit specific pluripotency genes to reprogram somatic cells and generate iPSC more efficiently than by using mouse or human genes. This work was supported by grants from Plurabbit, OMFB-00130/2010 ANR-NKTH; NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; EU FP7 (AniStem, PIAP-GA-2011-286264PartnErS, PIAP-GA-2008-218205; InduStem, PIAP-GA-2008-230675; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; PluriSys, HEALTH-2007-B-223485).

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