OCT4, NANOG and CTCF in human embryonic stem cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Justin Joseph Gnanakkan
PROVIDER: E-GEOD-21200 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We studied the genomic locations of three key regulatory proteins (OCT4, NANOG and CTCF) in human and mouse embryonic stem (ES) cells [see Series GSE20650]. To identify the conserved and unique human OCT4 targets, we performed an OCT4 RNAi knock-down experiment. We find that species-specific transposable elements have profoundly altered the transcriptional circuitry of pluripotent stem cells. To identify the mRNA targets of OCT4: 12 samples were collected in total. Each sample is done in triplicate and samples were collected at 2 different time point, 2 days and 3 days after transfection. Luciferase knockdown serves as negative control and OCT4 knockdown is the experimental sample.
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Project description:Transcription factors and their specific interactions with targets are crucial in specifying gene expression programs. To gain insights into the transcriptional regulatory networks in embryonic stem cells, we used chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing (ChIP-seq) to map the locations of OCT4, NANOG and CTCF. ChIP-enriched DNA from pooled ChIP samples were analyzed by Solexa sequencing.
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Project description:In our study, PRDM14 and NFRkB were found to enhance the reprogramming of human fibroblasts to hiPSCs in the presence of OCT4, SOX2, KLF4, with/without c-MYC (OSK/OSKC). To examnie if the obtained hiPSC share similar expression signature with hESC, we conducted the microarray analysis on the hiPSCs, hESCs and fibroblasts. The result showed that all of the examined hiPSCs resembled hESCs, but differed from fibroblasts MRC5. 4 hiPSC lines, 2 hESC lines and 1 type of fibroblasts were analyzed in total. Each sample is done in duplicates.
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Project description:The unique cellular state of embryonic stem cells is maintained by pluripotency-associated transcription factors such as OCT4. The transcriptional regulatory circuitries between human and mouse pluripotent stem cells exhibit notable differences. Transposable elements have altered the pluripotency network by contributing new cis-regulatory elements to mammalian genomes. In our study, we found that the expression of HERVH is enriched in human embryonic stem cells (hESCs) and depletion of HERVH in hESCs induced differentiation. To confirm the differentiation phenotype and identify potential downstream target genes of HERVH, we conducted the microarray analysis on hESCs treated with control shRNA against Luciferase and shRNA against HERVH. Total RNA was extracted from cells treated with control shRNA or HERVH shRNA for 5 days. 3 replicates each.
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Project description:Human endogenous retrovirus subfamily H (HERVH) is a class of transposable elements whose members are expressed preferentially in human pluripotent stem cells. Here, we report that the long-terminal repeat regions of HERVH function as enhancers which are regulated by OCT4. Strikingly, we found that HERVH is a nuclear-localized long non-coding RNA that is required to maintain the undifferentiated state of human embryonic stem cells. Furthermore, we showed that HERVH is associated with OCT4 and co-activators such as P300, CBP and mediator subunits. Together, these results uncover a new and unexpected role of species-specific transposable elements in specifying the pluripotency of human cells.
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Project description:PRDM14 belongs to the PR (PRDI-BF1 and RIZ) domain proteins (PRDM) family which is a subclass of the SET domain proteins, a common domain found in histone modifying enzymes. PRDM14 has been previously implicated to regulate self-renewal of hESCs as knock-down of PRDM14 induced expression of differentiation marker genes and altered the cellular morphology. We showed that PRDM14 directly regulates the expression of key pluripotency gene POU5F1. Genome-wide location profiling experiments revealed that PRDM14 co-localized extensively with other key transcription factors such as OCT4, NANOG and SOX2. More importantly, in a gain-of-function assay, we showed that PRDM14 is able to enhance the efficiency of reprogramming of human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Hence, PRDM14 exemplifies a key transcription factor that is required for the maintenance of human ESC identity and the reacquisition of pluripotency in human somatic cells. ChIP-seq of PRDM14 in human ESCs
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Project description:The mechanisms whereby the crucial pluripotency transcription factor Oct4 regulates target gene expression are incompletely understood. Using an assay system based on partially differentiated embryonic stem cells, we show that Oct4 opposes accumulation of local H3K9me2, and subsequent Dnmt3a-mediated DNA methylation. Upon binding DNA, Oct4 recruits the histone lysine demethylase Jmjd1c. ChIP timecourse experiments identify a stepwise Oct4 mechanism involving Jmjd1c recruitment and H3K9me2 demethylation, transient FACT complex recruitment, and nucleosome depletion. Genome-wide and targeted ChIP confirms binding of newly-synthesized Oct4, together with Jmjd1c and FACT, to the Pou5f1 enhancer and a small number of other Oct4 targets, including the Nanog promoter. Histone demethylation is required for both FACT recruitment and H3 depletion. Jmjd1c is required to induce endogenous Oct4 expression and fully reprogram fibroblasts to pluripotency, indicating that the assay system identifies functional Oct4 cofactors. These findings indicate that Oct4 sequentially recruits activities that catalyze histone demethylation and depletion. Examination of transcription factor occupancy in cells with newly synthesized Oct4.
2015-01-23 | E-GEOD-65192 | biostudies-arrayexpress
Project description:Human embryonic stem cells (hESCs) harbor the ability to undergo lineage-specific differentiation into clinically relevant cell types. Transcription factors and epigenetic modifiers are known to play important roles in the maintenance of pluripotency of hESCs. However, little is known about regulation of pluripotency through splicing. In this study, we identify the spliceosome-associated factor SON as a novel factor essential for the maintenance of hESCs. Depletion of SON in hESCs results in the loss of pluripotency and cell death. Using genome-wide RNA profiling, we identified transcripts that are regulated by SON. Importantly, we confirmed that SON regulates the proper splicing of transcripts encoding for pluripotency regulators such as PRDM14, OCT4, E4F1 and MED24. Furthermore, we show that SON is bound to these transcripts in vivo. In summary, we connect a splicing-regulatory network for accurate transcription production to the maintenance of pluripotency and self-renewal of hESCs.
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Project description:Human embryonic stem cells (hESCs) are isolated from the inner cell mass of the blastocysts. The pluripotent properties of hESCs enable the derivation of cell-types or tissues of different lineages for potential applications such as therapeutics discovery and regenerative medicine. Even though hESCs are pluripotent, differences have been observed when compared to the native pluripotent epiblast cells of the blastocyst. We use a chemical approach (3iL: 3 small molecule inhibitor and cytokine) to induce an expression signature that more closely resembles native pluripotent cells. This experiment is the transcription factor binding data of the study.
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