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Genome-wide DNA accessibility mapping using ATAC-seq in HEK293 in vector control, PC4 knockdown and PC4 phosphorylation specific mutant and mimic

ABSTRACT: To assess the mechanisms by which PC4 mediates chromatin compaction in cells and regulation of genome organization by its phosphorylation state, we measured the chromatin accessibility landscape using ATAC-seq of vector control 293 cells (Control/shNS), PC4 knockdown (KD/PC4 KD) and upon ectopic expression of Flag tagged PC4 (FP/PC4), Phosphomimic-PC4 (PM/PM-PC4) and phospho-mutant (MTP/MTP5) in PC4 knockdown cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE191333 | GEO | 2022/05/12

REPOSITORIES: GEO

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Project description:Spermatogonial stem cells (SSCs) can spontaneously dedifferentiate into embryonic stem cell (ESC)-like cells, which are designated as multipotent SSCs (mSSCs), without ectopic expression of reprogramming factors. SSCs express key OSKM reprogramming factors at some levels, and do not require ectopic expression of any gene for the acquisition of pluripotency during reprogramming to mSSCs. Therefore, we reasoned that additional factors are required to regulate SSC reprogramming. In this study, we first compared the expression of reprogramming signature genes among somatic cells, iPSC, SSCs, mSSCs, and partially reprogramed cells, and found that they appear to have similar pluripotency states, whereas their transcriptional program differs. We developed a systems biology approach to prioritise genes for pluripotency regulatory factors by integrating transcriptome and interactome data on the genome-wide functional network. Then, we performed a series of systematic gene prioritisation steps and identified 53 candidates, which included some known reprogramming factors. We experimentally validated one particular candidate, Positive cofactor 4 (Pc4), which was expressed in PSCs and yielded a positive RNA interference (RNAi) response in an Oct4 reporter assay. We demonstrated that Pc4 enhanced the efficiency of OSKM-mediated reprogramming by promoting the transcriptional activity of key pluripotency factors, and by regulating the expression of many protein- and miRNA-encoding genes involved in reprogramming and somatic cell-specific genes. Pc4-overexpressing mESC lines were established by Venus (YFP)-expressing lentiviral transfection. The mESCs were split at a density of 2 Â´ 104 cells onto fresh MEF feeder cells seeded into a 6 well dish (containing mESC growth medium) with virus particles, and 25 Î¼g/ml polybrene (Sigma Aldrich) was added. After 24 h, the medium was replaced with fresh growth medium. After 4 days later, mESC colonies expressing YFP were picked and replated. Three different Pc4-overexpressing mESC lines were established.

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Genome-wide DNA accessibility mapping using ATAC-seq in HEK293 in vector control, PC4 knockdown and PC4 phosphorylation specific mutant and mimic

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