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Project description:As somatic cells are converted to iPSCs, their chromatin undergoes wide-ranging rearrangements that affect the ratio of euchromatin-to-heterochromatin, DNA methylation patterns and the regulation of enhancers and promoters. The molecular machinery underlying this process remains largely unknown. Here, we show that Dppa2 and Dppa4, two thus far poorly characterized mES-specific factors, play a key role in resetting the epigenome to a pluripotent configuration. They function as a heterodimer, are induced in late reprogramming intermediates, and are required for reprogramming. When overexpressed with OSKM factors, Dppa2/4 yield reprogramming efficiencies exceeding 75% of the starting culture and accelerate reprogramming kinetics, generating iPSCs in as little as 4 days. When chromatinbound, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway, which subsequently activates mES promoters and enhancers and enables an efficient progression to pluripotency. Our work provides critical insights into how the epigenome is remodeled during cell fate transitions.
2018-08-30 | GSE117171 | GEO
Project description:As somatic cells are converted to iPSCs, their chromatin undergoes wide-ranging rearrangements that affect the ratio of euchromatin-to-heterochromatin, DNA methylation patterns and the regulation of enhancers and promoters. The molecular machinery underlying this process remains largely unknown. Here, we show that Dppa2 and Dppa4, two thus far poorly characterized mES-specific factors, play a key role in resetting the epigenome to a pluripotent configuration. They function as a heterodimer, are induced in late reprogramming intermediates, and are required for reprogramming. When overexpressed with OSKM factors, Dppa2/4 yield reprogramming efficiencies exceeding 75% of the starting culture and accelerate reprogramming kinetics, generating iPSCs in as little as 4 days. When chromatinbound, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway, which subsequently activates mES promoters and enhancers and enables an efficient progression to pluripotency. Our work provides critical insights into how the epigenome is remodeled during cell fate transitions.
2018-08-30 | GSE117172 | GEO
Project description:Chromatin-associated factors Dppa2 and Dppa4 guide epigenetic remodeling during reprogramming to pluripotency
| PRJNA481338 | ENA
Project description:As somatic cells are converted into induced pluripotent stem cells (iPSCs), their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratios, DNA methylation patterns, and enhancer and promoter status. The molecular machinery underlying this process is largely unknown. Here, we show that embryonic stem cell (ESC)-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer, and are required for efficient reprogramming of mouse and human cells. When co-expressed with Oct4, Klf4, Sox2, and Myc (OKSM) factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in 2 to 4 days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway and contribute to downregulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.
| S-EPMC6128737 | biostudies-literature
Project description:Chromatin-associated factors Dppa2 and Dppa4 guide epigenetic remodeling during reprogramming to pluripotency (RNA-seq)
| PRJNA481343 | ENA
Project description:Chromatin-associated factors Dppa2 and Dppa4 guide epigenetic remodeling during reprogramming to pluripotency (ChIP-seq)
| PRJNA481341 | ENA
Project description:In order to identify novel pluripotency-related oncogenes, an expression screen for oncogenic foci-inducing genes within a retroviral human embryonic stem cell cDNA library was conducted. From this screen, we identified not only known oncogenes but also intriguingly the key pluripotency factor, DPPA4 (developmental pluripotency-associated four) that encodes a DNA binding SAP domain-containing protein. DPPA4 has not been previously identified as an oncogene but is highly expressed in embryonal carcinomas, pluripotent germ cell tumors, and other cancers. DPPA4 is also mutated in some cancers. In direct transformation assays, we validated that DPPA4 is an oncogene in both mouse 3T3 cells and immortalized human dermal fibroblasts. Overexpression of DPPA4 generates oncogenic foci (sarcoma cells) and causes anchorage-independent growth. The in vitro transformed cells also give rise to tumors in immunodeficient mice. Furthermore, functional analyses indicate that both the DNA-binding SAP domain and the histone-binding C-terminal domain are critical for the oncogenic transformation activity of DPPA4. Downregulation of DPPA4 in E14 mouse embryonic stem cells and P19 mouse embryonic carcinoma cells causes decreased cell proliferation in each case. In addition, DPPA4 overexpression induces cell proliferation through genes related to regulation of G1/S transition. Interestingly, we observed similar findings for family member DPPA2. Thus, we have identified a new family of pluripotency-related oncogenes consisting of DPPA2 and DPPA4. Our findings have important implications for stem cell biology and tumorigenesis.
| S-EPMC3922068 | biostudies-literature
Project description:Environmental factors can trigger cellular responses that propagate across mitosis or even generations. Perturbations to the epigenome could underpin such acquired changes, however, the extent and contexts in which modified chromatin states confer heritable memory in mammals is unclear. Here, we exploit a precision epigenetic editing strategy and forced Xist activity to programme de novo heterochromatin domains (epialleles) at endogenous loci and track their inheritance in a developmental model. We find that naïve pluripotent phases systematically erase ectopic domains of heterochromatin via active mechanisms, which likely acts as an intergenerational safeguard against transmission of epialleles. Upon lineage specification, however, acquired chromatin states can be probabilistically inherited under selectively favourable conditions, including propagation of p53 silencing through in vivo development. Using genome-wide CRISPR screening, we identify molecular factors that restrict heritable memory of epialleles in naïve pluripotent cells, and demonstrate that removal of chromatin factor Dppa2 unlocks the potential for epigenetic inheritance uncoupled from DNA sequence. Our study outlines a mechanistic basis for how epigenetic inheritance is constrained in mammals, and reveals genomic and developmental contexts in which heritable memory is feasible.
| S-EPMC8982627 | biostudies-literature
Project description:The molecular regulation of zygotic genome activation (ZGA) in mammals remains an exciting area of research. Primed mouse embryonic stem cells contain a rare subset of "2C-like" cells that are epigenetically and transcriptionally similar to the two-cell embryo and thus represent an in vitro approximation for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA in either a Dux-dependent or Dux-independent manner. Here we performed a candidate-based overexpression screen, identifying, among others, developmental pluripotency-associated 2 (Dppa2) and Dppa4 as positive regulators of 2C-like cells and transcription of ZGA genes. In the germline, promoter DNA demethylation coincides with expression of Dppa2 and Dppa4, which remain expressed until embryonic day 7.5 (E7.5), when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during induced pluripotent stem cell (iPSC) reprogramming at the time that 2C-like transcription transiently peaks. Through a combination of overexpression, knockdown, knockout, and rescue experiments together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we teased apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilized. Dppa2 and Dppa4 require Dux to initiate 2C-like transcription, suggesting that they act upstream by directly regulating Dux. Supporting this, ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild-type cells but, in contrast to Dux, can no longer do so in Dppa2/4 double-knockout cells, suggesting that it may act to stabilize rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux up-regulation and activation of the 2C-like transcriptional program, which is subsequently reinforced by Zscan4c.
| S-EPMC6362816 | biostudies-literature
Project description:PARP1 and poly(ADP-ribosyl)ation (PARylation) have been shown to be essential for the initial steps of cellular reprogramming. However, the mechanism underlying PARP1/PARylation-regulated activation of pluripotency loci remains undetermined. Here, we demonstrate that CHD1L, a DNA helicase, possesses chromatin remodeling activity and interacts with PARP1/PARylation in regulating pluripotency during reprogramming. We found that this interaction is mediated through the interplay of the CHD1L macro-domain and the PAR moiety of PARylated-PARP1. Chromatin immunoprecipitation assays demonstrated the co-occupancy of CHD1L and PARP1 at Pou5f1, Nanog, and Esrrb pluripotency loci. Knockdown of CHD1L significantly blocked the binding activity of PARP1 at pluripotency loci and inhibited the efficiency of PARP1-driven reprogramming. Notably, we found that CHD1L-promoted reprogramming requires both a PARP1-interacting domain and DNA helicase activity, partly contributing to the chromatin-remodeling states of pluripotency loci. Taken together, these results identify CHD1L as a key chromatin remodeler involved in PARP1/PARylation-regulated early-stage reprogramming and pluripotency in stem cells.
| S-EPMC4832376 | biostudies-literature