Unknown,Transcriptomics,Genomics,Proteomics

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Pontin functions as an essential coactivator for Oct4 target genes and lincRNAs in embryonic stem cells.

ABSTRACT: The actions of transcription factors, chromatin modifiers, and noncoding RNAs are crucial for the programming of cellular states. Although chromatin remodeling factors regulate the functional status of cells including pluripotency and differentiation, how they cross-talk with embryonic stem (ES) cell-specific transcription factors and noncoding RNAs to coordinate networks controlling of ES cell identity remain unknown. Here, we find that Pontin chromatin remodeling factor plays an essential role as a coactivator for Oct4 target genes and large intergenic noncoding RNAs (lincRNAs) in ES cells. mRNA- and ChIP-sequencing analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in maintenance of ES cells. Intriguingly, Oct4-dependent coactivator function of Pontin extends to transcription of lincRNAs that are mainly involved in repression of differentiation in ES cells. Together, our findings demonstrate newly identified Oct4-Pontin-lincRNA module plays critical roles in the ES cell circuitry to orchestrate cell fate determination program. For mRNA-sequencing, we obtained mRNAs from 1) Pontinf/f; CreER ES cells at 0, 3, or 4 days post-treatment with 4-hydroxy tamoxifen (OHT) for Pontin-depleted ES cells without biological replicates (n=1), 2) ZHBTc4 ES cells at 2 days post-treatment with tetracycline (Tc) for Oct4-depleted ES cells (n=1), and 3) ZHBTc4 ES cells infected by pLKO control or pLKO-shlinc1253 lentivirus at 4 days post infection for knockdown of linc1253 (n=2). For ChIP-sequencing, chromatin extracts containing DNA fragments with an average size of 400bp were immmunoprecipitated by using antibodies against GFP (control) or Pontin. Eluted ChIP DNA (n=1).

ORGANISM(S): Mus musculus

SUBMITTER: Bhin Jin-hyuk

PROVIDER: E-GEOD-58206 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3b signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters.Upon withdrawal of 2i/LIF, naM-CM-/ve mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalency acquisition on lineage regulatory genes. The feasibility for establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in rodent ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo. Four chromatin marks H3K4me1, H3K4me3, H3K27ac and H3K27me3 were measured from 3 cell lines: C1 and WIBR3 (naM-CM-/ve and conventional/primed stem cells), and BGO1 (only naM-CM-/ve stem cells).

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Pontin functions as an essential coactivator for Oct4 target genes and lincRNAs in embryonic stem cells.

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