ChIP_Seq_DPY30-sc-2013-07-25T14:56:56Z-2712
ChIP_Seq_DPY30
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Activin Nodal signaling direct the activity of DPY30 to maintain the epigenetic status of human pluripotent stem cells.  Human Pluripotent Stem Cells derived from embryos at the blastocyst stage (human Embryonic Stem Cells or hESCs) or from reprogrammed somatic cells (human Induced Pluripotent Stem Cells or hIPSCs) have the capacity to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a broad number of cell types. This unique pluripotent state is maintained by the Activin/Nodal signalling pathway which controls directly trough its effectors Smad2/3 the expression of a broad number of factors blocking differentiation such as Nanog. In addition, hPSCs are characterised by a specific epigenetic profile identified by the present of bivalent histone marks on a broad number of genes. The function of these marks is likely to poise developmental genes to be rapidly expressed upon differentiation and thus this mechanisms has an essential role in the capacity of hPSCs to generate cell for potential clinical/commercial applications. Importantly, the relation between this specific epigenetic state and the signaling pathways maintening pluripotency such as Activin is not yet understood. To address this limitation, we recently performed coimmunoprecipitation and Mass Spectrometry analyses to identify Smad2/3 partners in hPSCs. These proteomics analyses reveal that Smad2/3 could interact with a broad number of epigenetic modifiers. Of particular interest, we uncovered that Samd2/3 could interact with DPY30 a core subunit of the SET1/MLL histone methyltransferase complexes which regulates chromosomal H3K4 trimethylation (H3K4me3) throughout the mammalian genome. We then knocked down and overexpressed DPY30 in hESCs and observed that the resulting phenotype is similar if not identical to the one observed after inhibition of Activin/Nodal signaling. Indeed, DPY30 appears to be necessary for maintaining the expression of pluripotency markers and for blocking neuroectoderm markers expression. Importantly, knock down of DPY30 also resulted in a decrease of H3Kme3 as shown by histone ChIP-QPCR on essential genes such as Nanog or Oct4. Therefore, these studies demonstrated for the first time a functional link between an epigenetic modifier complex and the Activin/Nodal signaling in hESCs. However, these observations were performed on limited number of genes and thus only gave limited information regarding the complexity of the transcriptional network downstream DPY30-Smad2/3 complexes. Furthermore, DPY30 is likely to interact with additional factors such as Nanog and specificity of action remain to be determined. For this reason, we propose to perform Histone ChIP-Seq (H3K4me3 and H3K27me3) analyses on DPY30 knock down hESCs, Activin/Nodal inhibited hESCs and Nanog knock down hESCs. These genome wide studies will reveal the transcriptional network not only controled by DPY30-Smad2/3 complexes but also the cross talk between Nanog and DPY30. Overall this study will uncover the mechanisms by which extra-cellular signaling pathways cooperate with  pluripotency factors to orchestrate the activity of epigenetic modifiers and to maintain the pluripotent status of hPSCs.