Project description:TG-interacting factor1 (Tgif1) maintains the identity of mouse ES cells by counterbalancing the expression of core pluripotency factors.
Project description:Abstract: The Promyelocytic Leukemia protein (PML) and its associated nuclear bodies have re-cently emerged as essential factors for maintaining the characteristics of embryonic stem (ES) cells. However, the full repertoire of PML driven gene regulatory events in ES cells is not resolved. In this report we have studied the role of PML in shaping the proteomic and SUMO proteomic landscape in ES cells. Our analysis of the PML KD proteome revealed a suppression of proteins related with self-renewal and an up-regulation of proteins vital for translation and proteasome functions, reflecting a cellular transition from pluripotency to differentiation. Major targets of PML-directed sumoylation include pluripotency factors, chromatin organizers and cell cycle regulators. We demonstrate that PML promotes the sumoylation of SALL1 and CDCA8, two proteins that are highly expressed in undifferentiated ES cells. SALL1 sumoylation increases the activation of the Wnt pathway, contributing to its ability to inhibit ES cell differentiation. Similarly, CDCA8 sumoylation enhances its capacity to promote cell proliferation. Our results demonstrate that PML maintains ES cell functions by modulating the abundance or sumoylation of key regulators involved in pluripotency and cell cycle progression.
Project description:The pluripotency of embryonic stem cells (ESCs) is maintained by a small group of master transcription factors including Oct4, Sox2 and Nanog. These core factors form a regulatory circuit controlling the transcription of a number of pluripotency factors including themselves. Although a lot of previous studies have identified key factors regulating this core network in trans, the contribution of cis-regulatory DNA sequences on the transcription of these key pluripotency factors remains elusive. We analyzed epigenomic data within the 1.5 Mb gene-desert regions around Sox2 gene and predicted only one 13kb-long enhancer located 100kb downstream of Sox2 in mouse ES cells. This enhancer is occupied by Oct4, Sox2, Nanog, and mediator complex and forms a long-range DNA looping to Sox2 locus. We hypothesized that this enhancer is critical for Sox2 gene expression and tested this hypothesis by deleting this entire 13-kb enhancer with a simple highly-efficient double-excision CRISPR strategy. Allele-specific of Sox2 transcripts in heterozygous enhancer-deletion clones showed that the enhancer affects expression through a cis-acting mechanism. Strikingly, although this distal enhancer is not conserved in other mammals including human, it is responsible for over 90% of Sox2 expression in mouse ESCs. Taken together, our results provide direct evidence that in mouse ESCs, Sox2 transcription is primarily driven by a species-specific distal enhancer, which may provide new perspectives explaining the physiological difference between human and mouse ES cells. This dataset include ChIP-seq of H3K4me3 and H3K27ac in a hybrid mouse ES cells (F123). H3K27ac in J1 mouse ES cells. And RNA-seq in F123 mESCs with complete Sox2 enhancer deletion or enhancer haploinsufficient clones.