Project description:We provide data from several targeted deletions of transcriptional enhancer clusters within mouse F1 embryonic stem (ES) cells. We targeted these regions for deletion with CRISPR/Cas9 genome editing tools. We demonstrate through heterozygous enhancer cluster deletion and allele specific RNA-seq that enhancer clusters differ in their regulatory activity as the magnitude of the observed change in transcription upon enhancer cluster deletion varies greatly.
Project description:In mouse embryonic stem cells SNPs disrupting closely-spaced hexanucleotide motifs are associated with lack of ZFP57 binding and H3K9me3 enrichment. Examination of ZFP57-KAP1 allele-specific binding in two lines of mouse embryonic stem cells JB1 and BJ1 generated from F1 hybrids derived from JF1 x B6 and B6 x JF1 crosses respectively.
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.
Project description:We have generated ATAC-sequencing datasets of the regulome of mouse neural stem and progenitor cells derived from embryonic stem cells, with allele-specific deletions of Sox2 enhancer cluster regions. ATAC-seq experiments were conducted to evaluate the alterations in chromatin accessibility at candidate regulatory elements genome-wide in neural stem and progenitor cells with Sox2 enhancer loss-of-function.
Project description:We used RNA-seq data from mouse embryonic fibroblasts from F1 reciprocal crosses to determine a biologically relevant allelic ratio cutoff, and define for the first time an entire allelome. Furthermore, we show that Allelome.PRO detects differential enrichment of H3K4me3 over promoters from ChIP-seq data validating the RNA-seq results. Defining allele-specific genome features from F1 E12.5 mouse embryonic fibroblasts
Project description:We have generated RNA-sequencing datasets of the regulome of mouse neural stem and progenitor cells derived from embryonic stem cells, with allele-specific deletions of Sox2 enhancer cluster regions. RNA-seq experiments were conducted to evaluate the regulatory function of Sox2 candidate enhancers in neural stem and progenitor cells.
Project description:We report the effect on genome-wide gene expression after deletion of an enhancer region downstream of Sox2 in F1 ES cells. The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Reporter assays reveal novel enhancers, including two enhancers over 100 kb downstream (SRR107 and SRR111) which, through the formation of chromatin loops, localise to the Sox2 promoter in ES cells. Using CRISPR/Cas9 we deleted a region containing these two enhancers, which we term the Sox2 control region (SCR). This deletion revealed that the SCR is required for Sox2 transcription in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together these data identify a distal control region essential for Sox2 transcription in ES cells. Examination of PolA+ RNA after heterozygous and homozygous enhancer deletion in F1 ES cells (M. musculus129 x M. castaneus).
Project description:We reported the allele-specific bulkl RNA sequencing in highly morphogenic (C57Bl6J-maternal x CASTEiJ-paternal) male F1 mouse embryonic stem cells (mESC) and in vitro mESC derived six cardiac lineage cell types. We demonstrated distinctive gene regulation based on the parental origin of the alleles. We showed that deterministic and stochastic monoalleleic gene classes are distinctive in regulation and are involved in unique processes. In this study we highlighted the importance of parental origin-specific gene expression in development, homeostasis and disease.
