Project description:Active enhancers are identified by H3K27ac ChIP-seq analysis. To determine the dynamics of active enhancers during spermatogenesis, we performed H3K27ac ChIP-seq and detected reagions of active enhancers during spermatogenesis. We analyzed four representative stages of spermatogenesis: Thy1+ undifferentiated spermatogonia, which contains spermatogonial stem cells and progenitor cells; c-Kit+ differentiating spermatogonia from P7 testes; purified pachytene spermatocytes (PS) undergoing meiosis; and postmeiotic round spermatids (RS) from adult testes.
Project description:Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. While enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4+ T cells as a model of differentiation, mapping the acquisition of cell-type-specific enhancer elements in T-helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the acquisition of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. Active enhancer elements were defined as p300-high/H3K4me1-high. Using ChIP-seq, we mapped active enhancer landscapes of two CD4+ T helper cells, Th1 and Th2. To investigate the effect of STAT proteins on this landscape, we profiled active enhancers in the absence of STATs. Overall, STATs deficiency had a major impact on recruitment of p300. In addition, ectopic expression of master regulators T-bet and GATA3 in STAT-deficient cells failed to recover most active enhancers.
Project description:Recent studies suggest a hierarchical model in which lineage-determining factors act in a collaborative manner to select and prime cell-specific enhancers, thereby enabling signal-dependent transcription factors to bind and function in a cell type-specific manner. Consistent with this model, TLR4 signaling primarily regulates macrophage gene expression through a pre-existing enhancer landscape. However, TLR4 signaling also induces priming of ~3000 enhancer-like regions de novo, enabling visualization of intermediates in enhancer selection and activation. Unexpectedly, we find that enhancer transcription precedes local mono- and di-methylation of histone H3 lysine 4 (H3K4me1/2). H3K4 methylation at de novo enhancers is primarily dependent on the histone methyltransferases Mll1, Mll2/4 and Mll3, and is significantly reduced by inhibition of RNA polymerase II elongation. Collectively, these findings suggest an essential role of enhancer transcription in H3K4me1/2 deposition at de novo enhancers that is independent of potential functions of the resulting eRNA transcripts. ChIP-Seq and Gro-Seq profiling was performed in thioglycollate-elicited peritoneal macrophages, PU.1-/- and PUER cells treated as indicated.
Project description:Regulatory T (Treg) cells, whose identity and function are defined by the transcription factor Foxp3, are indispensable for immune homeostasis. It is unclear whether Foxp3 exerts its Treg lineage specification function through active modification of the chromatin landscape and establishment of new enhancers or by exploiting a pre-existing enhancer landscape. Analysis of the chromatin accessibility of Foxp3-bound enhancers in Treg and Foxp3-negative T cells showed that Foxp3 was bound overwhelmingly to pre-accessible enhancers occupied by its cofactors in precursor cells or a structurally related predecessor. Furthermore, the bulk of Foxp3-bound Treg cell enhancers inaccessible in Foxp3- CD4+ cells became accessible upon T cell receptor activation prior to Foxp3 expression with only a small subset associated with several functionally important genes being exclusively Treg cell-specific. Thus, in a late cellular differentiation process Foxp3 defines Treg cell functionality in an “opportunistic” manner by largely exploiting the preformed enhancer network instead of establishing a new enhancer landscape. Array expression. Three cell types with 3-5 replicates each.
Project description:Here we characterized the transcriptome and epigenome of control keratinocytes during differentiation. Epigenomic analyses showed that the temporal enrichment of p63 motifs in dynamic enhancers underscores the key role of p63 in orchestrating the enhancer landscape during keratinocyte differentiation. The cooperation between p63 and its co-regulating factors, such as RUNX1, is important for the finetuning of gene expression.