Dynamics of chromatin marks and the role of JMJD3 during pancreatic endocrine cell fate commitment

Pancreatic endocrine lineages are derived from pancreatic progenitors that undergo a cell fate transition requiring a switch from low to high Ngn3 expression. However, the underlying chromatin regulatory mechanisms are unclear. Here, we performed epigenomic analysis of gene regulatory loci featuring histone marks in cells with low or high level of Ngn3 expression. In combination with transcriptomic analysis, we discovered that in Ngn3-high cells, the removal of H3K27me3 was associated with the activation of key transcription factors and the establishment of primed and active enhancers. Deletion of Jmjd3, a histone demethylase for H3K27me3, at the pancreatic progenitor stage impaired the efficiency of endocrine cell fate transition and thereafter islet formation. Curiously, single-cell RNA-seq revealed that the transcriptome and developmental pathway of Ngn3-high cells were not affected by the deletion of Jmjd3. Our study indicates sequential chromatin events and identifies a crucial role for Jmjd3 in regulating the efficiency of the transition from Ngn3-low to Ngn3-high cells. The overall goal of this study was to understand the process of pancreatic endocrine cell specification and the chromatin regulations of endocrine cell fate transition. Specifically, we performed RNA-seq analyses on Ngn3-low and Ngn3-high cells from E13.5 Ngn3-GFP pancreata, and on nascent beta-cells from E17.5 Ins1-RFP pancreata. To study the dynamic changes in the promoter and enhancer landscape during endocrine fate transitions, we performed ChIP-seq of promoter related histone modifications (H3K27me3 and H3K4me3) and enhancer related histone modifications (H3K4me1 and H3K27ac) in purified Ngn3-low, Ngn3-high and nascent beta cells. To investigate the occupancy of NeuroD1 in endocrine progenitors, we performed NeuroD1 ChIP-seq of E14.5 whole pancreatic cells. To test the role of a histone demethylase, Jmjd3, in regulating endocrine lineage specification, we analyzed single-cell transcriptomes of Ngn3-low and Ngn3-high cells from Jmjd3-deleted pancreas.胰腺内分泌谱系源自胰腺祖细胞，此类细胞需经历细胞命运转变，该过程伴随Ngn3（Neurogenin 3）表达从低水平向高水平的切换。然而，其背后的染色质调控机制仍未明确。本研究针对低或高表达Ngn3的细胞中带有组蛋白修饰标记的基因调控位点开展了表观基因组分析。结合转录组分析，我们发现，在高表达Ngn3的细胞中，H3K27me3的去除与关键转录因子的激活以及预激活型增强子、激活型增强子的建立密切相关。在胰腺祖细胞阶段敲除靶向H3K27me3的组蛋白去甲基化酶Jmjd3，会损害内分泌细胞命运转变以及后续胰岛形成的效率。有趣的是，单细胞RNA测序（single-cell RNA-seq）结果显示，Jmjd3敲除并未影响高表达Ngn3细胞的转录组与发育通路。本研究揭示了时序性的染色质事件，并明确了Jmjd3在调控Ngn3低表达向高表达细胞转变的效率中发挥关键作用。本研究的总体目标是解析胰腺内分泌细胞特化过程，以及内分泌细胞命运转变的染色质调控机制。具体而言，我们对来自E13.5 Ngn3-GFP小鼠胰腺的低、高表达Ngn3细胞，以及来自E17.5 Ins1-RFP小鼠胰腺的新生β细胞开展了RNA测序（RNA-seq）分析。为探究内分泌命运转变过程中启动子与增强子景观的动态变化，我们在纯化获得的低表达Ngn3、高表达Ngn3细胞以及新生β细胞中，针对启动子相关组蛋白修饰（H3K27me3与H3K4me3）以及增强子相关组蛋白修饰（H3K4me1与H3K27ac）开展了染色质免疫共沉淀测序（ChIP-seq）。为探究NeuroD1在内分泌祖细胞中的结合占据特征，我们对E14.5全胰腺细胞开展了NeuroD1染色质免疫共沉淀测序。为验证组蛋白去甲基化酶Jmjd3在内分泌谱系特化中的作用，我们对来自Jmjd3敲除小鼠胰腺的低、高表达Ngn3细胞开展了单细胞转录组分析。