Control of cell identity and early neuronal fate commitment by the enhancer module of Integrator [ChIP-Seq]

Cell fate specification occurs via coordinated transcriptional changes in pluripotent cells, resulting in progressive commitment towards distinct lineages. Lineage-specific transcription factors (TFs), through their intrinsic DNA-binding ability, operate as master orchestrators of early and late developmental processes by turning on select cis-regulatory enhancers and proximal promoter elements. TF binding ultimately drives recruitment of the basal transcriptional machinery that comprises RNA Polymerase II (RNAPII) and a host of polymerase-associated multiprotein complexes, including the metazoan-specific Integrator complex. Integrator is primarily known to modulate RNAPII processivity and to surveil RNA integrity. Here we show that a set of Integrator subunits (enhancer module) plays a direct role in cell fate specification by promoting epigenetic changes and TF binding at enhancer elements that drive neurogenesis and maintain neuronal cell identity. Depletion of a single Integrator subunit (INTS10) results in loss of molecular and morphological neuronal features, while diverting progenitor cells towards mesenchymal identity. Commissioning of developmental enhancers rely on Integrator's enhancer module, which stabilizes SOX2 binding at chromatin upon exit from pluripotency. Integrator therefore operates as a functional bridge between enhancers and target promoters and is a driver of early developmental processes, providing new insight into a growing family of neurodevelopmental syndromes. Overall design: INTS10/11/13, RNAPII, H3K27ac, H3K4me1 was ChIPed in WT and INTS10heterzyogote iPSCs/NPCs and WT Neurons. H3K27me3 was ChIPed in WT and INTS10heterzyogote NPCs. INTS10/11/13 and RNAPII were ChIP in WT SHSY5Y cells.

细胞命运特化过程通过多能干细胞内协同的转录调控变化实现，最终使细胞逐步定向分化为不同的细胞谱系。谱系特异性转录因子（Transcription Factors, TFs）凭借其内在的DNA结合能力，通过激活特定的顺式调控增强子与近端启动子元件，充当早期与晚期发育过程的核心调控因子。转录因子结合最终会招募基础转录机器，该机器包含RNA聚合酶II（RNA Polymerase II, RNAPII）以及一系列聚合酶相关多蛋白复合物，其中包括后生动物特异性整合酶复合物（Integrator Complex）。整合酶复合物的已知功能主要为调控RNAPII的延伸持续性以及监控RNA完整性。本研究发现，一组整合酶复合物亚基（增强子模块）可通过在驱动神经发生并维持神经元细胞身份的增强子区域促进表观遗传修饰与转录因子结合，直接参与细胞命运特化过程。单个整合酶亚基（INTS10）的敲除会导致神经元分子特征与形态特征的丧失，同时将祖细胞定向转向间充质细胞身份。发育增强子的激活依赖于整合酶的增强子模块，该模块可在细胞退出多能性阶段稳定SOX2在染色质上的结合。因此，整合酶复合物充当增强子与靶启动子之间的功能性桥梁，是早期发育过程的调控驱动因子，为日益受到关注的一类神经发育综合征提供了新的研究视角。
实验设计方案：在野生型（WT）与INTS10杂合诱导多能干细胞（iPSCs）/神经前体细胞（NPCs）以及野生型神经元中，对INTS10/11/13、RNAPII、H3K27ac、H3K4me1进行染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）实验；在野生型与INTS10杂合神经前体细胞中，对H3K27me3进行染色质免疫沉淀实验；在野生型SHSY5Y细胞中，对INTS10/11/13与RNAPII进行染色质免疫沉淀实验。