The chromatin remodeling enzyme Chd4 regulates genome architecture in the mouse brain

The development and function of the brain require tight control of gene expression. Genome architecture is thought to play a critical regulatory role in gene expression, but the mechanisms governing genome architecture in the brain in vivo remain poorly understood. Here, we report that conditional knockout of the chromatin remodeling enzyme Chd4 in granule neurons of the mouse cerebellum increases accessibility of gene regulatory sites genome-wide in vivo. Conditional knockout of Chd4 promotes recruitment of the architectural protein complex cohesin preferentially to gene enhancers in granule neurons in vivo. Importantly, in vivo profiling of genome architecture reveals that conditional knockout of Chd4 strengthens interactions among developmentally repressed contact domains as well as genomic loops in a manner that tightly correlates with increased accessibility, enhancer activity, and cohesin occupancy at these sites. Collectively, our findings define a role for chromatin remodeling in the control of genome architecture organization in the mammalian brain. Cerebella were dissected from P22 mice. For ChIP-seq, protein-DNA complexes were isolated from sonicated lysates. For Hi-C, nuclei were digested and blunt-end ligated in the presence of a biotinylated nucleotide. Ligation complexes were then isolated from sonicated DNA by streptavidin pull-down. For DNaseI-seq, nuclei were digested with DNaseI, then DNA under 500bp were selected. For nuclear RNA-seq, total RNA was extracted from nuclei.大脑的发育与功能离不开基因表达的精密调控。目前学界普遍认为，基因组架构（Genome Architecture）对基因表达具有关键的调控作用，但活体大脑中调控基因组架构的分子机制仍不甚明晰。本研究报道，在小鼠小脑颗粒神经元中条件性敲除染色质重塑酶Chd4，可在活体状态下提升全基因组范围内基因调控位点的染色质开放程度。条件性敲除Chd4可优先促进结构蛋白复合物黏连蛋白（cohesin）招募至活体小鼠小脑颗粒神经元的基因增强子区域。尤为关键的是，活体基因组架构表征分析显示，条件性敲除Chd4会增强发育抑制型接触结构域与基因组环之间的相互作用，且该效应与这些位点的染色质开放程度升高、增强子活性增强以及黏连蛋白结合占有率提升紧密相关。综上，本研究阐明了染色质重塑在哺乳动物大脑基因组架构组织调控中的作用。实验从小鼠P22龄个体中分离小脑组织。染色质免疫共沉淀测序（ChIP-seq）实验中，从超声裂解的样品中分离蛋白质-DNA复合物。高通量染色体构象捕获（Hi-C）实验中，对细胞核进行酶切，并在生物素标记核苷酸存在的条件下进行平末端连接；随后通过链霉亲和素下拉富集，从超声破碎的DNA中分离连接产物。DNase I敏感位点测序（DNaseI-seq）实验中，先用DNase I消化细胞核，再筛选长度小于500bp的DNA片段。细胞核RNA测序（nuclear RNA-seq）实验中，从细胞核中提取总RNA。