H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions. H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions

Gene repression and silencers are poorly understood. We reasoned that H3K27me3-rich regions (MRRs) of the genome defined from clusters of H3K27me3 peaks may be used to identify silencers that can regulate gene expression via proximity or looping. MRRs were associated with chromatin interactions and interact preferentially with each other. MRR component removal at interaction anchors by CRISPR led to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. The MRR knockout cells also showed changes in phenotype associated with cell identity, and altered xenograft tumor growth. MRR-associated genes and long-range chromatin interactions were susceptible to H3K27me3 depletion. Our results characterized H3K27me3-rich regions and their mechanisms of functioning via looping. Overall design: K562 DMSO/5μM GSK343 ChIP-seq have 2 biological replicates for each type of antibody. HAP1 Wild-type (WT)/EZH2 Knockout (EZH2KO) ChIP-seq have 2 biological replicates for each type of antibody. K562 DMSO/5μM GSK343 RNA-seq have 2 biological replicates for each condition. K562 DMSO/1μM GSK343 RNA-seq have 3 biological and 3 technical replicates for each condition. HAP1 WT/EZH2KO RNA-seq have 2 biological replicates for each condition. K562 CRISPR Empty Vector (EV)/Knockout (KO) RNA-seq have 2 biological replicates for each CRISPR clone. K562 EV/MRR2-A1 knockout (IGF1KO)ChIP-seq have 2 biological replicates for each type of antibody. 4C experiments have 2 biological replicates except TMCO4 4C, which only has 1 biological replicate.

基因阻遏与沉默子的功能机制至今仍未被充分阐明。我们推测，基于H3K27me3峰簇定义的基因组H3K27me3富集区域（H3K27me3-rich regions, MRRs），可用于识别可通过邻近作用或染色质环化调控基因表达的沉默子。MRRs与染色质相互作用存在显著关联，且优先发生彼此间的互作。通过CRISPR技术去除染色质互作锚点处的MRR组分，会导致互作靶基因的表达上调、互作区域的H3K27me3与H3K27ac水平发生改变，同时改变染色质互作模式。高H3K27me3水平区域的染色质互作未发生改变，但低H3K27me3且高H3K27ac水平的区域则出现了染色质互作的异常变化。MRR敲除细胞还呈现出与细胞身份相关的表型改变，以及异种移植瘤生长的异常。与MRRs相关的基因及长距离染色质互作，均对H3K27me3的缺失敏感。本研究明确了H3K27me3富集区域的特征，并解析了其通过染色质环化发挥功能的分子机制。
实验设计概况：
1. K562细胞经DMSO/5μM GSK343处理后的染色质免疫共沉淀测序（ChIP-seq）实验：每种抗体设置2个生物学重复；
2. HAP1野生型（Wild-type, WT）/EZH2敲除（EZH2 Knockout, EZH2KO）细胞的ChIP-seq实验：每种抗体设置2个生物学重复；
3. K562细胞经DMSO/5μM GSK343处理后的RNA测序（RNA-seq）实验：每种处理条件设置2个生物学重复；
4. K562细胞经DMSO/1μM GSK343处理后的RNA-seq实验：每种处理条件设置3个生物学重复与3个技术重复；
5. HAP1 WT/EZH2KO细胞的RNA-seq实验：每种条件设置2个生物学重复；
6. K562细胞转染空载体（Empty Vector, EV）/MRR2-A1敲除（IGF1KO）的ChIP-seq实验：每种抗体设置2个生物学重复；
7. 4C实验：除TMCO4的4C实验仅设置1个生物学重复外，其余4C实验均设置2个生物学重复。