Widespread impact of nucleosome remodelers on transcription at cis-regulatory elements [RNA-seq]

Nucleosome remodeling complexes and other regulatory factors work in concert to build a chromatin environment that directs the expression of a distinct set of genes in each cell using cis-regulatory elements (CREs) such as promoters and enhancers which drive transcription of both mRNAs and CRE-associated non-coding RNAs (ncRNAs). Two classes of CRE-associated ncRNAs include upstream antisense RNAs (uaRNAs), which are transcribed divergently from a shared mRNA promoter element, and enhancer RNAs (eRNAs), which are transcribed bidirectionally from active enhancers. The complicated network of CRE regulation by nucleosome remodelers remains only partially explored, with a focus on a select, limited number of remodelers. We endeavored to elucidate a remodeler-based regulatory network governing CRE-associated transcription (mRNA, eRNA, and uaRNA) in murine embryonic stem (ES) cells to test the hypothesis that many SNF2-family nucleosome remodelers collaborate to regulate the coding and non-coding transcriptome. Using depletion followed by transient transcriptome sequencing (TT-seq), we identified thousands of misregulated mRNAs and CRE-associated ncRNAs across the remodelers examined, identifying novel contributions by understudied remodelers in the regulation of coding and non-coding transcription. Our findings suggest that transcription of paired mRNAs and eRNAs are coordinately co-regulated, while mRNAs and uaRNAs sharing a promoter are independently regulated by remodelers. Our mechanistic studies suggest that while SRCAP and CHD8 modulate transcription through classical mechanisms such as transcription factors and histone variants, a broad set of remodelers including SMARCAL1 indirectly contribute to transcription regulation through maintenance of genomic stability and accurate Integrator complex activity. This study systematically examines the contribution of SNF2-remodelers to the CRE-associated transcriptome, identifying at least two classes for remodeler action. Overall design: rRNA depleted total RNA-seq samples. Wildtype murine embryonic stem cells transfected with an RNAi agent for 48 hours. Twenty nine targets profiled in biological duplicate, and seventeen control samples (Atrx, Btaf1, Chd1, Chd1l, Chd2, Chd3, Chd4, Chd5, Chd7, Chd8, Chd9, Ep400, Ercc6, Ercc6l, Hells, Hltf, Ino80, Radb54b, Radb54l, Rad54l2, Shprh, Smarca2, Smarca4, Smarca5, Smarcad1, Smarcal1, Srcap, Ttf2, Zranb3).

核小体重塑复合物（nucleosome remodeling complexes）与其他调控因子协同作用，借助顺式调控元件（cis-regulatory elements, CREs，包括启动子与增强子）构建染色质微环境，以指导每个细胞中特定基因集的表达——此类元件可驱动信使RNA（messenger RNA, mRNA）及与CRE相关的非编码RNA（non-coding RNAs, ncRNAs）的转录。与CRE相关的ncRNAs可分为两类：其一为上游反义RNA（upstream antisense RNAs, uaRNAs），从共享的mRNA启动子元件反向转录而来；其二为增强子RNA（enhancer RNAs, eRNAs），从活性增强子处双向转录获得。
目前，核小体重塑酶（nucleosome remodelers）对CRE调控的复杂网络仍仅得到部分解析，且研究焦点仅局限于少数有限的重塑酶类。本研究旨在阐明小鼠胚胎干细胞（murine embryonic stem cells, ES cells）中调控CRE相关转录（涵盖mRNA、eRNA及uaRNA）的、基于重塑酶的调控网络，以验证下述假说：众多SNF2家族核小体重塑酶可协同调控编码及非编码转录组。
本研究采用敲低联合瞬时转录组测序（transient transcriptome sequencing, TT-seq）技术，在所检测的各类重塑酶中鉴定出数千个表达失调的mRNA及CRE相关ncRNAs，揭示了此前研究不足的重塑酶在编码与非编码转录调控中的全新功能。
研究结果表明，配对的mRNA与eRNA的转录受到协同共调控，而共享同一启动子的mRNA与uaRNA则由重塑酶独立调控。
机制研究显示，尽管SRCAP与CHD8可通过转录因子、组蛋白变体等经典机制调控转录，但包括SMARCAL1在内的广谱重塑酶家族，可通过维持基因组稳定性及准确的Integrator复合物（Integrator complex）活性，间接参与转录调控。
本研究系统分析了SNF2家族重塑酶对CRE相关转录组的调控贡献，鉴定出至少两类重塑酶的作用模式。
总体实验设计：对总RNA进行核糖体RNA（ribosomal RNA, rRNA）去除处理的测序样本。将经RNA干扰试剂（RNAi agent）转染48小时的野生型小鼠胚胎干细胞进行测序，共检测29个靶点，设置生物学重复两份；同时设置17份对照样本，涵盖靶点包括：Atrx、Btaf1、Chd1、Chd1l、Chd2、Chd3、Chd4、Chd5、Chd7、Chd8、Chd9、Ep400、Ercc6、Ercc6l、Hells、Hltf、Ino80、Radb54b、Radb54l、Rad54l2、Shprh、Smarca2、Smarca4、Smarca5、Smarcad1、Smarcal1、Srcap、Ttf2、Zranb3。