Widespread impact of nucleosome remodelers on transcription at cis-regulatory elements [TT-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 Nascent RNA-seq (TT-seq) samples. Wildtype murine embryonic stem cells transfected with an RNAi agent for 48 hours. Twenty nine targets profiled in biological duplicate, and 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). Also included are TT-seq for DMSO or dTAG treated (3 hr) for Chd4-dTAG, Chd8-dTAG, or Hltf-dTAG endogenously tagged ES cells.