Recruitment of transcriptional effectors by Cas9 creates cis regulatory elements and demonstrates distance-dependent transcriptional regulation

Abstract

It is essential to regulate the expression of genes, such as those encoding the proteins of the cardiac sarcomere. This regulation is often mediated by cis regulatory elements termed enhancers and repressors that recruit transcription factors to gene-distal sites. However, the relationship between transcription factors recruitment to gene-distant sites and the regulation of gene expression is not fully understood. Specifically, it is unclear if such recruitment to any genomic site is sufficient to form an enhancer or repressor at the site, and what is the relationship between the cis regulatory element’s position and its ability to control the transcription of distant genes. Using dead Cas9 to recruit either viral or endogenous transcription factor activation domains, we demonstrate that targeting ‘naïve’ genomic sites lacking open chromatin or active enhancer marks is sufficient to alter the chromatin signature of the target site, the distant gene promoter, and significantly induce the distant gene expression, even across chromatin insulating loci. The magnitude of induction is affected by the distance between the activation site and the cognate gene in a non-linear manner. Dead Cas9 mediated recruitment of repression domains behave similarly to activation in that targeting of non-regulatory regions could repress gene expression with a nonlinear distance dependence and across chromatin insulating loci. These findings expand the models of enhancer generation and function by showing that an arbitrary genomic site can become a regulatory element and interact epigenetically and transcriptionally with a distant promoter. They also provide new fundamental insights into the rules governing gene expression.

What is an enhancer?

“…Other co-activators have catalytic activity and endow TFs, co-factors, and histones with posttranslational modifications that are thought to be activating. 24,25 The combined action of these co-activators results in a unique chromatin signature (Figure 1): while enhancers themselves have reduced histone density and increased accessibility, their surrounding nucleosomes are marked with the histone modifications H3K27ac and H3K4me1. 26 This allows for the identification of an epigenomic enhancer signature with methods such as ATAC-and/or ChIP-seq to map combinations of chromatin modification typically found at enhancers.…”

“…Among the co‐activators recruited by enhancers are chromatin remodelers that can move or displace histones, thus increasing the accessibility of DNA and facilitating the binding of additional regulatory factors. Other co‐activators have catalytic activity and endow TFs, co‐factors, and histones with post‐translational modifications that are thought to be activating 24,25 . The combined action of these co‐activators results in a unique chromatin signature (Figure 1): while enhancers themselves have reduced histone density and increased accessibility, their surrounding nucleosomes are marked with the histone modifications H3K27ac and H3K4me1 26 .…”

Long range regulation of transcription scales with genomic distance in a gene specific manner

“…Previous studies demonstrated that the requirement for native sequence-specific transcription factors (TFs) at enhancers can be bypassed by the synthetic recruitment of strong activation domains or specific coactivators to the enhancer DNA 14,[28][29][30] . In particular, catalytically inactive Cas9 (dCas9) fused to activation domains such as VP64, VPR or to the coactivator p300 has been shown to achieve gene activation at long-ranges 24,[30][31][32][33] . Indeed, VP64 and VPR robustly bind multiple coactivators such as Mediator and p300/CBP, providing a mechanistic explanation for their strong activation properties 34 .…”