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

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 behaves 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.对基因（例如编码心肌肌节（cardiac sarcomere）蛋白的基因）的表达进行调控至关重要。此类调控通常由被称为增强子（enhancers）和阻遏子（repressors）的顺式调控元件（cis regulatory elements）介导，这些元件可将转录因子（transcription factors）招募至基因远端位点。然而，转录因子被招募至基因远端位点与基因表达调控之间的关联尚未完全阐明。具体而言，目前尚不明确：招募至任意基因组位点（genomic site）是否足以在该位点形成增强子或阻遏子，以及顺式调控元件的位置与其调控远端基因转录的能力之间存在何种关联。 本研究利用失活Cas9（dead Cas9）招募病毒源性或内源性转录因子激活结构域（transcription factor activation domains），实验证明，靶向缺乏开放染色质（open chromatin）或活性增强子标记（active enhancer marks）的未修饰基因组位点（naïve genomic sites），足以改变靶位点及远端基因启动子（gene promoter）的染色质特征（chromatin signature），并可显著诱导远端基因的表达，即便该过程跨越染色质绝缘位点（chromatin insulating loci）亦可行。诱导幅度与激活位点及同源基因之间的距离呈非线性相关。 失活Cas9介导的阻遏结构域（repression domains）招募行为与激活过程类似：靶向非调控区域可通过非线性距离依赖效应，在跨越染色质绝缘位点的前提下抑制基因表达。 上述研究结果表明，任意基因组位点均可转变为调控元件，并与远端启动子发生表观遗传及转录水平的相互作用，这拓展了增强子生成与功能的相关模型；同时也为解析基因表达调控的内在规律提供了全新的基础认知。