CRISPR-ChIP to identify mechanisms of chromatin regulation [CRISPR-ChIP]

The regulation of all chromatin-templated processes involves the selective recruitment of chromatin factors to facilitate DNA repair, replication, and transcription. Chromatin immunoprecipitation (ChIP) is a critical experimental method used to provide spatiotemporal evidence for the coordination of these chromatin-based events including the dynamic regulation of chromatin modifications at cis-regulatory elements. However, getting a global appreciation of all the factors that influence a specific chromatin event has remained challenging. Here, as a proof of concept we demonstrate the utility of coupling unbiased functional genomics with ChIP to identify the factors associated with active transcription. Specifically, we use this method to identify the major chromatin factors associated with recruitment of RNA polymerase 2 and the catalysis of two evolutionarily conserved histone modifications; H3K4me3 present at the transcriptional start site and H3K79me2 present through the gene body of actively transcribed genes. With CRISPR-ChIP we identify all the non-redundant COMPASS complex members required for H3K4me3 and the major components of mediator and TFIID required for Pol II recruitment during the maintenance of gene expression. Importantly, using CRISPR-ChIP in leukaemia cells driven by MLL-translocations we uncover a functional partitioning of H3K79 methylation into two distinct regulatory units. An oncogenic DOT1L complex, where the malignant driver directs the catalytic activity of DOT1L at MLL-fusion target genes which is separate from the endogenous DOT1L complex where catalytic activity is directed by MLLT10 at actively expressed genes not controlled by the MLL-fusion protein. This functional demarcation has therapeutic implications and explains why Menin inhibition surprisingly controls methylation of H3K79 at a critical subset of genes that sustain MLL-fusion leukaemia. Coupling CRISPR screen with Chromatin immunoprecipitation (ChIP)所有以染色质为模板的过程的调控，均涉及染色质因子的选择性招募，以促进DNA修复、复制与转录。染色质免疫共沉淀（Chromatin immunoprecipitation, ChIP）是一项关键实验方法，可为这类基于染色质的事件的协同调控提供时空证据，包括顺式调控元件处染色质修饰的动态调控。然而，全面厘清影响某一特定染色质事件的全部因子，始终是一项极具挑战性的工作。本研究作为概念验证，展示了将无偏功能基因组学与ChIP技术联用，以鉴定与活跃转录相关的因子的实用价值。具体而言，我们利用该方法鉴定出了与RNA聚合酶Ⅱ（RNA polymerase 2）招募，以及两种进化保守的组蛋白修饰催化相关的主要染色质因子：存在于转录起始位点的组蛋白H3第4位赖氨酸三甲基化（H3K4me3），以及分布于活跃转录基因体的组蛋白H3第79位赖氨酸二甲基化（H3K79me2）。借助CRISPR-ChIP技术，我们鉴定出了H3K4me3形成所需的所有非冗余COMPASS复合物成员，以及在维持基因表达过程中Pol II招募所需的中介体复合物与转录因子IID（TFIID）的主要组分。值得注意的是，我们在MLL易位驱动的白血病细胞中开展CRISPR-ChIP实验，发现H3K79甲基化可被划分为两种截然不同的调控单元：一类为致癌性DOT1L复合物，其恶性驱动因子可在MLL融合蛋白靶基因处指导DOT1L的催化活性；另一类为内源性DOT1L复合物，其催化活性由MLLT10介导，作用于不受MLL融合蛋白调控的活跃表达基因。这种功能划分具有治疗学意义，并解释了为何Menin抑制竟可在维持MLL融合蛋白白血病的关键基因子集上调控H3K79的甲基化。本研究将CRISPR筛选与染色质免疫共沉淀（ChIP）技术联用