Chromatin binding of CFP1 mutants in C127 mouse cell lines. [C127_ChIPseq]. Mus musculus

Chromatin modifications and the promoter associated epigenome are thought to be important for the regulation of gene expression. However, the mechanisms by which chromatin modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed target gene promoters through CFP1 which engages in a unique form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed suggesting that the normal targeting and function of SET1 complex is central to creating an appropriately functioning vertebrate promoter associated epigenome. Overall design: C127 mouse cell line was used to investigate the occupancy of variuos chromatin binding proteins, including CFP1, KDM2B and RNA PolII, as well as the genome-wide distribution of H3K4me3. In addition, novel C127 cell lines were generated that expressed different forms of a GFP-tagged CFP1 protein to compare the binding properties of wild-type CFP1 with single point mutants within its binding domains. The cell lines generated include GFP-CFP1 wild-type , GFP-CFP1 CXXC mutant, GFP-CFP1 PHD mutant, a double GFP-CFP1 PHD and CXXC mutant, GFP-CPF1 SID mutant, along with a GFP control.

染色质修饰与启动子关联表观基因组（epigenome）被认为在基因表达调控中具有重要作用。然而，染色质修饰复合物如何被精准靶向至脊椎动物的特定基因启动子，以及其调控基因表达的具体机制，迄今仍未得到清晰阐明。本研究结合活细胞成像与功能基因组学技术手段，发现脊椎动物SET1复合物（SET1 complex）通过CFP1被靶向至活跃转录的靶基因启动子；CFP1能够以一种独特的多价染色质识别模式，同时识别非甲基化DNA与组蛋白H3赖氨酸4三甲基化（H3K4me3）。CFP1决定了SET1复合物在染色质上的结合分布，且其多价相互作用是SET1复合物催化生成H3K4me3的必要条件。当CFP1缺失时，基因表达会出现紊乱，这表明SET1复合物的正常靶向与功能，对于构建功能正常的脊椎动物启动子关联表观基因组至关重要。实验设计：本研究以C127小鼠细胞系为模型，检测包括CFP1、KDM2B及RNA聚合酶II（RNA PolII）在内的多种染色质结合蛋白的结合分布，以及H3K4me3的全基因组分布情况。此外，本研究构建了一系列可表达不同形式绿色荧光蛋白（GFP）标记CFP1蛋白的新型C127细胞系，用于对比野生型CFP1与其结合结构域内单点突变体的结合特性。所构建的细胞系包括野生型GFP-CFP1、CXXC结构域突变型GFP-CFP1、PHD结构域突变型GFP-CFP1、CXXC与PHD双突变型GFP-CFP1、SID结构域突变型GFP-CFP1，以及仅表达GFP的阴性对照细胞系。