PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression

Histone methylation occurs on both lysine and arginine residues and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD domain (PHDUHRF1), an important regulator of DNA CpG methylation, as an unanticipated histone H3 unmodified arginine 2 (H3R2)-recognition modality. This conclusion is based on binding studies and co-crystal structures of the PHDUHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1’s ability to repress its direct target gene expression is dependent on PHDUHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function. UHRF1 protein was depleted in HCT116 cells by shRNA treatment. Total RNA was purified and used to determine the global gene transcription profiles by microarray assays. The UHRF1-regulated genes were identified by comparing the gene expression profiles of control and UHRF1-depleted HCT116 cells.

组蛋白甲基化可发生于赖氨酸与精氨酸残基，其动态调控在染色质生物学中发挥关键作用。本研究鉴定出UHRF1 PHD结构域（PHDUHRF1）——一种重要的DNA CpG甲基化调控因子——作为一种此前未被发现的组蛋白H3未修饰精氨酸2（H3R2）识别模式。该结论基于结合实验与PHDUHRF1结合组蛋白H3肽段的共晶结构：未修饰R2的胍基可形成广泛的分子间氢键网络，而H3R2甲基化（而非H3K4或H3K9甲基化）会破坏复合物的形成。我们通过基因芯片与染色质免疫共沉淀（ChIP）实验鉴定了UHRF1的直接靶基因。重要的是，我们证实UHRF1抑制其直接靶基因表达的能力依赖于PHDUHRF1结合未修饰H3R2，从而证明了该识别事件的功能重要性，并支持组蛋白精氨酸甲基化与UHRF1功能间存在交叉串扰的潜在可能。研究人员通过短发夹RNA（shRNA）处理HCT116细胞以敲低UHRF1蛋白，纯化总RNA后通过基因芯片分析测定全基因组基因转录谱。通过对比对照组与UHRF1敲低HCT116细胞的基因表达谱，鉴定出UHRF1调控的基因。