The PHD Finger of Human UHRF1 Reveals a New Subgroup of Unmethylated Histone H3 Tail Readers

The human UHRF1 protein (ubiquitin-like containing PHD and RING finger domains 1) has emerged as a potential cancer target due to its implication in cell cycle regulation, maintenance of DNA methylation after replication and heterochromatin formation. UHRF1 functions as an adaptor protein that binds to histones and recruits histone modifying enzymes, like HDAC1 or G9a, which exert their action on chromatin. In this work, we show the binding specificity of the PHD finger of human UHRF1 (huUHRF1-PHD) towards unmodified histone H3 N-terminal tail using native gel electrophoresis and isothermal titration calorimetry. We report the molecular basis of this interaction by determining the crystal structure of huUHRF1-PHD in complex with the histone H3 N-terminal tail. The structure reveals a new mode of histone recognition involving an extra conserved zinc finger preceding the conventional PHD finger region. This additional zinc finger forms part of a large surface cavity that accommodates the side chain of the histone H3 lysine K4 (H3K4) regardless of its methylation state. Mutation of Q330, which specifically interacts with H3K4, to alanine has no effect on the binding, suggesting a loose interaction between huUHRF1-PHD and H3K4. On the other hand, the recognition appears to rely on histone H3R2, which fits snugly into a groove on the protein and makes tight interactions with the conserved aspartates D334 and D337. Indeed, a mutation of the former aspartate disrupts the formation of the complex, while mutating the latter decreases the binding affinity nine-fold.人类UHRF1蛋白（ubiquitin-like containing PHD and RING finger domains 1，泛素样含PHD与环指结构域1）因其参与细胞周期调控、复制后DNA甲基化维持及异染色质形成，已成为潜在的癌症靶点。UHRF1作为衔接蛋白，可结合组蛋白并招募组蛋白修饰酶（如HDAC1或G9a），在染色质层面发挥调控功能。本研究采用非变性凝胶电泳（native gel electrophoresis）与等温滴定量热法（isothermal titration calorimetry），阐明了人类UHRF1的PHD结构域（huUHRF1-PHD）与未修饰组蛋白H3 N末端尾肽的结合特异性。我们通过解析huUHRF1-PHD与组蛋白H3 N末端尾肽复合物的晶体结构，揭示了该相互作用的分子基础。该结构显示，其组蛋白识别模式具有新颖性：在传统PHD结构域区域之前，存在一个额外的保守锌指结构。这一额外锌指构成了大型表面空腔的组成部分，该空腔可容纳组蛋白H3赖氨酸K4（H3K4）的侧链，且不受其甲基化状态的影响。将特异性结合H3K4的Q330突变为丙氨酸后，并未对结合作用产生明显影响，这提示huUHRF1-PHD与H3K4之间的相互作用较为松散。另一方面，该识别过程似乎依赖于组蛋白H3R2：H3R2紧密嵌入蛋白表面的凹槽中，并与保守天冬氨酸残基D334和D337形成紧密相互作用。实验证实，突变前者（D334）会破坏复合物的形成，而突变后者（D337）则会使结合亲和力降低9倍。