作为一种新鉴定到的将代谢组与表观基因组关联的组蛋白赖氨酸酰基化修饰，组蛋白乳酸化在基因转录调节、巨噬细胞极化、细胞重编程以及肿瘤发生等生物学进程中发挥着重要作用。组蛋白酰基化修饰作为广泛分布的表观遗传标记可以被对应的效应因子即阅读器识别，调节特定靶基因的转录以及招募下游复合物发挥生理功能。因此，本研究希望鉴定到组蛋白乳酸化修饰的阅读器，从而更加深入得理解其调控的分子机制并寻求新的相关疾病治疗策略。 本课题采用候选蛋白的方法，对Bromodomain、DPF以及YEATS结构域三类人源组蛋白酰基化修饰阅读器家族蛋白进行了系统的筛选。有趣的是，体外结合实验表明只有AF9的YEATS结构域被鉴定为组蛋白乳酸化修饰的阅读器，结合能力远高于非修饰多肽但低于乙酰化修饰多肽，而其余检测的酰基化阅读器蛋白并不具备这种识别能力。随后，通过复合物晶体结构解析以及关键残基突变研究揭示了AF9识别组蛋白乳酸化修饰的分子基础，除多肽骨架对于识别的锚定作用外，其中修饰侧链的L型乳酰化基团贡献氢键互作可以更好得与含丝氨酸的芳香型三明治识别口袋结合。在功能研究方面，发现AF9识别H3K9la可以协作调节TGFβ1以及PHGDH的基因表达，促进乳腺癌MCF7细胞的增殖与侵袭能力。特别的是，AF9识别H3K9la可以上调转录因子KLF2的表达，而KLF2定位于AF9基因的启动子，调节AF9转录，从而形成一条正反馈调节环路，放大AF9与组蛋白乳酸化修饰之间的调控信号与下游效应。与此同时，本研究聚焦于催化组蛋白酰基化修饰建立与去除的动态调控因子书写器和擦除器。体内实验表明，HDAC3具有显著的组蛋白去乳酰化修饰酶活性。而以具有两个不同酰基转移酶亚基的人源ATAC复合物为研究对象，通过冷冻电镜结构解析与体外酶活研究初步对其组装机制和催化位点特异性有所认知，并提出了可能的作用机制。 综上所述，本论文围绕着组蛋白酰基化修饰的三大调控因子展开，主要揭示了一个AF9识别组蛋白乳酸化修饰的转录调控模型。二者协调作用维持一组特定基因表达的同时，可以干扰其余酰基化修饰的识别作用。同时随着代谢状态的改变，可以协同促进转录网络的重编程。
As a newly identified histone mark linking metabolism to epigenetics, histone lactylation regulates gene expression and plays an important role in biological processes such as macrophage polarization, cell reprogramming, and tumorigenesis. One working mechanism underlying histone modification is that they may serve as a docking mark for epigenetic readers, regulating the transcription of target genes and recruiting downstream complexes. Here we set out to find histone lactylation readers in order to understand the molecular mechanism of regulation and to find novel therapeutic strategies for diseases. Through a candidate approach, we screened Bromodomain, DPF, and YEATS family of histone acylation readers. Interestingly, our binding studies showed that most of these histone acylation readers could not recognize histone lactylation. The YEATS domain of AF9 could serve as a histone lactylation reader with a binding affinity higher than the unmodified peptide but lower than the H3K9 acetylated form. Then, the structural and metagenesis studies revealed the molecular basis for histone lactylation readout by AF9, in which the L-form lactylate group was shown to bind to a Ser-lined aromatic sandwich pocket and the peptide backbone displayed the contribution to the binding. Functionally, we found that AF9 and H3K9la cooperatively regulated gene expression of TGFβ1 and PHGDH, in accordance with the role of AF9-Kla recognition pair in MCF7 breast cancer cells proliferation and invasiveness. Besides, AF9 recognition of H3K9la could upregulate the expression of KLF2, which in turn was the transcription factor of AF9 gene, forming a positive feedback regulatory pathway. Meanwhile, we also focused on the dynamic regulators of histone acylation, writers, and erasers, which could catalyze the establishment and removal of acylation modifications, respectively. In vivo, enzymatic results suggested that HDAC3 is a potential histone Kla eraser protein. We studied the human ATAC complex with two different HAT subunits by Cryo-EM and enzymatic activity assay and provided preliminary insights into the assembly mechanism as well as the site-specificity of the enzymatic activity. Collectively, our work revealed a transcription attenuation model, in which histone lactylation perturbed the binding of most histone acetylation readers. Meanwhile, the unique histone lactylation readout by AF9 could sustain a particular set of gene expressions, and they cooperatively contribute to a reprogrammed transcriptional network following metabolic alternations.
