Structural analyses of the assembly, chromatin recognition and modification of the human KMT2D complex

人类 KMT2D 复合物的组装、染色质识别和修饰的结构分析

• 批准号: 504090354
• 负责人: Dr. Simon Pöpsel
• 金额: --
• 依托单位: Center for Molecular Medicine in Cologne (CMMC)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/504090354?language=en
• 关键词: Structural; analyses; assembly; chromatin; recognition

Enhancers are gene regulatory elements that control cellular differentiation and identity by establishing patterns of gene activity. The human KMT2D (MLL4) complex is a central enhancer co-activator and is essential for development and differentiation by enabling enhancer activation. Commonly, chromatin associated regulators function as multi-subunit complexes. The KMT2D complex (KMT2Dc) comprises nine subunits, two of which – KMT2D and UTX – are enzymes that catalyze histone post-translational modifications (PTMs) that are hallmarks of enhancers: KMT2D is a methyltransferase that mono-methylates lysine 4 of histone H3 (H3K4me1), and UTX demethylates trimethylated lysine 27 of histone H3 (H3K27me3). Genetic alterations of KMT2D and UTX lead to the developmental disorder Kabuki syndrome, and are frequently observed in human cancer. Despite key roles in development and disease, little is known about the mechanisms of KMT2Dc function, its chromatin interactions and activity regulation. Due to large and partially disordered subunits, KMT2Dc has so far resisted recombinant expression and structural analyses. We have established the purification of native KMT2Dc from human cells and obtained cross-linking mass spectrometry (CL/MS) data, a valuable resource of topological information on the endogenous complex, providing insights into the spatial relationship of protein subunits and domains. Based on this information, we have designed complexes for recombinant expression and purification. These are composed of UTX, the WRAD core (WDR5, RBBP5, ASH2L and DPY-30) and parts of the ~600 kDa large KMT2D protein that our CL/MS data suggest to be interaction hubs within endogenous KMT2Dc. Here, we will use recombinant sub-complexes to obtain high-resolution structures by single particle cryo-electron microscopy (cryo-EM). We will focus on the following questions: How are UTX and KMT2D domains integrated into the complex? What is the structural relationship of the catalytically active domains of UTX and KMT2D? Using recombinant nucleosomes, we will visualize chromatin engagement and the recognition of specific histone PTMs at high resolution. We will further study how the recognition of chromatin and histone PTMs affects the histone methylation and demethylation activities by KMT2D and UTX, respectively. Finally, we will optimize cryo-EM sample preparation for structural studies of endogenous KMT2Dc to incorporate our findings into the native, complete complex. Human K562 cells will be used to determine how the disruption of defined interfaces within KMT2Dc or between KMT2Dc and chromatin affects cellular KMT2Dc integrity, its recruitment to chromatin, and the distribution of histone PTMs at enhancers. Taken together, we hope to significantly enhance our understanding of KMT2Dc function by providing mechanistic models of the assembly, chromatin engagement and activity regulation of KMT2Dc based on structural, biochemical and cell biological approaches.

增强子是通过建立基因活性模式来控制细胞分化和身份的基因调控元件。人KMT 2D（MLL 4）复合物是一种中心增强子共激活剂，通过激活增强子对发育和分化至关重要。通常，染色质相关调节子作为多亚基复合物起作用。KMT 2D复合物（KMT 2Dc）包含9个亚基，其中两个-KMT 2D和UTX -是催化组蛋白翻译后修饰（PTM）的酶，这些修饰是增强子的标志：KMT 2D是一种甲基转移酶，可使组蛋白H3的赖氨酸4（H3 K4 me 1）单甲基化，UTX使组蛋白H3的三甲基化赖氨酸27（H3 K27 me 3）去甲基化。KMT 2D和UTX的遗传改变导致发育障碍Kabuki综合征，并且经常在人类癌症中观察到。尽管在发育和疾病中起着关键作用，但对KMT 2Dc功能，染色质相互作用和活性调节的机制知之甚少。由于大的和部分无序的亚基，KMT 2Dc到目前为止一直抵制重组表达和结构分析。我们已经建立了从人细胞中纯化天然KMT 2Dc的方法，并获得了交联质谱（CL/MS）数据，这是内源性复合物拓扑信息的宝贵资源，为蛋白质亚基和结构域的空间关系提供了见解。基于这些信息，我们设计了用于重组表达和纯化的复合物。这些由UTX、WRAD核心（WDR 5、RBBP 5、ASH 2L和DPY-30）和~600 kDa大KMT 2D蛋白的部分组成，我们的CL/MS数据表明这些蛋白是内源性KMT 2Dc内的相互作用中心。在这里，我们将使用重组子复合物通过单粒子冷冻电子显微镜（cryo-EM）获得高分辨率结构。我们将重点关注以下问题：UTX和KMT 2D域如何集成到复杂的？UTX和KMT 2D的催化活性结构域之间的结构关系是什么？使用重组核小体，我们将可视化染色质接合和识别特定的组蛋白PTM在高分辨率。我们将进一步研究染色质和组蛋白PTM的识别如何分别影响KMT 2D和UTX的组蛋白甲基化和去甲基化活性。最后，我们将优化cryo-EM样品制备内源性KMT 2Dc的结构研究，将我们的研究结果纳入本地，完整的复杂。人K562细胞将用于确定KMT 2Dc内或KMT 2Dc与染色质之间的限定界面的破坏如何影响细胞KMT 2Dc完整性、其向染色质的募集以及组蛋白PTM在增强子处的分布。总之，我们希望通过提供基于结构，生物化学和细胞生物学方法的KMT 2Dc组装，染色质参与和活性调节的机制模型，显着提高我们对KMT 2Dc功能的理解。