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

增强子是通过建立基因活性模式来控制细胞分化和特性的基因调控元件。人类KMT2D(MLL4)复合体是一个中央增强子共激活子，通过激活增强子对发育和分化是必不可少的。通常，染色质相关的调节因子以多亚单位复合体的形式发挥作用。KMT2D复合体(KMT2Dc)由九个亚基组成，其中两个亚基-KMT2D和UTX-是催化组蛋白翻译后修饰(PTM)的酶，这是增强剂的特征：KMT2D是一种甲基转移酶，使组蛋白H3的赖氨酸4(H3K4me1)甲基化，UTX去甲基化组蛋白H3的赖氨酸27(H3K27me3)。KMT2D和UTX的基因改变导致发育障碍歌舞伎综合症，在人类癌症中经常被观察到。尽管KMT2Dc在发育和疾病中起着关键作用，但人们对KMT2Dc的功能机制、染色质相互作用和活性调节知之甚少。由于KMT2Dc的亚基较大且部分无序，到目前为止，KMT2Dc一直拒绝重组表达和结构分析。我们已经建立了从人类细胞中纯化天然KMT2Dc的方法，并获得了交联质谱(CL/MS)数据，这是关于内源复合体的有价值的拓扑信息来源，为了解蛋白质亚单位和结构域的空间关系提供了见解。根据这一信息，我们设计了用于重组表达和纯化的复合体。它们是由UTX、WARD核心(WDR5、RBBP5、ASH2 L和DPY-30)和部分~600 kDa大的KMT2D蛋白组成的，我们的CL/MS数据表明它们是内源KMT2Dc内的相互作用中心。在这里，我们将使用重组亚复合体通过单粒子冷冻电子显微镜(Cryo-EM)获得高分辨率结构。我们将集中讨论以下问题：UTX和KMT2D结构域是如何整合到复合体中的？UTX和KMT2D的催化活性结构域的结构关系是什么？利用重组核小体，我们将在高分辨率下可视化染色质结合和特定组蛋白PTM的识别。我们将进一步研究染色质和组蛋白PTMS的识别如何分别影响KMT2D和UTX的组蛋白甲基化和去甲基化活性。最后，我们将优化冷冻EM样品制备，以进行内源KMT2Dc的结构研究，将我们的发现纳入到天然的、完整的复合体中。人类K562细胞将被用来确定KMT2Dc内或KMT2Dc与染色质之间明确的界面的破坏如何影响细胞KMT2Dc的完整性、它对染色质的募集以及组蛋白PTM在增强子上的分布。综上所述，我们希望通过基于结构、生化和细胞生物学的方法提供KMT2Dc的组装、染色质结合和活性调节的机制模型，显著提高我们对KMT2Dc功能的理解。

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.