Functional characterization of the trithorax protein Ash2l, a core component of histone H3 lysine 4 methyltransferase complexes

差异基因表达对于细胞的特性和控制细胞生理是必不可少的。染色质的调节，特别是核小体重塑和核心组蛋白修饰，有助于基因表达的调节。这些修饰之一是组蛋白H3在赖氨酸4(H3K4)上的甲基化。它的单甲基化和三甲基化(H3K4me3)分别与可访问和开放的增强子和启动子相关，转录基因在其启动子区域通常携带H3K4me3。这种修饰是由包括MLL1-4、SET1A和SET1B在内的KMT2甲基转移酶家族传递的。它们需要WDR5、RBBP5、ASH2L和DPY30(又名WRAD络合物)组成的核心络合物才能具有催化活性。我们正在研究ASH2 L，因为我们发现这个三胸蛋白是原癌蛋白c-myc的相互作用因子。我们发现，在小鼠的肝脏或造血系统中，Ash21基因的敲除(KO)是致命的，伴随着H3K4甲基化的丧失和基因表达的失控。从这些和其他发现中，很明显，H3K4的甲基化对正常的细胞生理是必要的，但H3K4me3是否对染色质有更普遍的影响，或者对基因转录有更多的指导作用，仍然是个未知数。因此，主要目标是(I)更多地了解Ash2l本身的功能及其相关的H3K4甲基转移酶活性和(Ii)解决H3K4甲基化在启动子中的功能。我们将使用我们的小鼠胚胎成纤维细胞，其带有表达可调控的CRE-ERTM2重组酶的Ash21等位基因。我们将通过测量KO小鼠胚胎成纤维细胞的补体增殖、基因转录和H3K4甲基化能力来进行ASH2 L的结构和功能分析，以确定相关的区域和结构域。此外，我们将使用无法与其他WRAD复杂成分结合的ASH2 L突变体来筛选新的相互作用伙伴，以解决ASH2 L的新功能。由于Ash2l KO相当缓慢，我们将在小鼠和人类细胞系中建立一个系统，允许通过与生长素诱导降解的融合来短期调节ASH2 L。这将用于ASH2 L的短期失活和重新激活，以确定对染色质和基因调控的直接影响。我们将使用核小体作图和ATAC-SEQ来探索依赖于Ash21的染色质紧致。这将通过使用dCas9融合将ASH2 L和突变体以及其他表观遗传调节因子靶向特定基因来评估H3K4在启动子和协作性上的甲基化来补充。通过深入的测序和染色质和基因表达分析，我们将能够评估H3K4甲基化和合作活动的后果。最后，我们将详述依赖于H3K4甲基化来控制基因表达的信号过程。综上所述，该提案涉及ASH2功能的基本方面以及H3K4甲基化在基因转录中的作用。

Differential gene expression is essential for cell identity and to control cell physiology. Modulating chromatin, in particular nucleosome remodeling and core histone modifications, contribute to the regulation of gene expression. One of these modifications is methylation of histone H3 at lysine 4 (H3K4). Its mono-methylation and tri-methylation (H3K4me3) is associated with accessible and open enhancers and promoters, respectively, with transcribed genes carrying typically H3K4me3 in their promoter regions. This modification is transferred by the KMT2 family of methyltransferases, including MLL1-4, SET1A and SET1B. They require a core complex of WDR5, RBBP5, ASH2L and DPY30 (aka WRAD complex) for catalytic activity. We are studying ASH2L as we identified this trithorax protein as an interactor of the proto-oncoprotein c-MYC. We find that the knockout (KO) of Ash2l in the liver or in the hematopoietic system in the mouse is lethal, accompanied by a loss of H3K4 methylation and deregulated gene expression. From these and other findings, it is evident that methylation at H3K4 is necessary for normal cell physiology, but whether H3K4me3 has a more general effect on chromatin or a more instructive role in gene transcription remains open. Therefore, the main objectives are (i) to learn more about the function of Ash2l itself together with its associated H3K4 methyltransferase activity and (ii) to address the function of H3K4 methylation at promoters. We will use our mouse embryo fibroblasts with floxed Ash2l alleles that express regulatable Cre-ERTM2 recombinase. We will perform a structure-function analysis of ASH2L to determine relevant regions and domains by measuring the ability to complement proliferation, gene transcription and H3K4 methylation in the KO mouse embryo fibroblasts. Moreover, we will screen for novel interaction partners using an ASH2L mutant that is unable to bind to other WRAD complex components to address novel functions of ASH2L. Because the Ash2l KO is rather slow, we will establish a system in mouse and human cell lines that allows short-term regulation of ASH2L by using fusions with an auxin inducible degron. This will be used for short-term inactivation and reactivation of ASH2L to determine direct effects on chromatin and gene regulation. We will probe chromatin compaction dependent on Ash2l using nucleosomal mapping and ATAC-seq. This will be complemented by targeting ASH2L and mutants as well as other epigenetic regulators to specific genes using dCas9 fusions to evaluate H3K4 methylation at promoters and cooperativities. With deep sequencing and chromatin and gene expression analyses we will be able to evaluate the consequences of H3K4 methylation and cooperating activities. Finally, we will expand on signaling processes that control gene expression dependent on H3K4 methylation. In summary, the proposal addresses fundamental aspects of ASH2L function and the role of H3K4 methylation in gene transcription.