组蛋白乙酰基转移酶MOF-NSL与甲基转移酶MLL/SET在基因转录调控中的相互作用及其机制研究

Currently, many types of histone modification and modified sites have been proven. Although the specific biological functions still need to be studied, research evidence about the crosstalk between different chromatin modifying enzymes has been increased, suggesting the importance of co-ordination between histone modifications in gene transcriptional regulation. In previous study, we purified and characterized the MOF-NSL histone acetyltransferase complex which included nine protein subunits in human cells. We also clarified that the MOF-NSL complex can acetylate histone H4K5/K8/K16. Furthermore, we verified that MOF-NSL complex could facilitate H3K4me2 by MLL/SET methyltransferase through acetylating histone H4 in vitro. In addtion, the MOF target gene ANKRD2 was co-regulated by MOF-NSL and MLL/SET complexes. In this study, based on RNA-Seq and ChIP-Seq data analysis, we will: 1) confirm the genome-wide distribution of the MOF-NSL and MLL/SET complexes and their corresponding histone modifications; 2) verify the binding mode between the complexes and the nucleosomal chromatin under different histone H4 modification conditions; 3) acquire genes that are regulated by both complexes and closely related to tumorigenesis. 4) further demonstrate the crosstalk between two complexes in gene transcriptional regulation and their mechanism. Our study will provide experimental evidence and theoretical basis for developing the gene-targeted medicine in future studies.

目前已被证实的组蛋白修饰位点和修饰类型有很多，尽管其确切的生物学意义尚不清楚，但是不同组蛋白修饰在基因转录调控中的相互协调作用越来越受关注。我们在之前研究中分离纯化了含9个亚基的人MOF-NSL组蛋白乙酰基转移酶，并鉴定其能够乙酰基化组蛋白H4上第5、8和16赖氨酸位点的底物特异性。在体外实验我们还证实NSL复合物可以通过乙酰基化H4来促进MLL/SET催化H3K4me2活性，而且这两种复合物在细胞中协同调控MOF的靶基因ANKRD2。本项目将延续前期研究内容，拟通过RNA-Seq和ChIP-Seq检测及分析，明确NSL和MLL/SET两种复合物及其相应组蛋白修饰在基因组上的分布情况，弄清MLL-SET复合物与不同H4乙酰基化修饰状态染色质的结合模式，筛选能被两种复合物共同调控、并与肿瘤发生密切相关的基因，阐明两种复合物对其转录调控的协同机制，为后续相关靶向治疗药物的研发提供实验基础。

MOF是组蛋白乙酰基转移酶，作为共享催化亚基参与组成MSL和NSL两个蛋白质复合物，两者都可以通过组蛋白H4K16的乙酰基化修饰参与细胞内的基因转录、DNA复制和细胞周期等调控过程。另外有研究数据证明MOF还参与一些非组蛋白蛋白质的乙酰基化修饰从而影响细胞内的一些信号传导通路。为了进一步探究MOF在细胞内的生物学功能，我们利用基因编辑技术构建了复合物中两个关键亚基MSL1和NSL3的稳敲293T细胞系，细胞迁移实验结果显示它可以影响293T正常肾胚细胞和MCF7乳腺癌细胞的侵袭能力，非常有趣的是MSL和NSL两种复合物对细胞侵袭和迁移具有相反的作用。利用上述293T稳敲细胞系并通过RNA-seq和Chip-seq实验的数据分析，证明MSL和NSL两种复合物在细胞中各自调控不同基因的表达以及影响不同的信号传导通路。进一步用免疫印记和定量PCR方法验证了NSL和MSL两种复合物在蛋白表达水平上各自调控了不同的EMT相关蛋白因子的表达。接下来，我们从NSL3敲除293T细胞的组蛋白H4K5/8/16乙酰基化、H3K4的1/2/3甲基化、H3K27ac/me3、MOF、NSL3和POLII的Chip-seq数据中研究MOF/NSL和MLL/SET两种复合物在基因组上分布及其协同关系，以及与转录组数据进行比对分析进一步挖掘NSL复合物调控的靶基因。进一步，探讨NSL-MOF和MLL/SET两种复合物共同调控的靶基因和信号通路，为后期深入探讨如何利用调控肿瘤侵袭转移相关关键基因表达的手段来靶向治疗相应肿瘤提供有力的实验基础。