Deciphering the role of the MOF complex and H4K16ac in the regulation of neuronal enhancers

组蛋白将DNA包裹成紧密的DNA-蛋白质结构，称为染色质。压缩DNA转录的可及性是由酶复合物调控的，酶复合物在组蛋白上添加共价修饰，如乙酰基和甲基。包括乙酰化在内的组蛋白共价修饰在细胞功能的许多方面起着至关重要的作用，包括大脑等复杂器官的正常发育。编码组蛋白修饰蛋白复合物的基因在发育障碍中经常发生突变。严重神经发育障碍最常见的原因是对中枢神经系统正常发育至关重要的基因发生了新的突变，其表型范围广泛，如全面发育迟缓、严重智力残疾和自闭症。据估计，在英国，每220名儿童中就有1名因基因突变而患有严重的神经发育障碍。明确的早期诊断和对疾病机制的更好理解将提高神经发育障碍患者的分层，为个性化治疗和支持提供帮助。编码表观遗传修饰因子的基因在发育障碍患者中经常发生突变。编码组蛋白乙酰转移酶复合物（MSL/MOF）成分的男性特异性致死3 （MSL3）基因在称为MSL3综合征的神经发育障碍中发生突变。MSL/MOF蛋白复合物负责组蛋白H4在赖氨酸16 （H4K16ac）上的乙酰化，已知这可以增加基因表达。然而，MSL/MOF复合物在神经系统发育中的特定作用以及MSL3突变如何促进特定的神经发育表型尚不清楚。只有约42%的严重发育障碍患者有可检测到的影响基因蛋白质编码区的突变。在称为增强子点的非编码远端调控元件中发现的序列变异对发育障碍中增强子功能障碍的贡献。增强子分布在98%的非编码基因组中，可以远距离调节基因，因此，由于增强子DNA序列的变化，描述疾病机制具有挑战性。组蛋白H3赖氨酸27位点乙酰化（H3K27ac）的全基因组谱分析是鉴定基因组中活性增强子的广泛方法。我们之前已经发现了一类新的基于H4K16ac存在而缺乏H3K27ac的活性增强子。在本项目中，我们将鉴定MOF和H4K16ac调控的基因和增强子，然后比较基于组蛋白乙酰化鉴定的增强子的活性。此外，我们将研究组蛋白乙酰转移酶复合体调节基因表达的机制。我们还将研究组蛋白乙酰化途径中编码蛋白质的基因突变如何导致特定的神经元表型。最后，我们将测试是否可以通过增加组蛋白乙酰化水平的药物治疗来挽救改变的细胞和基因表达表型。总体而言，我们将利用最先进的功能基因组学和基因组编辑方法，全面了解MSL/MOF和H4K16ac调控基因的机制。重要的是，这项工作将为导致组蛋白乙酰化水平改变的突变所导致的疾病机制提供重要的见解。这项工作将提高我们对MSL3和增强子突变导致神经发育障碍的机制的理解，此外，还提供了对患者进行分层治疗干预的功能标准。

Histone proteins package DNA into a compact DNA-protein structure termed chromatin. Accessibility of compacted DNA for transcription is regulated by enzymatic complexes that add covalent modifications like acetyl and methyl groups to histones. Covalent modifications on the histones including acetylation play essential roles in many aspects of cellular function, including normal development of complex organs including the brain. Genes encoding histone-modifying protein complexes are frequently mutated in developmental disorders. Severe neurodevelopmental disorders are most commonly caused by new mutations in genes important for normal development of the central nervous system, with a wide range of phenotypes such as global developmental delay, severe intellectual disability and autism. It is estimated that about 1 in 220 children in the UK have severe neurodevelopmental impairments as a result of new mutations. Clear and early diagnosis and a better understanding of disease mechanism will improve the stratification of neurodevelopmental disorder patients for personalized therapy and support. Genes encoding epigenetic modifiers are frequently mutated in patients with developmental disorders. Male specific lethal 3 (MSL3) gene encoding a component of histone acetyltransferase complex (MSL/MOF) is mutated in a neurodevelopmental disorder called MSL3 syndrome. The MSL/MOF protein complex is responsible for acetylation of histone H4 at lysine 16 (H4K16ac), which is known to increase gene expression. However, a specific role of MSL/MOF complex in the development of the nervous system and how mutations in MSL3 contribute to a specific neurodevelopmental phenotype is not known.Only about 42% of patients with severe developmental disorders have detectable mutations that affect the protein coding regions of genes. Sequence variants found in non-coding distal regulatory elements called enhancers points contribution of enhancer dysfunction in developmental disorders. Enhancers are scattered around 98% of the genome that is non-coding and can regulate genes from large distances, hence it is challenging to delineate disease mechanism due to sequence variation in the enhancer DNA. Genome-wide profiling of acetylation of histone H3 at lysine 27 (H3K27ac) is widely method to identify active enhancers in the genome. We have previously discovered a new class of active enhancers based on the presence of H4K16ac that lack H3K27ac. In this project, we will identify genes and enhancers regulated by MOF and H4K16ac, then compare the activity of enhancers identified based on histone acetylation. Furthermore, we will investigate the mechanism through which histone acetyltransferase complex function to regulate gene expression. We will also investigate how mutations in genes encoding proteins in histone acetylation pathway leads to a specific neuronal phenotype. Finally, we will test if we can rescue altered cellular and gene expression phenotype by treating with drugs that increases histone acetylation levels.Overall, we will use state-of-the-art functional genomics and genome editing methods to comprehensively understand the mechanism through which MSL/MOF and H4K16ac regulate genes. Importantly, this work will provide important insights into the disease mechanism which results from mutations that leads to altered level of histone acetylation. This work will improve our understanding of the mechanisms through which mutations in MSL3 and enhancers contribute to neurodevelopmental disorders and, in addition, provide afunctional criterion on which to stratify patients for therapeutic intervention.