Roles of KAT8 complexes in governing histone acylation and mouse cerebral development

表观遗传调控是所有真核生物的基本自然过程。我们采用小鼠遗传学的方法来了解三种赖氨酸(K)乙酰转移酶(KATS)及其激活剂(JBC 2015A，2015B；PLoS Genet 2015；JBC 2016；JCI 2016)。这些酶属于Myst家族的乙酰转移酶家族，在体内乙酰化组蛋白H3的两个特定的赖氨酸残基。作为一项独立的研究，我们分析了哺乳动物KAT8(又称KAT8)。MOF)，神秘族的另一个成员。它是果蝇MOF(第一只雄性果蝇缺失)的同源基因，之所以这样命名，是因为它在控制雄性果蝇的基因剂量补偿方面发挥了关键作用。像苍蝇MOF一样，哺乳动物KAT8不乙酰化组蛋白H3，但特异性靶向组蛋白H4的赖氨酸(K)16(H4K16)。KAT8是体内使该残基乙酰化的主要乙酰转移酶，因此它具有明显的底物特异性来赋予H4K16乙酰化作用。从酵母菌和植物到人类，这种表观遗传标记都是至关重要的。一个重要的问题是KAT8在动物和人类的发育中扮演着什么角色。小鼠基因的破坏在非常早期就会导致胚胎死亡，而小鼠小脑浦肯野神经元中该基因的条件性失活会导致共济失调。关于KAT8在哺乳动物前脑发育中的作用，人们知之甚少。我们最近产生了大脑特异的Kat8基因敲除小鼠，并发现了严重的大脑缺陷(提交给NAT Genet)。假设我们假设KAT8通过酰化组蛋白H4K16来调节大脑发育。在这个假设下，我们将通过结合分子和遗传方法来追求三个特定的目标。具体目的1)阐明KAT8如何催化组蛋白H4K16丙酰化。我们已发表的数据表明，KAT8在活体中组蛋白H4K16丙酰化反应中起关键作用。为了证实这一点，我们将分析KAT8及其配合物在体外对H4K16丙酰化反应的影响。2)研究KAT8对小鼠脑发育的调节作用。使用Cre-loxP系统，我们已经产生了大脑特异的基因敲除。他们表现出明显的大脑缺陷，从E12.5开始。为了证实这一点，我们将利用一个可诱导的Cre系来赋予时间依赖的大脑特定的失活，以研究KAT8在大脑发育的不同阶段中的作用。3)确定MSL1在小鼠脑发育中的作用。KAT8是两个多亚基复合体的催化亚基，但目前还不清楚MSL1等非催化亚基在体内对KAT8功能的贡献。我们将进行可诱导的大脑特异性失活MSL1，以评估其在大脑发育的不同阶段的作用。意义该计划将解决KAT8及其复合体如何调节大脑发育的问题。由于组蛋白H4乙酰化从酵母、苍蝇和植物到人类都是保守的，这些知识将揭示不同生物体的自然过程，从而直接为NSERC的发现和创新任务做出贡献。

Epigenetic regulation is a natural process fundamental to all eukaryotic organisms. We have taken a mouse genetic approach to understand three lysine (K) acetyltranferases (KATs) and their activators (JBC 2015A, 2015B; PLoS Genet 2015; JBC 2016; JCI 2016). The enzymes belong to the MYST family of acetyltransferases and acetylate two specific lysine residues of histone H3 in vivo. As an independent line of research, we have analyzed mammalian KAT8 (a.k.a. MOF), another member of the MYST family. It is an ortholog of fly Mof (males absent on the first), which is so named due to its crucial role in governing gene dosage compensation in male flies. Like fly Mof, mammalian KAT8 does not acetylate histone H3 but specifically targets histone H4 at lysine (K) 16 (H4K16). KAT8 is the major acetyltransferase acetylating this residue in vivo, so it has a distinct substrate specificity to confer H4K16 acetylation. This epigenetic mark is of fundamental importance from yeasts and plants to humans. An important question is what roles KAT8 has in animal and human development. Disruption of the mouse gene led to embryonic lethality at a very early stage, and conditional inactivation of the gene in Purkinje neurons of the mouse cerebellum causes ataxia. Little is known about the role of KAT8 in mammalian forebrain development. We have recently generated cerebrum-specific Kat8 knockout mice and found severe cerebral defects (to be submitted to Nat Genet). Hypothesis We hypothesize that KAT8 regulates cerebral development through acylating histone H4K16. With this hypothesis, we will pursue 3 specific aims by combining molecular and genetic approaches. Specific Aims 1) Elucidate how KAT8 catalyzes histone H4K16 propionylation. Our published data indicate that KAT8 is critical for histone H4K16 propionylation in vivo. To substantiate this, we will analyze H4K16 propionylation in vitro by KAT8 and its complexes. 2) Investigate how KAT8 regulates mouse cerebral development. Using the Cre-LoxP system, we have generated cerebrum-specific knockouts. They display striking cerebral defects, starting at E12.5. To substantiate this, we will utilize an inducible Cre line to confer time-dependent cerebrum-specific inactivation to investigate the role of KAT8 at different stages of cerebral development. 3) Determine the role of MSL1 in mouse cerebral development. KAT8 is the catalytic subunit of two multisubunit complexes, but it is unclear how the non-catalytic subunits such as MSL1 contribute to the functions of KAT8 in vivo. We will perform inducible cerebrum-specific inactivation Msl1 to assess its role at different stages of cerebral development. Significance The program will address how KAT8 and its complexes regulate brain development. As histone H4 acetylation is conserved from yeasts, flies and plants to humans, the knowledge will shed light on natural processes in diverse organisms, thus contributing directly to the NSERC's mission in discovery and innovation.