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方法来了解三种赖氨酸（K）乙酰转移酶（KAT）及其激活剂（JBC 2015 A，2015 B; PLoS Genet 2015; JBC 2016; JCI 2016）。这些酶属于MYST乙酰转移酶家族，在体内乙酰化组蛋白H3的两个特异性赖氨酸残基。作为一项独立的研究，我们分析了哺乳动物KAT 8（又名KAT 8）。MOF），MYST家族的另一个成员。它是果蝇Mof（雄性在第一次缺席）的直系同源物，由于其在雄性果蝇中控制基因剂量补偿的关键作用而如此命名。与果蝇Mof一样，哺乳动物KAT 8不乙酰化组蛋白H3，而是特异性靶向组蛋白H4的赖氨酸（K）16（H4 K16）。KAT 8是体内乙酰化该残基的主要乙酰转移酶，因此它具有独特的底物特异性来赋予H4 K16乙酰化。这种表观遗传标记从酵母和植物到人类都具有根本的重要性。一个重要的问题是KAT 8在动物和人类发育中发挥什么作用。小鼠基因的破坏导致胚胎在非常早期的阶段死亡，并且小鼠小脑浦肯野神经元中的基因的条件失活导致共济失调。关于KAT 8在哺乳动物前脑发育中的作用知之甚少。我们最近产生了大脑特异性Kat 8基因敲除小鼠，并发现了严重的大脑缺陷（将提交给Nat Genet）。** 假设 * 我们假设KAT 8通过酰化组蛋白H4 K16调节大脑发育。有了这个假设，我们将通过结合分子和遗传方法来追求3个具体目标。1）阐明KAT 8如何催化组蛋白H4 K16丙酰化。我们发表的数据表明，KAT 8是关键的组蛋白H4 K16丙酰化在体内。为了证实这一点，我们将通过KAT 8及其复合物在体外分析H4 K16丙酰化。**2）研究KAT 8如何调节小鼠大脑发育。使用Cre-LoxP系统，我们产生了大脑特异性敲除。他们表现出明显的大脑缺陷，从E12.5开始。为了证实这一点，我们将利用诱导型Cre系来赋予时间依赖性大脑特异性失活，以研究KAT 8在大脑发育不同阶段的作用。3)确定MSL 1在小鼠大脑发育中的作用。KAT 8是两个多亚基复合物的催化亚基，但目前还不清楚非催化亚基如MSL 1如何在体内促进KAT 8的功能。我们将进行可诱导的大脑特异性失活Msl 1，以评估其在大脑发育不同阶段的作用。该计划将解决KAT 8及其复合物如何调节大脑发育。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，因此这些知识将揭示不同生物体中的自然过程，从而直接有助于NSERC在发现和创新方面的使命。

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.**