Hdac2 and Hopx: Regulators of Cardiac Development

描述(申请人提供)：项目摘要/摘要：这项提议的基本假设，也是我的研究计划的驱动力之一，是表观遗传和染色质修饰在心脏发育过程中至关重要，并将成为心脏疾病的重要治疗靶点。虽然已经描述了许多在心脏发育过程中受到调控的单个基因，但这一过程的全球转录调节因子和表观遗传修饰物的特征还不是很好。组蛋白脱乙酰酶(HDAC)改变染色质结构，影响心脏和其他部位的局部和整体基因表达。最近，我发现在小鼠体内，HDAC2的全球缺失会导致部分围产期死亡，并伴有严重的发育心肌缺陷。有趣的是，HDAC2影响心肌细胞分化和增殖之间的平衡。此前，我们已经证明同源结构域蛋白(Hopx)在胚胎和成人心脏中表达，并至少部分地通过直接与HDAC2相互作用来调节心肌基因的抑制。在小鼠中，Hopx基因的全局缺失也会导致部分围产期死亡和类似HDAC2基因敲除的心脏缺陷。在这里，我们发现HDAC2和Hopx在发育中的心脏中共表达，HDAC2和Hopx的缺失会导致围产期完全死亡，并伴有严重的心脏缺陷，包括肌性室间隔缺陷和显著的心肌细胞增殖。微阵列分析显示，在HDAC2-Hopx缺失的心脏中，几个细胞周期特异性基因以及心脏结构基因调控失调。我们的机制分析表明，HDAC2和Hopx的丢失都会导致Gata4的激活，而Gata4已经被证明调节心肌细胞的增殖。HDAC2与Gata4相互作用，HDAC2-Hopx的缺失增加了发育中心肌中Gata4的乙酰化和激活。这些结果表明，HDAC2和Hopx之间的相互作用在心脏发育过程中具有功能性，因此，我将检验HDAC2和Hopx在心脏中协同作用，通过直接调节Gata4乙酰化来调节Gata4活性的假设，这解释了心肌细胞增殖的变化。具体地说，我将研究HDAC2-Hopx复合体在心肌细胞增殖过程中调节Gata4乙酰化和活性的机制，以及组织特异性HDAC2-Hopx功能丧失在发育中的心肌中的影响。这将通过追求以下特定目标来实现：目标1：确定并表征HDAC2和Hopx是否在体外和体内协同调节Gata4乙酰化和转录活性。A)表征Hopx-HDAC2-Gata4络合物。B)确定HDAC2-Hopx是否使GATA4脱乙酰化。C)确定并鉴定HDAC2-Hopx是否调节Gata4的转录活性。目的：通过对新产生的HDAC2的等位基因进行分析，研究HDAC2-Hopx复合体在心脏发育中的组织特异性作用。 公共卫生相关性：项目叙述：先天性心脏缺陷是人类最常见的发育缺陷。表观遗传和染色质修饰对先天性心脏病的作用在很大程度上是未知的。这项建议中概述的一系列实验对于理解心肌细胞增殖和心脏发育的基本机制具有广泛的意义。

DESCRIPTION (provided by applicant): Project Summery / Abstract: The underlying hypothesis of this proposal, and one that has become the driving force of my research program, is that epigenetic and chromatin modifications are critical during cardiac development and will emerge as important therapeutic targets for cardiac diseases. While numerous individual genes that are regulated during cardiac development have been described, global transcriptional regulators and epigenetic modifiers of this process have been less well characterized. Histone deacetylases (HDACs) modify chromatin structure and affect local and global gene expression in the heart and elsewhere. Recently, I have discovered that global loss of Hdac2 in mice results in a partial perinatal lethality with severe developmental myocardial defects. Interestingly, Hdac2 affects the balance between differentiation and proliferation of cardiomyocytes. Previously, we have shown that Homeodomain only protein (Hopx) is expressed in the embryonic and adult heart and functions, at least in part, by directly interacting with Hdac2 to mediate the repression of myocardial genes. Global loss of Hopx in mice also results in a partial perinatal lethality and cardiac defects that resemble Hdac2 knockouts. Here, we show that Hdac2 and Hopx are co-expressed in the developing heart and loss of both Hdac2 and Hopx results in complete perinatal lethality with severe cardiac defects including muscular ventricular septal defects and markedly increased myocyte proliferation. Microarray analysis reveals dysregulation of several cell-cycle specific genes as well as cardiac structural genes in Hdac2- Hopx-null hearts. Our mechanistic analysis indicates that loss of both Hdac2 and Hopx leads to activation of Gata4, which has been shown previously to regulate myocyte proliferation. Hdac2 interacts with Gata4 and loss of Hdac2-Hopx increases Gata4 acetylation and activation in developing myocardium. These results suggest that the interaction between Hdac2 and Hopx is functional during cardiac development and therefore, I will test the hypothesis that Hdac2 and Hopx coordinately function in the heart to regulate Gata4 activity by directly regulating Gata4 acetylation and that this accounts for changes in myocyte proliferation. Specifically, I will investigate the mechanism by which Hdac2-Hopx complex regulates Gata4 acetylation and activity during myocyte proliferation and the effects of tissue specific loss of Hdac2-Hopx function in the developing myocardium. This will be accomplished by pursuing the following specific aims: Aim 1: Determine and characterize whether Hdac2 and Hopx function coordinately to regulate Gata4 acetylation and transcriptional activity in vitro and in vivo. A) Characterize the Hopx-Hdac2-Gata4 complex. B) Determine whether Hdac2-Hopx deacetylates Gata4. C) Determine and characterize whether Hdac2-Hopx regulates Gata4 transcriptional activity. Aim 2: Characterize the tissue specific role of Hdac2-Hopx complex in cardiac development through analysis of a newly generated floxed allele of Hdac2. PUBLIC HEALTH RELEVANCE: Project Narrative: Congenital heart defects are the most commonly occurring developmental defects in humans. Contributions of epigenetic and chromatin modifications to congenital heart diseases are largely unknown. The set of experiments outlined in this proposal have broad significance for understanding the fundamental mechanisms underlying myocyte proliferation and heart development.