Hdac2 and Hopx: Regulators of Cardiac Development

项目概述/摘要：本提案的基本假设是，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，这一假设已成为我研究项目的推动力。虽然已经描述了许多在心脏发育过程中受到调节的个体基因，但这一过程的全局转录调节因子和表观遗传修饰因子尚未得到很好的表征。组蛋白去乙酰化酶（hdac）修饰染色质结构并影响心脏和其他部位的局部和全局基因表达。最近，我发现Hdac2在小鼠中的整体缺失会导致部分围产期死亡，并伴有严重的发育性心肌缺陷。有趣的是，Hdac2影响心肌细胞分化和增殖之间的平衡。在此之前，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，并且至少部分地通过直接与Hdac2相互作用来介导心肌基因的抑制。小鼠中Hopx的整体缺失也会导致部分围产期死亡和心脏缺陷，类似于Hdac2基因敲除。在这里，我们发现Hdac2和Hopx在发育中的心脏中共同表达，Hdac2和Hopx的缺失会导致严重心脏缺陷（包括肌性室间隔缺陷）和心肌细胞增殖明显增加的完全围产期死亡。微阵列分析揭示了Hdac2- hopx缺失心脏中几个细胞周期特异性基因以及心脏结构基因的失调。我们的机制分析表明，Hdac2和Hopx的缺失导致Gata4的激活，而Gata4先前已被证明可以调节肌细胞增殖。Hdac2与Gata4相互作用，Hdac2- hopx的缺失增加了发育心肌中Gata4的乙酰化和活化。这些结果表明，Hdac2和Hopx之间的相互作用在心脏发育过程中起作用，因此，我将验证Hdac2和Hopx在心脏中通过直接调节Gata4乙酰化来协调调节Gata4活性的假设，这解释了心肌细胞增殖的变化。具体来说，我将研究Hdac2-Hopx复合物在肌细胞增殖过程中调控Gata4乙酰化和活性的机制，以及Hdac2-Hopx功能在发育心肌中组织特异性丧失的影响。这将通过追求以下具体目标来实现：目的1：确定和表征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复合体。B)确定Hdac2-Hopx是否脱乙酰Gata4。C)确定并表征Hdac2-Hopx是否调控Gata4的转录活性。目的2：通过分析Hdac2新产生的floxed等位基因，表征Hdac2- hopx复合物在心脏发育中的组织特异性作用。

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.