Hdac2 and Hopx: Regulators of Cardiac Development

项目摘要/摘要： 这一提议的基本假设，以及已经成为我的 研究计划，是表观遗传和染色质修饰在心脏 这种药物的研发将成为心脏病的重要治疗靶点。虽然数量众多 在心脏发育过程中被调控的单个基因已经被描述，全球 这一过程的转录调节因子和表观遗传修饰因子还没有得到很好的描述。 组蛋白脱乙酰酶(HDAC)修饰染色质结构，影响局部和整体基因表达 在心脏和其他地方。最近，我发现小鼠体内HDAC2基因的全球缺失导致了 严重发育心肌缺陷儿围产期部分死亡。有趣的是，HDAC2会影响 心肌细胞分化与增殖之间的平衡。在此之前，我们已经证明 同源结构域仅限蛋白(Hopx)在胚胎和成人心脏中表达，至少具有功能 在一定程度上，通过直接与HDAC2相互作用来调节心肌基因的抑制。全球损失 在小鼠中，Hopx还导致部分围产期死亡和类似HDAC2的心脏缺陷 淘汰赛。在本研究中，我们发现HDAC2和Hopx在心脏的发育和缺失中共同表达。 HDAC2和Hopx都会导致围产期完全死亡，并伴有严重的心脏缺陷，包括 肌性室间隔缺损，明显增加心肌细胞增殖。微阵列分析 揭示了HDAC2中几个细胞周期特异性基因以及心脏结构基因的异常调节 霍普克斯-零心脏。我们的机理分析表明，HDAC2和Hopx的损失都会导致 激活Gata4，先前已经表明它可以调节心肌细胞的增殖。HDAC2交互 随着Gata4和HDAC2-Hopx的缺失，在发育过程中Gata4乙酰化和激活增加 心肌。这些结果表明，HDAC2和Hopx之间的相互作用在 心脏发育，因此，我将检验HDAC2和Hopx协调发挥作用的假设 在心脏中通过直接调节Gata4乙酰化来调节Gata4的活性，这解释了 心肌细胞增殖的变化。具体地说，我将研究HDAC2-Hopx 复合体调节心肌细胞增殖过程中Gata4乙酰化和活性及其对组织的影响 发育中心肌中HDAC2-Hopx功能的特异性丧失。这将通过以下方式实现 追求以下具体目标： 目的1：确定和表征HDAC2和Hopx是否协同调节Gata4 体外和体内的乙酰化和转录活性。 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2：研究HDAC2-Hopx复合体在心脏发育中的组织特异性作用 新产生的HDAC2等位基因的分析。

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.