Histone Chaperones in Angiogenesis

描述(由申请人提供)：多年来，大量研究表明，许多转录因子在内皮分化和功能中扮演着重要角色。然而，许多问题仍然没有得到回答。例如，在血管生成过程中调节血管内皮生长因子受体(VEGFRs)表达的转录机制在很大程度上是未知的。早些时候，我们发现在成纤维细胞生长因子2和表皮生长因子的介导下，血管生成信号会迅速诱导血管内皮细胞中VEGFR1的转录。我们还发现，转录因子ETS1和缺氧诱导因子2-α以组合方式发挥作用，直接介导血管内皮细胞中VEGFR1的转录诱导。为了进一步了解内皮细胞的转录机制，我们研究了介导VEGFR1染色质区域核蛋白结构变化的分子机制。特别是，我们对组蛋白修饰模式的改变感兴趣。有趣的是，我们发现血管生成信号诱导VEGFR1基因的转录依赖于组蛋白伴侣蛋白，组蛋白细胞周期调节缺陷同源A(HIRA)的功能。我们的分析表明，作为对血管生成信号的响应，Hira介导了乙酰化组蛋白H3(AcH3)在VEGFR1染色质区域的掺入。有趣的是，通过RNA干扰(RNAi)在内皮细胞中敲除Hira可以抑制血管生成信号诱导的VEGFR1和其他血管生成调控基因的诱导，也可以抑制Matrigel上内皮细胞网络/管的形成。此外，在Matrigel Plug实验中，我们发现Hira的缺失也会抑制体内的血管生成。因此，在本提案中，我们将进一步测试Hira在内皮基因调控和出生后/病理性血管生成中的功能。提出了两个具体目标。目的1验证Hira功能对VEGFR1基因座转录活性核蛋白结构的诱导或维持具有重要作用的假设。我们将测试血管生成信号是否改变了VEGFR1基因座染色质的可及性和转录因子的占有率以及VEGFR1基因座的亚核定位，以及Hira对调节这些机制是否重要。此外，利用小鼠胚胎干细胞分化系统和分离的内皮细胞，我们将确定Hira功能是否对组蛋白乙酰化、染色质可及性、从而对内皮祖细胞和成熟内皮细胞中血管生成调控基因的转录起到差异调节作用。在目标2中，我们将确定Hira在病理性血管生成调节中的作用。我们将使用激光诱导的脉络膜新生血管模型和乳腺癌原位异种移植模型中的肿瘤血管生成来实施RNAi方法来测试Hira在病理性血管生成中的功能。这些研究将表明组蛋白伴侣蛋白是否可能是调节血管生成的重要靶点。 公共卫生相关性：血管生成，即从现有的血管系统发展出新的血管，是许多生理过程中的关键事件，如器官生长和发育、伤口愈合和生殖。血管生成在某些病理疾病中也是至关重要的，包括肿瘤生长/转移和老年性黄斑变性。血管内皮细胞基因调控是血管发育的关键，也是血管生成的重要环节。因此，从这些研究中收集到的信息将有助于我们理解血管生成的分子机制，并有望为血管再生和抗血管生成治疗提供新的治疗方式。

DESCRIPTION (provided by applicant): Over the years, numerous studies have implicated roles of many transcription factors in endothelial differentiation and function. However, many questions remain unanswered. For example, transcriptional mechanisms that regulate vascular endothelial growth factor receptors (VEGFRs) expression during angiogenesis are largely unknown. Earlier, we found that Vegfr1 transcription is rapidly induced in endothelial cells in response to angiogenic signals, mediated by fibroblast growth factor 2 and epidermal growth factor. We also found that transcription factors ETS1 and hypoxia inducible factor 2-alpha function in a combinatorial fashion to directly mediate the transcriptional induction of Vegfr1 in endothelial cells. To further understand transcription mechanisms in endothelial cells, we examined molecular mechanisms that mediate alteration of the nucleoprotein structure at the Vegfr1 chromatin domain. In particular, we were interested about the alteration of histone modification patterns. Interestingly, we found that angiogenic signal-induced transcription at the Vegfr1 locus is dependent on the function of a histone chaperone, histone cell cycle regulation defective homolog A (HIRA). Our analyses revealed that in response to angiogenic signals, HIRA mediates incorporation of acetylated histone H3 (acH3) at the Vegfr1 chromatin domain. Intriguingly, knockdown of HIRA by RNA interference (RNAi) in endothelial cells inhibits angiogenic signal-induced induction of Vegfr1 and other angiogenesis regulatory genes and also inhibits endothelial network/tube formation on matrigel. Furthermore, in a matrigel plug assay we found that depletion of HIRA also inhibits angiogenesis in vivo. Therefore, in this proposal, we will further test function of HIRA in endothelial gene regulation and postnatal/pathological angiogenesis. Two specific aims are proposed. Aim 1 will test the hypothesis that HIRA function is important for the induction or maintenance of the transcriptionally active nucleoprotein structure of the Vegfr1 locus. We will test whether the chromatin accessibility and transcription factor occupancy at the Vegfr1 locus as well as the subnuclear localization of the Vegfr1 locus is altered by angiogenic signals and whether HIRA is important for regulating these mechanisms. In addition, using mouse embryonic stem cell differentiation system and isolated endothelial cells, we will determine whether HIRA function differentially regulates histone acetylation, chromatin accessibility, and thereby transcription of angiogenesis regulatory genes in endothelial progenitors vs. in matured endothelial cells. In Aim 2, we will determine HIRA function in the regulation of pathological angiogenesis. We will implement an RNAi approach for testing HIRA function in pathological angiogenesis by using a laser-induced choroidal neovascularization model and also analyzing tumor angiogenesis in a orthotopic xenograft model of breast cancer. These studies will indicate whether histone chaperones could be an important target for modulating angiogenesis. PUBLIC HEALTH RELEVANCE: Angiogenesis, the development of new blood vessels from existing vasculature, is a key event in many physiological processes, like organ growth and development, wound healing, and reproduction. Angiogenesis is also critical for certain pathological disorders including tumor growth/metastasis and age-related macular degeneration. Endothelial gene regulation is critical for blood vessel development and is also important during angiogenesis. Therefore, Information gleaned from these studies will contribute to our understanding of the molecular mechanisms of angiogenesis and hopefully will lead to novel therapeutic modalities for vascular regeneration and anti-angiogenesis therapy.