Histone Chaperones in Angiogenesis

描述（由申请人提供）：多年来，许多研究表明许多转录因子在内皮细胞分化和功能中的作用。然而，许多问题仍然没有答案。例如，在血管生成过程中调节血管内皮生长因子受体（VEGF）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Vegfr 1转录在内皮细胞中响应于血管生成信号被快速诱导，由成纤维细胞生长因子2和表皮生长因子介导。我们还发现，转录因子ETS 1和缺氧诱导因子2-α的功能在一个组合的方式直接介导的内皮细胞中的Vegfr 1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Vegfr 1染色质结构域核蛋白结构改变的分子机制。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的转录在Vegfr 1基因座是依赖于组蛋白伴侣，组蛋白细胞周期调控缺陷同源物A（HIRA）的功能。我们的分析表明，在响应血管生成信号，HIRA介导的乙酰化组蛋白H3（ACH 3）在Vegfr 1染色质结构域的掺入。有趣的是，在内皮细胞中通过RNA干扰（RNAi）敲低HIRA抑制血管生成信号诱导的Vegfr 1和其他血管生成调节基因的诱导，并且还抑制基质胶上的内皮网络/管形成。此外，在基质胶塞测定中，我们发现HIRA的耗尽也抑制体内血管生成。因此，在本研究中，我们将进一步检测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将测试的假设，HIRA功能是重要的诱导或维持转录活性的Vegfr 1基因座的核蛋白结构。我们将测试是否染色质的可及性和转录因子占用的Vegfr 1基因座以及亚核定位的Vegfr 1基因座是改变血管生成信号和HIRA是否是重要的调节这些机制。此外，使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功能是否差异调节组蛋白乙酰化，染色质可及性，从而在内皮祖细胞与成熟内皮细胞中的血管生成调控基因的转录。在目标2中，我们将确定HIRA在病理性血管生成调节中的功能。我们将实施一种RNAi方法，通过使用激光诱导的脉络膜新生血管模型来测试HIRA在病理性血管生成中的功能，并分析乳腺癌原位异种移植模型中的肿瘤血管生成。这些研究将表明组蛋白伴侣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，即从现有血管系统发育出新血管，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。血管生成对于某些病理性疾病也至关重要，包括肿瘤生长/转移和年龄相关性黄斑变性。内皮基因调控对血管发育至关重要，在血管生成过程中也很重要。因此，从这些研究中收集的信息将有助于我们了解血管生成的分子机制，并有望导致新的治疗方式，血管再生和抗血管生成治疗。

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.