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细胞分化和功能中的作用。然而，许多问题仍未得到解答。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。之前，我们发现Vegfr1转录在内皮细胞响应血管生成信号时被快速诱导，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ETS1和缺氧诱导因子2- α以组合方式直接介导内皮细胞中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染色质结构域核蛋白结构改变的分子机制。我们特别感兴趣的是组蛋白修饰模式的改变。有趣的是，我们发现血管生成信号诱导的Vegfr1位点的转录依赖于组蛋白伴侣的功能，组蛋白细胞周期调节缺陷同源物a （HIRA）。我们的分析显示，为了响应血管生成信号，HIRA介导了Vegfr1染色质结构域乙酰化组蛋白H3 （acH3）的结合。有趣的是，在内皮细胞中通过RNA干扰（RNAi）敲低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表达，也可以抑制基质上内皮网络/管的形成。此外，在基质堵塞试验中，我们发现HIRA的消耗也会抑制体内血管生成。因此，在本研究中，我们将进一步测试HIRA在内皮基因调控和出生后/病理血管生成中的功能。提出了两个具体目标。目的1将验证HIRA功能对于诱导或维持Vegfr1位点的转录活性核蛋白结构很重要的假设。我们将测试血管生成信号是否会改变Vegfr1位点的染色质可及性和转录因子占用以及Vegfr1位点的亚核定位，以及HIRA是否在调节这些机制中起重要作用。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功能是否在内皮祖细胞和成熟内皮细胞中对组蛋白乙酰化、染色质可及性以及血管生成调节基因的转录有差异。在目的2中，我们将确定HIRA在病理性血管生成调控中的功能。我们将通过激光诱导脉络膜新生血管模型和原位乳腺癌异种移植模型的肿瘤血管生成分析，实施RNAi方法来检测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是否可能是调节血管生成的重要靶点。

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.