Regulation of Venous and Lymphatic Identity by Chromatin-Remodeling

描述（由申请人提供）：表观遗传调节因子在多种发育过程中发挥着重要作用，但人们对它们如何调节血管规格知之甚少。申请人实验室的长期目标是了解ATP依赖性染色质重塑复合物如何表观遗传地调节导致发育和出生后血管病理的基因转录，例如由于血管和淋巴管的错误指定而引起的血管病理。该提案的目的是了解染色质重塑酶 BRG1 和 CHD4 如何影响胚胎发育过程中的静脉和淋巴管特性。中心假设是 BRG1 和 CHD4 通过调节核受体 Coup-TFII 的血管表达来影响静脉和淋巴管的特性。该假设基于申请人实验室产生的初步数据以及血管 Brg1、Chd4 和 Coup-TFII 突变小鼠胚胎之间的表型相似性。 Coup-TFII 在静脉和淋巴管规范中发挥重要作用，但对于促进其血管表达的因素尚不清楚。因此，鉴定调节其表达的表观遗传机制将阐明 COUP-TFII 如何影响血管发育。将采用两个具体目标来检验中心假设。首先，将使用一系列遗传工具来辨别 BRG1 和 CHD4 对静脉和淋巴管特性的时间/空间影响。我们将在整个发育过程中对突变胚胎中的静脉和淋巴标记进行分析，以准确确定 BRG1 和 CHD4 影响这些血管的时间和地点。其次，将检查 BRG1 和 CHD4 对静脉和淋巴管中 Coup-TFII 表达的表观遗传影响。 Brg1 和 Chd4 突变体中的 COUP-TFII 表达将与目标 1 中定义的静脉和淋巴管规范表型相关。此外，原代和培养的内皮细胞将用于测试以下假设：BRG1 和 CHD4 通过 Coup-TFII 启动子处的核小体重塑和组蛋白乙酰化直接促进血管 Coup-TFII 表达。拟议的研究意义重大，因为它将提供有关染色质重塑酶如何影响 Coup-TFII 表达的新信息，并将作为开发药理学策略来操纵 Coup-TFII 表达以正面或负面影响血管发育的第一步。这项研究具有创新性，因为它强调血管发育的表观遗传调控，这偏离了处理该课题的传统方法。

DESCRIPTION (provided by applicant): Epigenetic regulators play important roles in a variety of developmental processes, but little is understood about how they modulate vascular specification. A long-term goal of the applicant's laboratory is to understand how ATP-dependent chromatin-remodeling complexes epigenetically regulate transcription of genes that con- tribute to developmental and postnatal vascular pathologies, such as those that arise from misspecification of blood and lymphatic vessels. The objective of this proposal is to understand how the chromatin-remodeling enzymes BRG1 and CHD4 influence venous and lymphatic identity during embryonic development. The cen- tral hypothesis is that BRG1 and CHD4 affect venous and lymphatic identity by modulating vascular expression of the nuclear receptor Coup-TFII. This hypothesis is based on preliminary data produced in the applicant's laboratory and on phenotypic similarities between vascular Brg1, Chd4, and Coup-TFII mutant mouse em- bryos. Coup-TFII plays important roles in venous and lymphatic specification but nothing is known about what promotes its vascular expression. Therefore identification of epigenetic mechanisms for regulating its expres- sion would elucidate how COUP-TFII influences vessel development. Two specific aims will be employed to test the central hypothesis. First, the temporal/spatial impact of BRG1 and CHD4 on venous and lymphatic identity will be discerned using an array of genetic tools. Venous and lymphatic markers in mutant embryos will be analyzed throughout development to determine precisely when and where BRG1 and CHD4 affect these vessels. Secondly, the epigenetic impact of BRG1 and CHD4 on Coup-TFII expression in veins and lymphatics will be examined. COUP-TFII expression in Brg1 and Chd4 mutants will be correlated with the ve- nous and lymphatic specification phenotypes defined in Aim 1. In addition, primary and cultured endothelial cells will be used to test the hypothesis that BRG1 and CHD4 directly promote vascular Coup-TFII expression through nucleosome remodeling and histone acetylation at the Coup-TFII promoter. The proposed research is significant because it will provide novel information about how chromatin-remodeling enzymes affect Coup-TFII expression and will serve as a first step toward developing pharmacological strategies to manipulate Coup-TFII expression in order to positively or negatively influence vascular development. This research is innovative be- cause it emphasizes epigenetic regulation of vascular development, which deviates from traditional ways of approaching this subject.