Chromatin Regulation of Heart Valve Development

项目概要： 半月瓣（SLV）疾病，包括二叶式主动脉瓣（BAV），非常常见，但其 遗传和发育起源知之甚少。同样，目前仍不清楚如何破坏 胚胎瓣膜发育进展为显性瓣膜疾病。我们的长期目标是了解 基因调控驱动连续的发育过程，最终产生复杂的，有图案的瓣膜 以及这些过程在SLV疾病中是如何出错的。这些基因调控事件需要转录因子 与染色质化的基因组连接，这表明染色质调节因子是SLV的关键组成部分 发展网络。一个重要的事件是心内膜间质转化（EMT）， 发生在瓣膜发育早期，以填充内膜垫（EC），包括近端流出道 （pOFT）为SLV提供组织的垫。我们的目标是：1）了解染色质重塑 与EMT期间的细胞信号传导整合，以及2）确定瓣膜破坏的机制， 发展成患病的SLV。我们的中心假设是内皮细胞Brg 1相关因子 (BAF)染色质重塑复合物与Wnt信号传导效应物相互作用以促进pOFT EMT。作为 结果，当内源性Brg1缺失时，OFT间充质的亚型被耗尽。如果没有这些细胞， 过度生长和融合导致包括BAV在内的SLV变厚和图案不良。我们努力的理由 在EMT中确定染色质重塑的作用将有助于阐明SLV疾病的起源。此外，本发明还 我们的SLV疾病小鼠模型将使我们能够理解SLV疾病的细胞和分子进展， 瓣膜病我们的具体目标是：1）确定BAF复合物界面的分子网络 指导EMT和2）确定缺乏内皮细胞的小鼠中SLV疾病进展的机制。 谱系Brg1.在追求第一个目标，我们将比较细胞和分子pOFT缺陷中看到的， 破坏Brg1和Wnt信号传导的未发表的遗传模型。我们将应用一种变革性的新TU标记 技术来定义动态的，依赖于每个途径的内源性转录组。使用新的细胞培养 方法中，我们将测试EC细胞中BAF，Wnt效应子和染色质之间的生物化学相互作用。为 第二个目标，我们将使用遗传谱系追踪来确定EMT衍生细胞对不同的细胞分化的贡献。 SLV区域，定义SLV间充质亚群之间的相互作用，表征错误表达 转录，可能驱动SLV疾病的进展，并描述了一种新的成年SLV疾病的小鼠模型， 在临床前试验中的潜在效用。我们提出的研究使用新的技术和范式 解决SLV发展和疾病的未解决问题的方法。这些贡献将是 意义重大，因为它们将揭示SLV疾病的人类遗传学，并为再生医学提供信息 接近。我们新发现的与BAV模型相关的转录本可能代表了BAV的生物标志物。 疾病诊断或治疗目标，以防止先天性异常瓣膜患病。

PROJECT SUMMARY: Semilunar valve (SLV) diseases, including bicuspid aortic valves (BAV), are remarkably common and yet their genetic and developmental origins are poorly understood. Likewise, it remains unclear how disrupted embryonic valve development progresses into overt valve disease. Our long-term goal is to understand how gene regulation drives sequential developmental processes that ultimately produce complex, patterned valves and how these processes go awry in SLV disease. These gene regulatory events require transcription factors to interface with a chromatinized genome, suggesting that chromatin regulators are key components of SLV developmental networks. One important event is an endocardial-to-mesenchymal transformation (EMT) that occurs early in valve development to populate endocardial cushions (ECs), including the proximal outflow tract (pOFT) cushions that contribute tissue to SLVs. Our objectives are to 1) understand how chromatin remodeling integrates with cell signaling during EMT, and 2) determine mechanisms by which disruptions of valve development progress into diseased SLVs. Our central hypothesis is that endocardial Brg1-associated factor (BAF) chromatin remodeling complexes interact with Wnt signaling effectors to promote pOFT EMT. As a result, when endocardial Brg1 is deleted a subtype of OFT mesenchyme is depleted. Without these cells, cusp overgrowth and fusion results in thickened and malpatterned SLVs, including BAV. The rationale for our efforts is that defining chromatin remodeling roles during EMT will shed light on how SLV disease originates. Further, our mouse models of SLV disease will enable an understanding of the cellular and molecular progression of valve disease. Our specific aims are: 1) Determine the molecular networks that the BAF complex interfaces with to direct EMT and 2) Determine mechanisms of SLV disease progression in mice lacking endocardial- lineage Brg1. In pursuit of the first Aim, we will compare cellular and molecular pOFT defects seen in unpublished genetic models disrupting Brg1 and Wnt signaling. We will apply a transformative new TU-tagging technology to define dynamic, endocardial transcriptomes dependent on each pathway. Using new cell culture approaches, we will test biochemical interactions between BAF, Wnt effectors, and chromatin in EC cells. For the second Aim, we will use genetic lineage tracing to determine contributions of EMT-derived cells to distinct SLV regions, define interactions between SLV mesenchyme sub-populations, characterize misexpressed transcripts that may drive SLV disease progression, and describe a new mouse model of adult SLV disease of potential utility in preclinical trials. Our proposed research uses novel technological and paradigmatic approaches to pursue unresolved questions of SLV development and disease. These contributions will be significant as they will shed light on the human genetics of SLV disease and inform regenerative medicine approaches. Our newly identified transcripts associated with a BAV model may represent biomarkers for disease diagnostics or therapeutic targets to prevent congenitally abnormal valves from becoming diseased.