Chromatin Regulation of Heart Valve Development

描述(申请人提供)：半月瓣(SLV)疾病，包括二尖瓣(BAV)，是非常常见的，但其发生和发育的起源却知之甚少。同样，目前尚不清楚胚胎瓣膜发育受阻如何进展为显性瓣膜疾病。我们的长期目标是了解基因调控如何驱动最终产生复杂、有模式的瓣膜的顺序发育过程，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基因组相互作用，这表明染色质调节因子是SLV发育网络的关键组成部分。一个重要的事件是心内膜向间充质转化(EMT)，它发生在瓣膜发育的早期，以填充心内膜垫(ECs)，包括为SLV贡献组织的近端流出道垫(POFT)。我们的目标是1)了解在EMT过程中染色质重塑如何与细胞信号整合，以及2)确定瓣膜发育中断进展为病变SLV的机制。我们的中心假设是，心内膜BRG1相关因子(BAF)染色质重塑复合体与Wnt信号效应分子相互作用，促进pOFT EMT。结果，当心内膜BRG1缺失时，OFT间质的一个亚型被耗尽。如果没有这些细胞，牙尖过度生长和融合会导致SLV增厚和图案错误，包括BAV。我们努力的基础是，定义EMT中染色质重塑的角色将有助于阐明SLV疾病的起源。此外，我们的SLV疾病的小鼠模型将使我们能够了解瓣膜疾病的细胞和分子进展。我们的具体目标是：1)确定BAF复合体与之连接的分子网络以指导EMT；2)确定在缺乏心内膜BRG1系的小鼠中SLV疾病进展的机制。在追求第一个目标的过程中，我们将比较在未发表的破坏BRG1和Wnt信号的遗传模型中看到的细胞和分子pOFT缺陷。我们将应用一种变革性的新TU标记技术来定义依赖于每条途径的动态心内膜转录本。使用新的细胞培养方法，我们将在EC细胞中测试BAF、WNT效应器和染色质之间的生化相互作用。对于第二个目标，我们将使用遗传谱系追踪来确定EMT来源的细胞对不同SLV区域的贡献，定义SLV间充质亚群之间的相互作用，表征可能导致SLV疾病进展的错误表达的转录本，并描述一种新的成年SLV疾病的小鼠模型，该模型在临床前试验中具有潜在的实用价值。我们的 拟议的研究使用新的技术和范例方法来探索SLV发展和疾病的悬而未决的问题。这些贡献将是重大的，因为它们将阐明SLV疾病的人类遗传学，并为再生医学方法提供信息。我们新发现的与BAV模型相关的转录本可能是疾病诊断的生物标记物，或者是防止先天性异常瓣膜疾病的治疗靶点。

DESCRIPTION (provided by applicant): 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.