Genetic regulation of atrial gene expression in development and disease

摘要 对心房基因表达的精确调控是维持心房内稳态的关键，而失调或基因 影响心房基因表达的突变可导致房颤，这是一种严重的心律失常，影响 据估计，全球有3300万人。然而，我们对心房的认识还存在许多差距。 基因调控，包括小室选择性基因表达的基础机制。人类基因 研究表明，心脏转录因子基因TBX5附近的序列变异是相关的 有更大的房颤风险，Tbx5缺乏的小鼠会发生房颤。尽管最近的研究发现了一些 Tbx5调节的基因导致房颤易感性，以Tbx5为中心的转录网络是 没有完全探索过。阐明该网络及其对TBX5剂量的敏感性将揭示 房颤的发病机制，并可能为预防或治疗房颤提供新的方法。这次重启的首要目标是-- 研究方案是阐明心房基因调控网络及其在房颤中是如何被干扰的。这项建议 Pu实验室开发的新试剂和技术用于询问转录机制 体内，包括高度敏感和可重复性的心脏转录因子ChIP-Seq通过体内生物素- (生物芯片-序列)，大规模平行体内测量顺式调节元件(Cre)活性(AAV- MPRA分析)，以及镶嵌基因失活策略，以磨练基因inac的直接细胞自主效应- 激活。在目标1中，我们使用生物芯片-SEQ和AAV-MPRA来确定心房CRs，并对其序列fea-mpra进行分析。 它们的小室选择性活动所需的培养物。在目标2中，我们确定了TBX5缺乏对 其他转录因子和p300的占有率、Cres的活性以及心房基因的表达。我们使用这些数据 确定以TBX5为中心的心房基因调控网络，并确定该网络是如何被干扰的 Tbx5单倍体缺失或基因敲除。在目标3中，我们检验了我们的初步数据所提出的假设 Tbx5通过与TEAD1在功能和物理上的相互作用来调节心房基因。成功完成 这一建议将导致对房颤时心房基因调控及其扰动的新见解。

SUMMARY Precise regulation of atrial gene expression is crucial to maintain atrial homeostasis, and disorders or gene mutations that impact atrial gene expression can cause atrial fibrillation (AF), a serious arrhythmia that affects an estimated 33 million people worldwide. However, there remain many gaps in our understanding of atrial gene regulation, including the mechanisms that underlie chamber-selective gene expression. Human genetic studies have shown that sequence variants near the cardiac transcription factor gene TBX5 are associated with greater AF risk, and mice with Tbx5 deficiency develop AF. Although recent work has identified some TBX5-regulated genes that contribute to AF susceptibility, the atrial TBX5-centered transcriptional network is incompletely explored. Elucidation of this network and its sensitivity to TBX5 dose would reveal nodal points in AF pathogenesis and may suggest new approaches to prevent or treat AF. The overarching goal of this re- search proposal is to elucidate the atrial gene regulatory network and how it is perturbed in AF. The proposal buillds on novel reagents and techniques developed in the Pu lab to interrogate transcriptional mechanisms in vivo, including highly sensitive and reproducible cardiac transcription factor ChIP-seq through in vivo biotinyla- tion (bioChIP-seq), massively parallel in vivo measurement of cis-regulatory element (CRE) activity (AAV- MPRA assay), and mosaic gene inactivation strategies to hone in direct, cell autonomous effects of gene inac- tivation. In Aim 1, we use bioChIP-seq and AAV-MPRA to define atrial CREs and to dissect the sequence fea- tures required for their chamber selective activity. In Aim 2, we determine the effect of TBX5 deficiency on the occupancy of other TFs and p300, the activity of CREs, and the expression of atrial genes. We use these data to define the TBX5-centered atrial gene regulatory network, and to determine how this network is perturbed by TBX5 haploinsufficiency or knockout. In Aim 3, we test the hypothesis, suggested by our preliminary data, that TBX5 regulates atrial genes through functional and physical interaction with TEAD1. Successful completion of this proposal will lead to new insights into atrial gene regulation and its perturbation in AF.