Dynamics of cardiac nuclei in heart disease

项目总结/摘要 表观遗传过程与心脏发育的控制和心脏病的发生和发展有关。 疾病的进展。特别是心力衰竭已被证明涉及染色质重塑的作用 酶和进行组蛋白修饰和基因表达的时间重组。科学 本申请的前提是理解染色质结构-功能的原理，包括 特定的分子靶点，如连接组蛋白H1家族，是重新设计健康转录组的关键 在疾病的背景下。基于初步数据暗示其在成纤维细胞表型中的作用，我们将 机械地研究连接组蛋白H1.0在染色质组织中的作用，使用增益和损失- 在细胞和体内的功能方法。我们假设基因组的精确结构方向 由细胞类型特异性分子过程支撑，疾病是由全球 基因组结构，从而使病理基因表达。为了验证这个假设，我们将执行 有史以来第一次在不同的细胞类型-成纤维细胞和肌细胞-上重建基因组拓扑结构， 器官.我们将精确测量男性和女性成纤维细胞核染色质结构的差异， 小鼠，研究性别差异在基因组组织中的作用，作为一个潜在的未探索的贡献者， 性别之间基因表达和心血管表型的差异。我们将使用DCas CLOuD9- 基于染色质环重组工具，以明确测试染色质相互作用在 转录和成纤维细胞活化。基于初步数据暗示连接组蛋白的特权作用 H1.0在心脏成纤维细胞中的表达，我们将研究H1.0控制成纤维细胞基因的分子机制， 表达和基因座特异性染色质可及性。我们将使用增益和损失的功能的方法， 分离成纤维细胞，以检查组蛋白H1.0调节核凝聚的作用，成纤维细胞凝胶 收缩、增殖和肌成纤维细胞蛋白表达。我们将最终确定在体内的作用， 组蛋白H1.0在基础条件下和压力超负荷条件下心脏成纤维细胞表型中的表达，β 异丙肾上腺素引起的肾上腺素能应激或缺血性损伤。这种方法将使我们能够测试组蛋白H1.0的作用 调节我们在基因组结构研究中发现的特定染色质邻域的组装， 以检测这些邻近区域是否以组蛋白H1.0依赖的方式在体内重组 伴随着疾病的发展。拟议的实验将提供机制的见解，如何 组蛋白H1.0有助于体内成纤维细胞活化和心脏功能，揭示分子细节 支持心脏对损伤的反应的表观遗传控制。

PROJECT SUMMARY/ABSTRACT Epigenetic processes have been implicated in control of cardiac development and in the incidence and progression of disease. Heart failure in particular has been shown to involve the actions of chromatin remodeling enzymes and to proceed by temporal reorganization of histone modifications and gene expression. The scientific premise of this application is that understanding the principles of chromatin structure-function, including the roles of specific molecular targets such as the linker histone H1 family, is key to re-engineering healthy transcriptomes in the setting of disease. Based on preliminary data implicating its role in fibroblast phenotype, we will mechanistically investigate the role of linker histone H1.0 in chromatin organization, using gain- and loss-of- function approaches in cells and in vivo. We hypothesize that precise structural orientation of the genome is underpinned by cell type-specific molecular processes and that disease results from reorganization of global genome architecture, thereby enabling pathologic gene expression. To test this hypothesis, we will perform the first ever reconstruction of genome topology on distinct cell types—fibroblasts and myocytes—from the same organ. We will precisely measure differences in chromatin architecture in fibroblast nuclei from male and female mice, examining the role of sex differences in genome organization as a potential unexplored contributor to differences in gene expression and cardiovascular phenotype between the sexes. We will use dCas CLOuD9- based chromatin loop reorganization tools to definitively test the causative role of chromatin interactions in transcription and fibroblast activation. Based on preliminary data implicating a privileged role for linker histone H1.0 in cardiac fibroblasts, we will examine the molecular mechanisms whereby H1.0 controls fibroblast gene expression and locus specific chromatin accessibility. We will use gain- and loss-of-function approaches in isolated fibroblasts to examine the role of histone H1.0 to regulate nuclear condensation, fibroblast gel contraction, proliferation and myofibroblast protein expression. We will conclusively determine the in vivo role of histone H1.0 in cardiac fibroblast phenotype under basal conditions and in the setting of pressure overload, beta adrenergic stress by isoproterenol, or ischemic injury. This approach will allow us to test the role of histone H1.0 to regulate assembly of specific chromatin neighborhoods identified in our genome structure studies as well as to examine whether these neighborhoods are reorganized in a histone H1.0-dependent manner in vivo concomitant with development of disease. The proposed experiments will provide mechanistic insights into how histone H1.0 contributes to fibroblast activation and cardiac function in vivo, revealing molecular details underpinning epigenetic control of the heart’s response to injury.