Role of Linker Histone H1 Variants in Cardiac Phenotype in Health and Disease

描述（申请人提供）：成人心脏是一个动态器官；然而，心肌细胞在出生后不久就开始终末分化。随着时间的推移，成年心肌细胞暴露于多种病理性应激源，可诱导肥厚生长和功能障碍。疾病的进展导致各种适应性反应，包括胎儿蛋白的重新表达。尽管对控制肥厚的途径的了解有所进展，但对基因调控的修饰因子如何影响心脏的解剖重编程知之甚少。我假设组蛋白变异是控制患病和衰竭心脏中基因表达变化的重要调节因子。为了研究心肌疾病发生时染色质结合蛋白的变化，通过收缩小鼠横主动脉诱导心肌肥厚。我们实验室的蛋白质组学分析鉴定了来自细胞核的1048种蛋白质，包括54种组蛋白变体，并揭示了核小体化学计量学的明显改变，这些改变与器官水平上心脏病的表现相关。此外，对个体组蛋白H1变异（所谓的“连接组蛋白”，负责高阶染色质结构的形成）的分析显示，心肌肥厚期间表达的比率发生了明显变化：H1.0和H1.1变异增加，同时H1.2、H1.3、H1.4和H1.5亚型减少。我们假设组蛋白H1变异对染色质不同的物理区域有不同的调节。我们进一步推断，核心组蛋白（H2a, H2b， H3和H4）与连接组蛋白H1的比例可能是控制整体染色质包装的重要参数。为了验证这一假设，我提出了两个目标。首先，我将使用自上而下和自下而上的质谱分析确定H1亚型的心脏特异性化学计量学。我将检查核心和连接组蛋白变异，以揭示与心脏病表型表现相对应的化学计量学变化。其次，我将确定含有心脏H1亚型的核小体的基因组位置，并确定疾病发展过程中占据模式的变化。含有核小体的H1变异将通过免疫沉淀从细胞核中选择，定位将通过微阵列/DNA测序确定。这些研究将首次深入了解组蛋白H1家族成员在心脏肥厚中的作用，并将为对其体内作用的全基因组研究奠定基础。

DESCRIPTION (provided by applicant): The adult heart is a dynamic organ; however cardiomyocytes become terminally differentiated shortly after birth. Over time, adult cardiomyocytes are exposed to multiple pathological stressors that can induce hypertrophic growth and dysfunction. The progression of disease leads to a variety of adaptive responses including the re-expression of fetal proteins. Despite advances in the knowledge of pathways that control hypertrophy, little is known about how modifiers of gene regulation influence anatomical reprogramming of the heart. I hypothesize that histone variants are important regulators that govern changes in gene expression in the diseased and failing heart. To study the changes in chromatin-bound proteins that occur as cardiac disease develops, cardiac hypertrophy was induced in mice by constricting the transverse aorta. Our lab's proteomic analyses identified 1048 proteins from cardiac nuclei including 54 histone variants and revealed distinct alterations in nucleosome stoichiometry which correlate with the manifestation of heart disease at the organ level. Moreover, analysis of individual histone H1 variants-the so-called 'linker histone', responsible for the formation of higher order chromatin structures-revealed distinct alterations of the ratios expressed during cardiac hypertrophy: H1.0 and H1.1 variants increased concomitant with a decrease in the H1.2, H1.3, H1.4 and H1.5 isoforms. We hypothesize that histone H1 variants differentially regulate physically distinct regions of chromatin. We further reason that the ratio of core histones (H2a, H2b, H3 and H4) to linker histone H1 may be an important parameter controlling global chromatin packaging. I propose two aims to test this hypothesis. First, I will determine the cardiac-specific stoichiometry of H1 isoforms using top-down and bottom-up mass spectrometry analyses. I will examine core and linker histone variants to reveal changes in stoichiometry corresponding to the phenotypic presentation of heart disease. Second, I will identify the genomic location of nucleosomes containing cardiac H1 isoforms and determine changes in the occupancy pattern during the development of disease. The H1 variant containing nucleosomes will be selected from nuclei by immunoprecipitation and positioning will be determined by microarrays/DNA sequencing. These studies will provide the first insights into the role of the histone H1 family members in cardiac hypertrophy and will lay the groundwork for a genome-wide interrogation of their in vivo actions. PUBLIC HEALTH RELEVANCE: Heart disease is the leading cause of death in the USA and novel insights into the progression of complications such as heart failure are much needed. This application tests the hypothesis that a family of specific proteins called histone H1 control expression of genes in the normal heart and that alterations in this family underlie heart hypertrophy and failure. These studies will provide the first data on the histone H1 family in the heart including isoform-specificity and details on genomic targets-key insights for future therapeutic approaches.