The role of histone H2Az in cardiac gene expression

描述(由申请人提供)：细胞的基因表达谱的变化，是其表型变化的基础，以响应广泛的生理或病理刺激。这一过程在从膜到核的多个层面上受到非常严格的调控。许多调节转录机制的上游信号通路和第二信使仍在进行大量研究。最终结果不仅包括转录因子的修饰，还包括组蛋白的重塑、翻译后改变和/或变异替换。虽然组蛋白的主要作用是构造性的，涉及染色质包装，但越来越明显的是，它也在调节基因转录方面发挥着交互作用。然而，在哺乳动物细胞中，尤其是在心肌细胞中，所涉及的机制仍然知之甚少。我们以前曾报道，组蛋白H_2A的一种特殊亚型，称为H_2Az，在心肌肥厚过程中上调。我们的初步结果表明，在新生小鼠中，这种组蛋白在所有器官中的表达水平都相对较高，但仅在正常的成年心脏和骨骼肌中表达水平下降到无法检测到的水平。H_2Az的功能是发育所必需的、非冗余的、高度保守的，其机制仍有待在哺乳动物细胞中进行研究。然而，在低等真核生物中，它与核小体重塑和转录激活有明确的关系。这项提议将研究它在心肌肥厚期间在心脏中的作用。根据我们的初步结果，我们假设，通过直接调控生长相关基因的子集的表达，H_2Az是肥大生长所必需的。这项资助的具体目的是：1.利用突变和RNA干扰技术，确定H_2Az在心肌肥厚中的作用和机制。2.利用染色质免疫沉淀(ChIP)和消减杂交技术鉴定受H_2Az调控的基因。3.利用酵母双杂交系统鉴定H_2Az调节/效应蛋白及其功能。4.鉴定在心脏过度表达H_2Az的转基因小鼠模型。

DESCRIPTION (provided by applicant): Changes in the gene expression profile of a cell, underlie changes in its phenotype, in response to a broad range of physiological or pathological stimuli. This process is very tightly regulated at multiple levels, from the membrane to the nucleus. Many of the upstream signaling pathways and second messengers that regulate the transcriptional machinery continue to be heavily investigated. The end result does not only include modification of transcription factors, but in addition, remodeling, post-translational alterations, and/or variant replacements of histones. While the main role of histones is architectural, involving chromatin packaging, it is becoming increasingly evident that it also plays an interactive role in regulating gene transcription. The mechanisms involved, though, remain poorly understood in mammalian cells in general or in cardiac myocytes in particular. We have previously reported that a specific isoform of histone H2A, termed H2Az, is upregulated during cardiac hypertrophy. Our preliminary results show that in the neonatal mouse this histone is expressed at relatively high levels in all organs but drop to undetectable amounts in the normal adult heart and skeletal muscle only. The functions of H2Az are essential for development, non-redundant, and highly conserved, the mechanisms of which remain to be investigated in mammalian cells. In lower eukaryotes though, it has been specifically implicated in nucleosomal remodeling and transcriptional activation. This proposal will investigate its role in the heart during cardiac hypertrophy. Built on our preliminary results, we hypothesize that H2Az is necessary for hypertrophic growth through directly regulating the expression of a subset of growth-related genes. The specific aims of this grant are: 1. To determine the role and mechanism of function of H2Az in cardiac hypertrophy, using mutagenesis and RNA interference. 2. To identify genes that are regulated by H2Az, using chromatin immunoprecipitation (ChIP) and subtractive hybridization. 3. To identify H2Az regulatory/effector proteins and their functions, using the yeast two-hybrid system. 4. Characterize a transgenic mouse model over-expressing H2Az in the heart.