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.
