Functions of Mammalian Histone Modifiers

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DESCRIPTION (provided by applicant): The Gcn5 histone acetyltransferase (HAT) is implicated in gene activation in organisms from yeast to humans. Although the structure of Gcn5 and the components of Gcn5-containing complexes (e.g. STAGA/TFTC) are well defined, the functions of Gcn5 in mammalian cells are still largely unknown. We previously reported that Gcn5 knock out mice die soon after gastrulation. We have now determined that Gcn5 null cells contain telomere end-to- end fusions. However, Gcn5hat/hat embryos, which bear mutations in the catalytic site of Gcn5, do not exhibit a telomere dysfunction phenotype, and they develop much further than do Gcn5 null embryos. Our findings indicate that Gcn5 has important functions in telomere maintenance and mouse development that are independent of its acetyltransferase activity. Our proposed experiments will build upon these previous findings to define these functions. The telomere dysfunction phenotype we observe in Gcn5 null cells is reminiscent of that which occurs upon mutation of components of the Shelterin complex, which protects telomeres and distinguishes them from double-stranded DNA breaks. Preliminary data indicate that Gcn5 is not needed for transcription of the genes encoding Shelterin component proteins, but Gcn5 is required for normal levels of at least one of these proteins, TRF1. We hypothesize that Gcn5 affects TRF1 levels at a post-transciptional or post-translational step that does not involve acetyltransferase activity, and that lowered levels of TRF1 (and possibly other Shelterin components) contribute to the telomere dysfunction phenotype we observe in the absence of Gcn5. We also hypothesize that Gcn5 may affect TRF1 stability through effects on a deubiquitinating module within the STAGA/TFTC complex. We will test these hypotheses and further define the functions of Gcn5 through three Specific Aims: 1) To determine whether loss of Gcn5 affects localization or stability of components of the telomeric Shelterin complex. 2) To determine whether Gcn5 loss affects association or activity of a deubiquitinase module within STAGA/TFTC and 3) To define the genomic locations of Gcn5 and Gcn5-dependent histone modifications. Collectively, our studies will provide important and novel insights to the functions of Gcn5 and STAGA/TFTC in mammalian cells. Mutations in STAGA/TFTC components are linked to pediatric cancers, neurodegenerative disease, and poor prognosis for cancer survival. In the long term, then, our studies are likely to have important ramifications for human health.