Functions of Mammalian Histone Modifiers

描述（由申请人提供）：Gcn 5组蛋白乙酰转移酶（HAT）与从酵母到人类的生物体中的基因激活有关。虽然Gcn 5的结构和Gcn 5复合物（如STAGA/TFTC）的成分已经很好地定义，但Gcn 5在哺乳动物细胞中的功能仍然是未知的。我们以前报道过Gcn 5基因敲除小鼠在原肠胚形成后不久死亡。我们现在已经确定，Gcn 5空细胞含有端粒端到端融合。然而，Gcn 5 hat/hat胚胎，其在Gcn 5的催化位点中具有突变，不表现出端粒功能障碍表型，并且它们比Gcn 5无效胚胎发育得更远。我们的研究结果表明，GCN 5在端粒维持和小鼠发育中具有重要的功能，这些功能独立于其乙酰转移酶活性。我们提出的实验将建立在这些以前的发现，以定义这些功能。我们在Gcn 5空细胞中观察到的端粒功能障碍表型让人想起在保护端粒并将其与双链DNA断裂区分开的Shelterin复合物的组分突变后发生的端粒功能障碍表型。初步数据表明，Gcn 5是不需要的编码Shelterin组分蛋白质的基因的转录，但Gcn 5是这些蛋白质中的至少一个，TRF 1的正常水平所必需的。我们假设Gcn 5在不涉及乙酰转移酶活性的转录后或翻译后步骤影响TRF 1水平，并且TRF 1（以及可能的其他Shelterin组分）水平降低有助于我们在Gcn 5不存在的情况下观察到的端粒功能障碍表型。我们还假设Gcn 5可能通过影响STAGA/TFTC复合物内的去泛素化模块来影响TRF 1的稳定性。我们将测试这些假设，并通过三个具体目的进一步定义Gcn 5的功能：1）确定Gcn 5的缺失是否影响端粒Shelterin复合物组分的定位或稳定性。2)确定Gcn 5缺失是否影响STAGA/TFTC内去泛素化酶模块的缔合或活性，以及3）确定Gcn 5和Gcn 5依赖性组蛋白修饰的基因组位置。总的来说，我们的研究将为Gcn 5和STAGA/TFTC在哺乳动物细胞中的功能提供重要和新颖的见解。STAGA/TFTC组分中的突变与儿科癌症、神经退行性疾病和癌症生存预后不良有关。从长远来看，我们的研究可能会对人类健康产生重要影响。

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.