Functions of Mammalian Histone Modifiers

描述（由申请人提供）：Gcn5组蛋白乙酰转移酶（HAT）涉及从酵母到人类的生物体的基因激活。尽管Gcn5的结构和含有Gcn5复合物的组分（如STAGA/TFTC）已经明确，但Gcn5在哺乳动物细胞中的功能仍在很大程度上未知。我们之前报道过Gcn5敲除小鼠在原肠胚形成后很快死亡。我们现在已经确定Gcn5零细胞包含端粒端到端融合。然而，Gcn5催化位点突变的Gcn5hat/hat胚胎没有表现出端粒功能障碍表型，并且它们比Gcn5无基因胚胎发育得更远。我们的研究结果表明，Gcn5在端粒维持和小鼠发育中具有独立于乙酰转移酶活性的重要功能。我们提出的实验将以这些先前的发现为基础来定义这些功能。我们在Gcn5零细胞中观察到的端粒功能障碍表型与保护端粒并将其与双链DNA断裂区分开来的庇护蛋白复合物组分突变时发生的表型相似。初步数据表明，编码Shelterin成分蛋白的基因的转录不需要Gcn5，但这些蛋白中至少有一种TRF1的正常水平需要Gcn5。我们假设Gcn5在转录后或翻译后不涉及乙酰转移酶活性的步骤中影响TRF1水平，并且在Gcn5缺失的情况下，TRF1（可能还有其他Shelterin成分）水平的降低导致了端粒功能障碍表型。我们还假设Gcn5可能通过影响STAGA/TFTC复合物中的去泛素化模块来影响TRF1的稳定性。我们将通过三个具体目标来验证这些假设，并进一步定义Gcn5的功能：1)确定Gcn5的丢失是否会影响端粒Shelterin复合物组分的定位或稳定性。2)确定Gcn5缺失是否影响STAGA/TFTC中去泛素酶模块的关联或活性；3)确定Gcn5和Gcn5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Gcn5和STAGA/TFTC在哺乳动物细胞中的功能提供重要而新颖的见解。STAGA/TFTC成分突变与儿童癌症、神经退行性疾病和癌症生存不良预后有关。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.