Functions of Mammalian Histone Modifiers

描述(由申请人提供)：Gcn5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Gcn5的结构和含有Gcn5的复合体的组成(如STAGA/TFTC)已经很好地确定了，但Gcn5在哺乳动物细胞中的功能仍然很大程度上是未知的。我们之前曾报道过Gcn5基因敲除的小鼠在原肠后不久死亡。我们现在已经确定Gcn5缺失细胞包含端粒端到端融合。然而，带有Gcn5催化位点突变的Gcn5hat/HAT胚胎并不表现出端粒功能障碍的表型，而且它们比Gcn5缺失胚胎发育得更远。我们的发现表明，Gcn5在端粒维持和小鼠发育中具有重要的功能，这种功能不依赖于其乙酰转移酶的活性。我们提议的实验将基于这些先前的发现来定义这些功能。我们在Gcn5缺失细胞中观察到的端粒功能障碍表型使人联想到保护素复合体成分突变时发生的端粒功能障碍，这种复合体保护端粒并将其与双链DNA断裂区分开来。初步数据表明，Gcn5不是编码Shelterin组分蛋白的基因转录所必需的，但至少其中一种蛋白质TRF1的正常水平需要Gcn5。我们假设Gcn5在转录后或翻译后影响TRF1水平，不涉及乙酰转移酶活性，并且TRF1水平降低(可能还有其他保护素成分)导致我们在没有Gcn5的情况下观察到的端粒功能障碍表型。我们还假设Gcn5可能通过影响STAGA/TFTC复合体中的去泛素化模块来影响TRF1的稳定性。我们将验证这些假说，并通过三个具体目标进一步定义Gcn5的功能：1)确定Gcn5的缺失是否影响端粒保护素复合体的定位或稳定性。2)确定Gcn5缺失是否影响STAGA/TFTC中脱泛素酶模块的结合或活性；3)确定Gcn5和依赖于Gcn5的组蛋白修饰的基因组位置。总之，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和新颖的见解。STAGA/TFTC组分的突变与儿童癌症、神经退行性疾病和癌症存活率的预后不良有关。因此，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.