Histone Gene Expression in Yeast

描述(申请人提供)：真核细胞核小体的主体由四个核心组蛋白组成：H_2A、H_2B、H_3和H_4。编码这些蛋白质的基因一直是近20年来遗传学实验的主要焦点，我们已经了解了大量关于它们在基因转录和沉默中的作用。然而，到目前为止，所有研究的真核生物也表达一个或多个组蛋白变体，这些蛋白的丰度较低，与主要的组蛋白家族之一有关，但已沿着不同的谱系进化。尽管这些变异对后生动物的生存能力通常是必不可少的，但人们对它们的功能、遗传相互作用或组装途径知之甚少。我们发现，在发芽酵母中，HTZ1基因编码的组蛋白变异体H2A.Z是正常基因转录和染色体分离所必需的。因此，组蛋白H_2A.Z在至少两种基本的染色体功能中起着关键作用。我们将解决三个关于H_2A.Z功能的基本问题：(1)H_2A.Z如何调节转录？通过与RNA聚合酶II突变的新的遗传相互作用，我们已经发现HTZ1是转录延伸所必需的，但涉及的机制和分子途径目前尚不清楚。我们将通过基因筛查新的htzl点突变和其延伸缺陷的基因外抑制物来解决这个问题。利用染色质免疫沉淀，我们将确定H_2A.Z依赖的伸长步骤及其功能后果。(2)H_2A.Z在染色体分离中起什么作用？我们发现HTZL是人工致死的，着丝粒和纺锤体的成分发生了突变。它导致染色体倍性增加的频率很高，物理上位于第三染色体的着丝粒。然而，我们不知道Htz1在着丝粒上如何发挥作用，或者它针对的是什么蛋白质。为了解决这些问题，我们将进一步研究htzl突变体的纺锤体/着丝粒功能。我们将使用芯片分析来鉴定动粒的Htzl依赖成分。我们将分离HTZL的新的IPL等位基因，并表征它们与其他组蛋白、纺锤体和动粒突变的相互作用。(3)Htzl是如何被招募到特定的染色体位置的？我们发现，Htz1并不是均匀地分布在整个染色质中，而是优先结合在特定的基因座上。此外，这些基因座包括一些基因的启动子，其他基因的开放阅读框架，以及着丝粒的着丝粒周围染色质。为了了解这些组装模式的规则和机制，我们将在特定的顺式和反式作用因子中设计突变，以测试Htz1招募的替代模型。

DESCRIPTION (provided by applicant): The bulk of eukaryotic nucleosomes are made up of four core histones: H2A, H2B, H3, and H4. The genes encoding these proteins have been the primary focus of almost two decades of genetic experiments and we have learned a great deal about their roles in gene transcription and silencing. However, all eukaryotes studied to date also express one or more histone variants, less abundant proteins that are related to one of the major histone families but have evolved along separate lineages. Despite the fact that these variants are generally essential for viability in metazoans, little is known about their functions, genetic interactions, or assembly pathways. We have discovered that the histone variant H2A.Z, encoded by the HTZ1 gene in budding yeast, is required for normal gene transcription and chromosome segregation. Thus, histone H2A.Z is a critical player in at least two basic chromosomal functions. We will address three fundamental questions regarding H2A.Z function: (1) How does H2A.Z regulate transcription? Through novel genetic interactions with mutations in RNA polymerase II, we have found that HTZ1 is necessary for transcription elongation, but the mechanisms and molecular pathways involved are currently unknown. We will address this question through genetic screens for new htzl point mutations and for extragenic suppressors of their elongation defects. Using chromatin immunoprecipitation, we will determine the H2A.Z-dependent steps in elongation and their functional consequences. (2) What is the role of H2A.Z in chromosome segregation? We have discovered that htzl is synthetic lethal with mutations in components of the centromere and spindle apparatus. It causes high frequencies of increased chromosome ploidy and is physically located at the centromere of chromosome III. However, we do not know how Htzl functions at the centromere or what proteins it targets. To address these questions, we will further characterize spindle/kinetochore function in htzl mutants. We will use ChIP assays to identify Htzl-dependent components of the kinetochore. We will isolated novel Ipl- alleles of htzl and characterize their interactions with other histone, spindle, and kineotchore mutations. (3) How is Htzl recruited to specific chromosomal locations? We have found that Htz1 is not uniformly distributed throughout the chromatin but is preferentially incorporated at specific loci. Furthermore, these loci include the promoter for some genes, the open reading frame for other genes, and pericentric chromatin at centromeres. To understand the rules and mechanisms for these assembly patterns, we will engineer mutations in specific cis- and trans-acting factors to test alternative models of Htz1 recruitment.