Heterochromatin-Mediated Repression in Yeast

[00:9 . 98]大卫·格罗斯异染色质是一种浓缩形式的染色质，存在于所有真核（有核）细胞中。它负责哺乳动物雌性的x染色体失活，果蝇的位置效应变异以及出芽酵母的细胞类型决定。虽然异染色质长期以来一直与基因失活有关，但它抑制基因转录的确切机制尚不清楚。为了研究这个问题，我们开发了一个强大的模型系统。在这个系统中，一个动态调节的应激诱导酵母基因（HSP82）被置于异染色质诱导蛋白复合物SIR （Silent Information Regulator）的控制之下。该模型系统将用于解决以下问题。首先，哪些蛋白质与sir抑制基因的启动子DNA结合？使用一种强大的新技术，染色质免疫沉淀，阳性和阴性调节蛋白的存在将在活细胞中进行检查。这些包括序列特异性激活因子、热休克因子（HSF）、基本转录机制的组成部分（包括RNA聚合酶）以及异染色质结合蛋白Sir2p和Sir3p。其次，将研究HSP82沉默是否发生在人群中的每个细胞中。沉默是稳定地从亲代遗传到子代细胞，还是异染色质介导的抑制是杂色的（在种群的某些细胞中可见，但在其他细胞中没有），就像果蝇的情况一样？第三，HSF的合作者是什么，当它被压力激活时，允许它覆盖由SIR建立的抑制性异色结构？基因敲除方法将被用来回答这个问题。最后，由Ssn6-Tup1蛋白复合物介导的第二种全球抑制系统的机制是什么？一株酵母菌将经过基因工程改造，使HSP82基因启动子被Ssn6-Tup1蛋白复合物靶向。然后将进行上述平行表达、结构和遗传分析，从而对两种抑制系统进行直接比较。鉴于转录机制在进化上保守的程度，这些研究有可能揭示异染色质不仅在酵母中起作用，而且在包括人类在内的其他真核生物中也起作用。

0091898 David GrossHeterochromatin is a condensed form of chromatin found within all eukaryotic (nucleated) cells. It is responsible for X-chromosome inactivation in mammalian females, position-effect variegation in the fruit fly, and cell-type determination in budding yeast. While heterochromatin has long been associated with genetic inactivation, the precise mechanism by which it represses gene transcription is unclear. To investigate this, a powerful model system has been developed. In this system, a dynamically regulated, stress-inducible yeast gene (HSP82) has been placed under control of a heterochromatin-inducing protein complex termed SIR (Silent Information Regulator). This model system will be used to address the following questions. First, which proteins bind to the promoter DNA of the SIR-repressed gene? Using a powerful new technique, chromatin immunoprecipitation, the presence of both positive and negative regulatory proteins will be examined in living cells. These include the sequence-specific activator, heat shock factor (HSF), components of the basic transcriptional machinery (including RNA polymerase), and the heterochromatin-binding proteins Sir2p and Sir3p. Second, the question of whether silencing of HSP82 occurs in each cell in the population will be investigated. Is silencing stably inherited from parent to daughter cell, or is heterochromatin-mediated repression variegated (seen in some cells of a population but not in others), as is the case in fruit fly? Third, what are the collaborators of HSF which permit it, when activated by stress, to override the repressive heterochromatic structure established by SIR? A gene knock-out approach will be taken to answer this question. Finally, what is the mechanism of a second global repression system, mediated by the Ssn6-Tup1 protein complex? A yeast strain will be genetically engineered so that the HSP82 gene promoter is targeted by the Ssn6-Tup1 protein complex. Parallel expression, structural, and genetic analyses to those described above will then be carried out, allowing a direct comparison between the two repression systems. Given the degree to which transcriptional mechanisms are evolutionarily conserved, these studies have the potential to unravel the means by which heterochromatin works not only in yeast but also in other eukaryotes, including human.