Heterochromatin-Mediated Repression in Yeast

大卫格罗斯异染色质是在所有真核（有核）细胞中发现的染色质的浓缩形式。 它负责哺乳动物雌性中的X染色体失活，果蝇中的位置效应杂色，以及芽殖酵母中的细胞类型确定。 虽然异染色质长期以来与基因失活有关，但它抑制基因转录的确切机制尚不清楚。 为了研究这一点，一个强大的模型系统已经开发出来。 在该系统中，一个动态调节的、应激诱导的酵母基因（HSP 82）已被置于称为SIR（沉默信息调节器）的异染色质诱导蛋白复合物的控制之下。 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剂、热休克因子（HSF）、基本转录机制（包括RNA聚合酶）的组分以及异染色质结合蛋白Sir 2 p和Sir 3 p。 其次，将研究HSP 82沉默是否发生在群体中的每个细胞中的问题。 沉默是稳定地从亲本遗传到子细胞，还是异染色质介导的抑制是多样化的（在种群的某些细胞中看到，但在其他细胞中看不到），就像果蝇的情况一样？ 第三，什么是HSF的合作者，当它被压力激活时，允许它超越SIR建立的抑制性异染色质结构？ 基因敲除方法将被用来回答这个问题。 最后，由Ssn 6-Tup 1蛋白复合物介导的第二个全局阻遏系统的机制是什么？ 酵母菌株将被遗传工程化，使得HSP 82基因启动子被Ssn 6-Tup 1蛋白复合物靶向。 然后将进行与上述那些平行的表达、结构和遗传分析，从而允许两种阻遏系统之间的直接比较。 考虑到转录机制在进化上的保守程度，这些研究有可能揭示异染色质不仅在酵母中，而且在其他真核生物（包括人类）中起作用的方式。

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.