ALTERATIONS OF CHROMATIN ORGANIZATION BY NON-DNA BINDING PROTEINS AND THEIR ROLES IN TRANSCRIPTIONAL REGULATION

真核生物的跨环激活或抑制被认为伴随着染色质结构的改变。近年来，可能影响核小体或高阶染色质结构的因素开始在包括酿酒酵母在内的几种生物中被揭示。在本研究中，我们重点研究了酿酒酵母的SIN4因子。我们以前的研究已经定义了两类启动子，一类是被sin4-突变激活的，另一类是不被sin4-突变激活的。我们分析了这两类启动子对in4突变的不同反应机制。我们的分析结果表明，sin4突变激活了核心启动子依赖的基础转录，而不是激活剂介导的转录。从这一点和其他观察，我们认为基础转录的激活是通过一种不同于激活剂介导的转录增强的机制发生的。在后续研究中，我们确认了…进一步证实Tup1和SSN6是酿酒酵母减数分裂的转录激活子IME1的负转录调控因子。已经证实，Tup1和SSN6被一种基因特异性的DNA结合蛋白招募，通过促进核小体结构的刚性组织来发挥各种基因的一般抑制作用。Rind是一种DNA结合蛋白，与IME1的上游结合，抑制IME的转录。然而，IME1启动子上Rme1结合位点的缺失并没有导致IME1表达的激活，表明Rme1不是一个DNA结合蛋白来招募Tup1-SSN6抑制物复合体。我们还研究了除Tup1和Sin4外，Sir2、SIR3和HHO1基因的干扰对酿酒酵母VI号染色体上所有基因转录的影响。结果表明，sir2和sir3干扰物对HSP12和HXK1的转录有促进作用。然而，这些基因的转录增强发生在没有邻近基因的情况下，这表明染色质介导的抑制是基因特有的事件。微球菌核酸酶对sir3干扰物中HSP12和HXK1启动子区域的可及性增加，表明除了众所周知的HM基因座和端粒区域外，VI号染色体的内部区域还存在异染色质样结构。较少

Transcirpional activation or repression in eukaryotes is believed to be accompanied by an alteration of chromatin structure. In recent years, factors which may affect nucleosome or higher-order chromatin structure begun to be unveiled in several organisms including yeast Saccharomyces cerevisiae. In this study, we focused on one of such factors, SIN4 of S.cerevisiae. Our previous study has defined two classes of promoters, those which are activated by the sin4-mutation and those which are not activated by the sin4-mutation. We analyzed the mechanisms of this differential response of the two classes of promoters to thesin4 mutation. Results of our analysis revealed that the sin4 mutation activates core promoter-dependent basal transcription but not activator-mediated transcription. From this and other observations, we suggest that activation of the basal transcription occurs through a mechanism different from activator-mediated transcriptional enhancement. In sebsequent study, we identi … More fied TUP1 and SSN6 as negative regulators of transcription of IME1, a transcriptional activator of meiosis in S.cerevisiae. It has been well established that the TUP1 and SSN6 is recruited by a gene-specific DNA-bining protein to act as a general repressor of various genes by facilitating the rigid organization of nucleosome structure. Rind is known to be a DNA-binding protein which binds to upstream of IME1 to repress the IME] transcription. However, deletion of the Rme1-binding site from IME1 promoter did not result in activation of the expression of IME1, indicating that Rme1 does not function as a DNA-binding protein to recruit the Tup1-Ssn6 repressor complex. We also examined the effect of disruption of SIR2, SIR3 and HHO1 in addition to TUP1 and Sin4, all of which are thought to repress transcription through altering chromatin structure, on transcription of all genes on chromosome VI of S.cerevisiae. Results showed that enhancement of transcription of the HSP12 and HXK1 was seen in the sir2 and sir3 disruptant. However, transcriptional enhancement of those genes ocurred without that of their adjacent genes, suggesting that chromatin-mediated repression is gene-specific event. Increased accessibility of Micrococcal nuclease to the promoter regions of HSP12 and HXK1 in the sir3 disruptant suggested that heterochromatin-like structure exists in the internal regions of the chromosome VI in addition to well-known HM loci and telomeric regions. Less