Silent Chromatin: Mechanisms of Transcriptional Repression

染色质在真核生物基因表达调控中起着重要作用。 在第一种近似中，它以两种形式存在：一种是未折叠的10 nm样细丝，由串珠状的核小体阵列组成，另一种是紧凑的30 nm样纤维。 前者是转录活性常染色质的特征，而后者是沉默染色质（异染色质）的特征，其以区域而非启动子特异性方式抑制转录。 沉默染色质如何抑制基因表达还不清楚，尽管传统观点认为它是通过空间阻碍转录的正调控因子进入底层DNA来实现的。 在芽殖酵母（Saccharomyces cerevisiae）中，沉默染色质由沉默信息调节蛋白（SIR）形成。 先前的工作已经非常出乎意料地揭示，沉默的染色质允许基因特异性激活因子和一般转录因子（GTF）两者的结合。 事实上，RNA聚合酶II（Pol II）的起始能力形式存在于超阻遏启动子中。 相比之下，下游因子的占据，包括加帽新生mRNA的酶和促进Pol II延伸的因子，实际上被废除，导致Pol II在基因的5'末端停滞。 这种差异的可及性是如何建立的尚不清楚，但对于理解转录沉默的机制至关重要。 本研究将探讨沉默染色质调控基因转录的两个重要机制：1。 Sir蛋白如何抑制基础基因转录？ 这个问题将通过以下方式解决：（i）检验沉默染色质中差异因子可及性反映转录周期中因子募集到基因的时间顺序的假设;（ii）通过突变和功能分析表征关键下游因子在触发沉默中的作用;（iii）评估因子去乙酰化在触发沉默中的作用。 2. 沉默染色质中的基因激活是如何发生的？这将通过以下方式进行研究：（i）表征沉默染色质中基因激活期间核小体的动态变化;（ii）确定Sir蛋白在不同沉默程度的转基因中的扩散程度;（iii）比较常染色质和异染色质基因的共激活因子需求。 这个项目的好处，超出了预期的科学发现，包括两个全职博士的培训。研究生和两名本科生。

Chromatin plays a critical role in regulating the expression of eukaryotic genes. To a first approximation, it exists in two forms: as an unfolded, 10-nm-like filament, comprised of arrays of nucleosomes in a beads-on-a-string configuration, or as a compacted 30-nm-like fiber. The former is characteristic of transcriptionally active euchromatin, while the latter is characteristic of silent chromatin (heterochromatin), which represses transcription in a regional rather than promoter-specific fashion. How silent chromatin inhibits gene expression is not well understood, although the traditional view is that it does so by sterically hindering access of positive regulators of transcription to the underlying DNA. In the budding yeast, Saccharomyces cerevisiae, silent chromatin is formed by Silent Information Regulator (SIR) proteins. Previous work has revealed, very unexpectedly, that silent chromatin is permissive to the binding of both gene-specific activators and general transcription factors (GTFs). Indeed, an initiation-competent form of RNA polymerase II (Pol II) is present at hyperrepressed promoters. By contrast, occupancy of downstream factors, including enzymes that cap the nascent mRNA and factors that facilitate Pol II elongation, is virtually abolished, resulting in Pol II stalling at the gene's 5' end. How this differential accessibility is established is unknown, but is central to understanding the mechanism of transcriptional silencing. In this project, two important issues concerning the fundamental mechanisms by which silent chromatin regulates gene transcription will be investigated: 1. How do Sir proteins silence basal gene transcription? This question will be addressed by (i) Testing the hypothesis that differential factor accessibility in silent chromatin reflects the temporal order of factor recruitment to a gene during the transcription cycle; (ii) Characterizing the role of key downstream factors in triggering silencing through mutational and functional analyses; and (iii) Evaluating the role of factor deacetylation in triggering silencing. 2. How does gene activation take place in silent chromatin? This will be investigated by (i) Characterizing the dynamic alterations of nucleosomes during gene activation in silent chromatin; (ii) Determining the extent of spread of Sir proteins at transgenes subject to different degrees of silencing; and (iii) Comparing the coactivator requirements of euchromatic and heterochromatic genes. Benefits of this project, beyond the anticipated scientific discoveries, include the training of two full-time Ph.D. graduate students and two undergraduates.