Regulation of Expression of MHC Class I Genes

主要组织相容性复合体(MHC)I类基因的转录受组织特异性(基础)和激素/细胞因子(激活)机制的调节。虽然启动子-近端调控元件已经被广泛地表征，但核心启动子、下游元件和染色质结构在介导调节中的作用在很大程度上还不清楚。MHC I类基因的基础转录和激活转录以不同的核心启动子区域为靶点，形成不同的转录起始复合体，并在启动子内的不同位置启动。基础和激活的转录途径招募不同的转录因子复合体到核心启动子元件，并靶向不同的转录起始点。基础转录完全依赖于一般转录因子TAF1，而激活的转录起始不依赖于TAF1。为了进一步研究I类基因表达的调控，我们开展了体内核心启动子元件的鉴定和新的下游启动子元件的鉴定工作。令人惊讶的是，在核心启动子区域引入突变并没有显著影响基因在体内的表达，这表明转录不需要单一的核心启动子元件。在翻译起始的32bp内，我们已经确定了另外三个相互作用的元件，它们在体内和体外都能实现对上游核心启动子的正向和负向调控。我们提出核心启动子的转录启动是一个动态过程，核心启动子的作用机制因细胞环境的不同而不同。虽然染色质结构和重塑在许多诱导基因的调控中起着关键作用，但这种水平的调控在I类基因表达中的作用要小得多。因此，基于芯片分析和不同组织中I类基因周围的核小体定位，我们发现启动子是结构性的表达状态，与转录速度无关。在与表达水平相关的组织中观察到精确的核小体定位的微小变化。我们假设染色质结构在调节组成性表达基因的表达方面的作用不同于可诱导或组织限制性基因。除了发生在启动子周围的调控事件外，调控还通过下游边界元件介导，这是体内持续基因表达所必需的。具体地说，我们已经确定并表征了I类基因基因间隔区3中的一个边界元件。利用转基因小鼠和稳定转基因的细胞系，我们证明了3个片段起到了屏障元件的作用，保护MHC I类基因免受沉默。相应地，3个序列的截短导致基因沉默，核小体密度增加，组蛋白H3K9乙酰化和H3K4甲基化减少。此外，我们还发现组蛋白修饰酶p300和PCAF与3个边界元件相关。因此，动态的和组织特异性的变化发生在开放的染色质结构的背景下，但取决于功能边界元件。

Transcription of major histocompatibility complex (MHC) class I genes is regulated by both tissue-specific (basal) and hormone/cytokine (activated) mechanisms. Although promoter-proximal regulatory elements have been characterized extensively, the roles of the core promoter, downstream elements and chromatin structure in mediating regulation have been largely undefined. Basal and activated transcriptions of an MHC class I gene target distinct core promoter domains, nucleate distinct transcription initiation complexes and initiate at distinct sites within the promoter. Basal and activated transcription pathways recruit distinct transcription factor complexes to the core promoter elements and target distinct transcription initiation sites. Basal transcription is completely dependent upon the general transcription factor TAF1 whereas activated transcription initiates is TAF1 independent. To further characterize regulation of class I gene expression, we have undertaken to characterize core promoter elements in vivo and to identify novel downstream promoter elements. Surprisingly, introduction of mutations within the core promoter region do not markedly affect gene expression in vivo, indicating that no single core promoter element is necessary for transcription. Within 32 bp of translation initiation we have identified three additional, interacting elements that act in concert to achieve both positive and negative regulation of the upstream core promoter, both in vivo and in vitro. We have proposed that transcription initiation at the core promoter is a dynamic process in which the mechanisms of core promoter function differ depending on the cellular environment. While chromatin structure and remodeling play pivotal roles in the regulation of many inducible genes, this level of regulation has a much smaller role in class I expression. Thus, based on both ChIP analysis and nucleosomal mapping around the class I gene in various tissues, we find that the promoter is constitutively poised for expression, independent of the rate of transcription. Small changes in precise nucleosomal positioning are observed among the tissues that do correlate with level of expression. We hypothesize that the role of chromatin structure in regulating expression of constitutively expressed genes is distinct from that of inducible or tissue-restricted genes. In addition to the regulatory events occurring around the promoter, regulation is also mediated through a downstream boundary element which is necessary for sustained gene expression in vivo. Specifically, we have identified and characterized a boundary element within the intergenic region 3 of the class I gene. Using transgenic mice and stably transfected cell lines, we demonstrate that a 3 segment functions as a barrier element, protecting the MHC class I gene from silencing. Accordingly, truncation of the 3 sequences results in gene silencing, increased nucleosomes density and decreased histone H3K9 acetylation and H3K4 methylation across the gene. In addition, we show that histone modifying enzymes p300 and pCAF are associated with the 3 boundary element. Thus, dynamic and tissue-specific changes occur within the context of an open chromatin structure but depend upon a functional boundary element.