Regulation of Expression of MHC Class I Genes

MHC I类基因转录的调控是由基础启动子和上游调控元件的协调活动介导的，以达到组织特异性水平的表达，并根据细胞外信号进一步动态调节。实验室最近的研究重点是确定基础启动子的关键序列组织，转录启动子转录所需的转录起始复合物，以及调节基础启动子活性的上游元件。I类基础启动子由三个元件组成：一个TATAA盒、一个引发子（Inr）和一个新的S-box元件。TATAA和Inr元素的相对用法在不同的细胞类型中有所不同，但在任何测试的细胞类型中都不是绝对需要的元素。两个位点，TATAA盒的3‘和Inr盒的5’，似乎确实是基础启动子活性所必需的。这些元素在体内的功能正在一系列转基因小鼠构建中进行检测。基础I类启动子的转录-无论是通过TATAA还是Inr元件-取决于功能性TAFII250， TAFII250是TFIID复合物的一个组成部分，由TATA结合蛋白（TBP）和一系列TBP相关因子（TAFs）组成，这些因子共同参与转录起始前复合物的组装。TAFII250是一组基因转录所必需的，包括那些参与细胞周期和MHC i类的基因。tsBN462细胞系含有TAFII250的温度敏感突变，并在限制性温度下差异地阻止许多基因的转录，但不是所有基因。我们发现MHC I类基因的核心启动子需要TAFII250。这种依赖性可以通过选择上游调控元件来克服，但不能通过核心启动子元件来克服。因此，共激活子CIITA将基础启动子从TAFII250的要求中解救出来，而引入规范的TATAA盒子则没有。类似地，SV40核心启动子需要TAFII250，但72bp增强子的存在克服了这一要求。此外，当SV40 72bp增强子位于核心I类启动子的上游时，使其独立于TAFII250。这些数据表明，转录起始复合物的组装是动态的，可以被特定的转录因子调节。为了支持这一假设，我们发现细胞类型特异性增强体有助于调节I类转录。特别是干扰素诱导的MHC II类表达调节剂CIITA，是一种有效的I类表达共激活剂。CIITA的共激活需要干扰素反应元件和CRE；相比之下，CBP、PCAF和p300都不影响I类转录。已知TAFII250具有乙酰转移酶（AT）活性。我们已经证明AT活性对于MHC I类基因的转录是必要的：抑制AT活性会抑制转录。为了确定可能调节TAFII250 AT活性的潜在细胞因子，我们筛选了一个酵母双杂交文库，其中TAFII250片段（848-1279 aa）跨越AT结构域（848-1120）和RAP74结合结构域（1120-1279）。分离出TFIID成分TAFII55，发现它与rap74结合域相互作用。令人惊讶的是，TAFII55结合TAFII250抑制其AT活性。此外，在体外转录实验中加入重组TAFII55可抑制tafii250依赖性转录。因此，TAFII55通过调节其AT活性来调节TAFII250的功能。

Regulation of MHC class I gene transcription is mediated by the coordinate activities of the basal promoter and upstream regulatory elements, to achieve tissue-specific levels of expression which are further dynamically modulated in response to extracellular signals. The recent research focus of the laboratory has been to define the critical sequence organization of the basal promoter, the transcription initiation complexes that are required for transcription of the basal promoter, and upstream elements that modulate basal promoter activity. The class I basal promoter consists of three elements: a TATAA box, an initiator (Inr), and a novel S-box element. The relative usage of TATAA and Inr elements varies among different cell types, but neither element is absolutely required in any cell type tested. Two sites, 3' of the TATAA box and 5' of the Inr, do appear to be necessary for basal promoter activity. The function of these elements in vivo is being examined in a series of transgenic mouse constructs. Transcription of the basal class I promoter -whether through the TATAA or Inr elements - depends on a functional TAFII250, a component of the TFIID complex which consists of the TATA binding protein (TBP) associated with a series of TBP-associated factors (TAFs), that together participate in the assembly of the transcription preinitiation complex. TAFII250 is required for the transcription of a subset of genes, including those invovled in cell cycle and MHC class I. The tsBN462 cell line contains a temperature sensitive mutation of TAFII250, and differentially arrests transcription of many, but not all genes, at the restrictive temperature. We have found that the core promoter of MHC class I gene requires TAFII250. This dependence can be overcome by select upstream regulatory elements but not by core promoter elements. Thus, the coactivator CIITA rescues the basal promoter from the TAFII250 requirement, whereas introduction of a canonical TATAA box does not. Similarly, the SV40 core promoter requires TAFII250, but the presence of the 72bp enhancer overcomes this requirement. Further, the SV40 72 bp enhancer when placed upstream of the core class I promoter renders it independents of TAFII250. These data suggest that the assembly of transcription initiation complexes is dynamic and can be modulated by specific transcription factors. In support of this hypothesis, we find that cell type specific enhanceosomes contribute to the regulation of class I transcription. In particular, the interferon inducible regulator of MHC class II expression, CIITA, is a potent co-activator of class I expression. Coactivation by CIITA requires both the interferon response element and the CRE; in contrast, neither CBP, PCAF nor p300 affect class I transcription.TAFII250 is known to have acetyl transferase (AT) activity. We have shown AT activity is necessary for transcription of MHC class I genes: inhibition of the AT activity represses transcription. To identify potential cellular factors that might regulate the AT activity of TAFII250, a yeast two-hybrid library was screened with a TAFII250 segment (848-1279 aa) that spanned both the AT domain (848-1120) and the RAP74 binding domain (1120-1279). The TFIID component, TAFII55, was isolated and found to interact with the RAP74-binding domain. Surprisingly, TAFII55 binding to TAFII250 inhibits its AT activity. Furthermore, addition of recombinant TAFII55 to in vitro transcription assays inhibits TAFII250-dependent transcription. Thus, TAFII55 regulates TAFII250 function by modulating its AT activity.