CHROMATIN STRUCTURE IN REGULATION OF MAMMALIAN GENE EXPRESSION

我们研究增强子如何激活染色质中的转录 真核细胞在发育过程中的环境， 分化 珠蛋白基因提供了一个丰富的系统来研究 这个问题 基因座控制区（LCR），包括四个 红细胞特异性DNA酶I超敏位点，调节表达 的BETA-珠蛋白基因的染色质结构的解压缩， 整个基因座，并激活珠蛋白基因的转录 在发展过程中。 我们用微型染色体系统 看看在染色质中的一个模型ε-珠蛋白基因的结构， 转录活性或非活性状态。 没有 激活元件（HS 2或muLCR）， 珠蛋白在转录上是无活性的， 而不是ε-珠蛋白基因 然而，当muLCR或HS 2是 包括，ε-珠蛋白转录的激活发生， 位于近端启动子上方的核小体丢失，与 转录因子可以排除一个典型的 来自DNA的核小体。 当ε-珠蛋白转录活跃时， 事实上，所有的微型染色体分子都被DNA酶I在两个位点切割， 启动子和HS 2位点支持增强子的循环模型- 启动子相互作用，这些调控元件 相互影响。 珠蛋白启动子和HS位点含有 限制性激活蛋白的结合位点， 介导HS位点的形成，并被认为是直接相互作用或 间接影响增强子-启动子相互作用。 为了剖析 HS位点形成并与启动子通讯，我们突变了基序 对于这些DNA结合基序单独和成组。 转录 激活和启动子重塑，以及HS 2的形成 超敏结构本身，依赖于NF-E2的存在， HS 2中的结合基序。 其他的突变有更微妙的影响 这与基因组中转录因子的组合作用一致， activation. 我们继续探索LCR的作用机制 和体内染色质结构的调节作用， 珠蛋白基因的表达。

We study how enhancers activate transcription in the chromatin environment of eukaryotic cells during development and differentiation. Globin genes provide a rich system to investigate this question. The locus control region (LCR), encompassing four erythroid-specific DNase I hypersensitive sites, regulates expression of the BETA-globin genes by decondensing the chromatin structure of the entire locus, and activating transcription of the globin genes sequentially during development. We used a minichromosome system to look at the structure of a model epsilon-globin gene in chromatin in either a transcriptionally active or inactive state. Without an activation element (HS2 or muLCR) in the minichromosome, epsilon- globin is transcriptionally inactive and nucleosomes are positioned over the epsilon-globin gene. However, when the muLCR or HS2 is included, activation of epsilon-globin transcription occurs and the nucleosome positioned over the proximal promoter is lost, consistent with the idea that transcription factors can exclude a canonical nucleosome from DNA. When epsilon-globin transcription was active, virtually all minichromosome molecules were cleaved by DNase I at both the promoter and the HS2 site supporting a looping model for enhancer- promoter interaction in which these regulatory elements physically interact with one another. Globin promoters and HS sites contain binding sites for a restricted group of activator proteins which mediate HS site formation and are thought to interact directly or indirectly to effect enhancer-promoter interaction. To dissect how the HS sites form and communicate with promoters, we mutated motifs for these DNA binding motifs singly and in groups. Transcriptional activation and promoter remodeling, as well as formation of the HS2 hypersensitive structure itself, depended on the presence of the NF-E2 binding motif in HS2. Other mutations had more subtle effects consistent with a combinatorial role for transcription factors in gene activation. We continue to explore the mechanism of action of the LCR and the regulatory role in vivo of chromatin structure in the expression of globin genes.