CHROMATIN STRUCTURE IN REGULATION OF MAMMALIAN GENE EXPRESSION

我们研究增强剂如何激活染色质中的转录。 真核细胞发育和分化的环境。 珠蛋白基因为研究这个问题提供了一个丰富的系统。这个 包含四个红系特异性DNA酶的基因座控制区(LCR) I超敏部位，通过调节e-珠蛋白基因的表达 解聚整个基因座的染色质结构，并激活 珠蛋白基因在发育过程中的顺序转录。我们 使用微型染色体系统观察模型的结构 E-珠蛋白基因在染色质中的转录活性或 非活动状态。中没有激活元素(HS2或：lcr) 微染色体，e-珠蛋白转录不活跃，核小体 位于e-珠蛋白基因之上。但是，当：lcr或hs2 被包括在内，e-珠蛋白转录的激活发生，并且 位于近端启动子上方的核小体丢失，一致 认为转录因子可以排除一个典型的 来自DNA的核小体。当e-珠蛋白转录活跃时，虚拟 所有的微染色体分子都被DNase I在 启动子和支持环模型的HS2位点 增强子-启动子相互作用中这些调控元件 身体上相互影响。珠蛋白启动子和HS站点 包含一组受限制的激活蛋白的结合位点 调节HS站点的形成，并被认为直接或 间接影响增强子-启动子相互作用。要仔细分析 HS站点形成启动子并与启动子通信，我们突变了 这些DNA结合基序可以单独使用，也可以组合使用。转录的 HS2的激活和启动子重塑以及形成 超敏结构本身，依赖于核因子-E2的存在 HS2中的结合基序。其他突变有更微妙的影响 与转录因子在基因中的组合作用一致 激活。我们继续探讨LCR的作用机制 以及染色质结构在体内表达的调节作用 珠蛋白基因。

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 e-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 e-globin gene in chromatin in either a transcriptionally active or inactive state. Without an activation element (HS2 or :LCR) in the minichromosome, e-globin is transcriptionally inactive and nucleosomes are positioned over the, e-globin gene. However, when the: LCR or HS2 is included, activation of e-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 e-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.