Function of active chromatin domains in erythropoiesis

描述（由申请人提供）：红细胞的终末分化与广泛的异染色质化和基因抑制有关，反映在细胞核大小减小和染色质结构明显更紧密，红细胞在去核前成熟。然而，在这种压抑的环境中，一组精选基因的高水平表达——尤其是？-然后呢？-球蛋白-必须建立和维持。实现这一目标的机制代表了一个基本问题，对组织特异性基因调控研究和临床基因治疗都有影响，在临床基因治疗中，转基因表达必须在分化细胞中同样保持。大量的证据表明，共价组蛋白修饰（如乙酰化）在基因激活过程中的作用。在大多数情况下，组蛋白超乙酰化局限于启动子-近端区域，在那里它可能有助于染色质去致密化，组蛋白- dna相互作用的放松，以及转录因子相互作用的稳定。然而，在某些基因上，组蛋白超乙酰化延伸到更广泛的区域。在红细胞中，？-然后呢？-珠蛋白位点表现出这种超乙酰化的“结构域”，并且我们已经表征了其他几个具有它们的红细胞特异性基因位点。我们的研究结果以及对其他基因座的研究表明，这些结构域是其激活的关键特征，并且它们反映了高水平表达所需的远端增强元件的活性。然而，到目前为止，这些结构域形成的机制以及它们在终末分化过程中基因表达中的确切作用尚不清楚。我们拟对红细胞中p-珠蛋白和其他位点结构域形成所需的顺式作用DNA序列进行表征，并最终鉴定与这些序列结合并介导结构域形成的反式作用因子。通过这种方式，我们将建立可用于阐明超乙酰化结构域的形成和功能的系统。公共卫生声明：这项研究虽然本质上是高度基础的，但其目的是阐明终端细胞分化过程中基因表达的机制。因此，它与组织特异性基因表达被破坏的疾病（包括许多癌症）以及涉及在特定细胞类型中稳定的转基因表达的基因治疗方法有关。

DESCRIPTION (provided by applicant): Terminal differentiation of erythroid cells is associated with generalized heterochromatinization and gene repression, reflected in decreased nuclear size and visibly more compact chromatin structure as red cells mature prior to enucleation. In the midst of this repressive environment, however, high-level expression of a select group of genes - especially the ?- and ?-globins - must be established and maintained. The mechanisms by which this is accomplished represent a basic question with implications both for studies of tissue-specific gene regulation and for clinical gene therapies, in which transgene expression must similarly be maintained in differentiating cells. Abundant evidence has been presented to establish the role of covalent histone modifications, such as acetylation, in the process of gene activation. For the most part, histone hyperacetylation is confined to promoter-proximal regions, where it presumably contributes to chromatin decondensation, relaxation of histone-DNA interactions, and stabilization of transcription factor interactions. At certain genes, however, histone hyperacetylation extends across much broader regions. In erythroid cells, the ?- and ?-globin loci exhibit such "domains" of hyperacetylation, and we have characterized several additional erythroid-specific gene loci that have them as well. Our results, along with studies of other gene loci, suggest that such domains are a crucial feature of their activation, and that they reflect the activity of distal enhancer elements required for high-level expression. As yet, however, the mechanisms by which such domains are formed, and the precise role they play in gene expression during terminal differentiation, are unknown. We propose to characterize cis-acting DNA sequences required for domain formation at the p-globin and other loci in erythroid cells, and eventually to identify the trans-acting factors that bind to these sequences and mediate domain formation. In this way, we will establish systems that can be used to elucidate the formation and function of hyperacetylated domains. Public Health Statement: This research, while highly basic in nature, is designed to elucidate mechanisms that underlie gene expression during terminal cellular differentiation. As such, it has relevance to disorders in which tissue-specific gene expression is disrupted - which include a number of cancers - as well as gene therapy approaches involving stable transgene expression in specific cell types.