Function of active chromatin domains in erythropoiesis

描述(申请人提供)：红系细胞的终末分化与广泛的异染色质和基因抑制有关，反映在核尺寸减小和明显更紧密的染色质结构，因为红细胞在去核之前成熟。然而，在这种压抑的环境中，必须建立和维持一组选定的基因的高水平表达，特别是？和？珠蛋白。实现这一点的机制代表了一个基本问题，对组织特异性基因调控的研究和临床基因治疗都有意义，在临床基因治疗中，转基因表达同样必须在分化细胞中保持。大量的证据表明，组蛋白的共价组蛋白修饰，如乙酰化，在基因激活过程中起着重要作用。在大多数情况下，组蛋白的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的解缩，组蛋白-DNA相互作用的松弛，以及转录因子相互作用的稳定。然而，在某些基因上，组蛋白的超乙酰化延伸到更广泛的区域。在红系细胞中，？-和？-珠蛋白基因座表现出这种高乙酰化的“区域”，我们已经鉴定了其他几个红系特有的基因座也具有这些区域。我们的结果以及对其他基因座的研究表明，这些结构域是它们激活的关键特征，并且它们反映了高水平表达所需的远端增强子元件的活性。然而，到目前为止，这些结构域的形成机制以及它们在末端分化过程中在基因表达中所起的确切作用尚不清楚。我们建议鉴定红系细胞中p-珠蛋白和其他基因座的结构域形成所需的顺式作用DNA序列，并最终确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，但旨在阐明在细胞终末分化过程中基因表达的基础机制。因此，它与组织特异性基因表达中断的疾病有关--包括一些癌症--以及涉及特定细胞类型中稳定转基因表达的基因治疗方法。

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.