Regulation of insulator function and globin gene expression by USF and associated

说明(申请人提供)：红血球携带和交换氧气的主要功能依赖于血红蛋白，一种由两个α和两个β-珠蛋白链和相关的铁结合的血红素基团组成的异四聚体。珠蛋白基因突变是最常见的遗传性疾病之一，在人类群体中会导致轻度或严重的贫血。目前对严重贫血的治疗在很大程度上并不令人满意，预计了解珠蛋白基因是如何调控的将有助于开发新的治疗方法。红系特异性表达的珠蛋白基因需要位于基因近端或远端的顺式调控DNA元件。类珠蛋白基因受一个基因座控制区(LCR)的调控，该基因座控制区由几个DNase I超敏(HS)位点组成，位于基因的上游。HS2可能是LCR中最强大的调控元件。它由几个与造血细胞和普遍表达的转录因子结合的位点组成。其中一个位点是E-box，它与螺旋-环-螺旋蛋白USF相互作用。USF还与珠蛋白基因启动子中的E-box元件相互作用，先前的工作表明，USF是RNA聚合酶II(Pol II)高效募集到LCR元件HS2和珠蛋白基因启动子所必需的。此外，我们还发现USF介导了鸡2-珠蛋白绝缘体HS4的边界活性，在红系细胞中，HS4在珠蛋白基因上保持着可接近的染色质构象。我们的初步数据表明，USF1与含有两个组蛋白甲基转移酶PRMT1和hSET1的大的共激活复合体相互作用。我们假设USF招募组蛋白修饰酶来建立和/或维持边界元件和珠蛋白基因座调控元件的开放染色质结构，这反过来又控制红系特异性和发育阶段特异性珠蛋白的表达。我们将测试USF和相关的组蛋白修饰酶在建立和维持染色质屏障功能和组织特异性珠蛋白基因转录调控方面的功能。最后，我们将研究红系细胞分化过程中USF的稳定性是如何被调节的。我们对珠蛋白基因座表观遗传学改变的研究有望为珠蛋白基因的转录控制提供新的见解，并可能为贫血的分子治疗带来新的策略。此外，解决USF在珠蛋白基因调控中的作用可能有助于阐明增强子启动子相互作用的机制。公共卫生相关性：项目说明：珠蛋白是血红蛋白的重要组成部分，在红细胞的氧气运输和交换中发挥着关键作用，该基因的遗传缺陷与轻度至重度贫血有关。在这项建议中，我们将研究核蛋白USF1/2调控珠蛋白基因表达和珠蛋白基因座染色质屏障功能的表观遗传学机制。这些研究将为USF在调节发育阶段特异性珠蛋白基因表达中的功能提供新的见解，并将为贫血的分子治疗提供新的策略。

DESCRIPTION (provided by applicant): The main function of red blood cells, which carry and exchange oxygen, depends on hemoglobin, a heterotetramer composed of two a and two ¿-globin chains and associated iron-binding heme groups. Mutations of globin genes are among the most common inherited diseases and cause mild or severe anemia in the human population. Current treatments of severe anemia are largely unsatisfactory and it is anticipated that knowledge of how the globin genes are regulated will aide in the development of novel therapies. Erythroid-specific expression of the globin genes requires cis-regulatory DNA elements located in gene proximal or distal regions. The ¿-like globin genes are regulated by a locus control region (LCR), which is composed of several DNase I hypersensitive (HS) sites and located far upstream of the genes. HS2 is perhaps the most powerful regulatory element in the LCR. It consists of several binding sites for hematopoietic and ubiquitously expressed transcription factors. One of these sites is an E-box that interacts with the helix-loop-helix protein USF. USF also interacts with E-box elements in the ¿-globin gene promoter and previous work has shown that USF is required for efficient recruitment of RNA polymerase II (Pol II) to LCR element HS2 and to the ¿-globin gene promoter. In addition, we have shown that USF mediates the boundary activity of the chicken 2-globin insulator HS4, which maintains an accessible chromatin conformation over the globin genes in erythroid cells. Our preliminary data demonstrate that USF1 interacts with large co-activator complexes containing two histone methyltransferases PRMT1 and hSET1. We hypothesize that USF recruits histone modifying enzymes to establish and/or maintain an open chromatin structure at boundary elements and at regulatory elements in the ¿-globin locus, which in turn controls erythroid-specific and developmental stage-specific globin expression. We will test the function of USF and associated histone modifying enzymes in establishing and maintaining chromatin barrier function and tissue specific transcriptional regulation of the ¿-globin locus. Finally, we will investigate how the stability of USF is regulated during differentiation of erythroid cells. Our studies on epigenetic alterations in the ¿-globin gene locus are anticipated to provide new insight into the transcriptional control of globin genes and may lead to novel strategies for the molecular therapy of anemia. Furthermore, addressing the role of USF in ¿-globin gene regulation may shed light on the mechanisms involved in enhancer promoter interactions. PUBLIC HEALTH RELEVANCE: Project Narrative: ¿-globin, an important component of hemoglobin, plays a critical function in the transport and exchange of oxygen in red blood cells in which genetic defects of this gene have been implicated in mild to severe anemia. In this proposal, we will investigate the epigenetic mechanisms by which nuclear proteins USF1/2 regulate globin gene expression and chromatin barrier function in the globin loci. The studies will provide a novel insight into USF function in the regulation of developmental stage-specific globin gene expression and will lead to novel strategies for the molecular therapy of anemia.