Functional proteomics in differentiating erythroid cells

描述（由申请人提供）：人？-珠蛋白基因座由五个基因组成，这些基因以发育阶段和红系特异性方式表达，并由位于基因远上游的基因座控制区（LCR）调节。基因突变？-珠蛋白基因座在人群中相当常见，并与轻度至重度贫血相关。这些突变大多位于成人？珠蛋白基因或影响成体基因表达的调节序列内。其中最突出的是？球蛋白相关疾病是镰状细胞性贫血（SCA）和？地中海贫血目前对这些疾病的治疗是不令人满意的，许多努力正在投入开发新形式的疗法。我们预计，进一步了解珠蛋白基因在发育过程中是如何被激活的，将为血红蛋白病的治疗创造新的机会。例如，最近的证据表明，珠蛋白基因座的调节元件，包括LCR和基因启动子，在基因表达的激活过程中非常接近。如果能理解基因是如何与LCR紧密相连的，那么将来就有可能以这样一种方式调节这些相互作用，即？珠蛋白基因比成年人更受青睐？定型红系细胞珠蛋白基因。我们建议进行一个全面的分析的组成和功能的蛋白质复合物含有转录因子参与珠蛋白基因调控。在这些研究中，我们将利用新的方法，包括在分化的红系细胞和转基因小鼠中表达生物素标记的蛋白质，研究转录因子和相关辅因子在调节染色质可及性，珠蛋白基因座构象变化和转录复合物的募集中的作用。将使用链霉亲和素包被的磁珠从细胞中分离生物素化的转录因子，并进行质谱分析以鉴定相关蛋白质，进行染色质免疫沉淀（ChIP）以鉴定相关DNA序列，以及用于鉴定在其中LCR和启动子非常接近的球蛋白基因座构型的背景下起作用的蛋白质的组合ChIP和染色体构象捕获（3C）测定。 公共卫生相关性：本研究的目的是全面了解转录因子在？珠蛋白基因调控的新方法。生物素标记的转录因子将在红系细胞中表达，使用链霉亲和素包被的磁珠分离，并进行各种生物化学和分子测定，以鉴定相互作用的蛋白质和它们结合的DNA区域，以及确定蛋白质是否是形成“环状”染色质构型的一部分和所需的“环状”染色质构型，所述染色质构型将？珠蛋白基因座紧密靠近。

DESCRIPTION (provided by applicant): The human ?-globin gene locus consists of five genes that are expressed in a developmental stage- and erythroid-specific manner and regulated by a locus control region (LCR) located far upstream of the genes. Mutations in the ?-globin gene locus are quite frequent in the human population and associated with mild to severe anemias. Most of these mutations are located within the coding region of the adult ?-globin gene or within regulatory sequences affecting expression of the adult gene. Among the most prominent of ?-globin associated diseases are sickle cell anemia (SCA) and ?- thalassemias. Current treatments for these diseases are unsatisfactory and much effort is being invested in developing novel forms of therapies. We anticipate that further knowledge of how the globin genes are activated during development will create new opportunities for the treatment of hemoglobinopathies. For example, recent evidence suggests that the regulatory elements of the globin locus, including the LCR and the gene promoters, come in close proximity during activation of gene expression. If it is understood how genes are brought into close proximity to the LCR it may be possible in the future to modulate these interactions in such a way that associations of the ?-globin genes are favored over that of the adult ?-globin gene in definitive erythroid cells. We propose to perform a comprehensive analysis of the composition and function of protein complexes containing transcription factors involved in globin gene regulation. In these studies we will utilize novel methodology, including expression of biotin tagged proteins in differentiating erythroid cells and in transgenic mice, to examine the role of transcription factors and associated co-factors in the regulation of chromatin accessibility, globin locus conformational changes, and recruitment of transcription complexes. Biotinylated transcription factors will be isolated from cells using streptavidin coated magnetic beads and subjected to mass-spectrometry for identifying associated proteins, to chromatin immunoprecipitation (ChIP) for identifying associated DNA sequences, and to a combined ChIP and chromosome conformation capture (3C) assay for identifying proteins that function in the context of a globin locus configuration in which the LCR and promoters are in close proximity. PUBLIC HEALTH RELEVANCE: The goal of this study is to provide a comprehensive understanding of the role of transcription factors involved in ?-globin gene regulation using novel methodology. Biotin-tagged transcription factors will be expressed in erythroid cells, isolated using streptavidin coated magnetic beads, and subjected to various biochemical and molecular assays to identify interacting proteins and DNA regions they bind to as well as to determine whether the proteins are part of and required for the formation of "looped" chromatin configurations that bring regulatory elements of the ?-globin locus in close proximity.