Functional proteomics in differentiating erythroid cells

描述（由申请人提供）：人？-globin基因座由5个基因组成，这些基因以发育阶段和红细胞特异性的方式表达，并由位于基因上游的基因座控制区（locus control region， LCR）调控。基因突变？-珠蛋白基因位点在人群中相当常见，与轻度至重度贫血有关。这些突变大多位于成人的编码区。-珠蛋白基因或在影响成人基因表达的调控序列内。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并不令人满意，人们正在投入大量精力开发新的治疗方法。我们预计，进一步了解球蛋白基因在发育过程中是如何被激活的，将为治疗血红蛋白病创造新的机会。例如，最近的证据表明，珠蛋白位点的调控元件，包括LCR和基因启动子，在基因表达的激活过程中非常接近。如果了解了基因是如何接近LCR的，那么将来就有可能以这种方式调节这些相互作用-珠蛋白基因比成人的更受青睐？-珠蛋白基因在确定的红细胞。我们建议对含有参与珠蛋白基因调控的转录因子的蛋白质复合物的组成和功能进行全面的分析。在这些研究中，我们将利用新的方法，包括生物素标记蛋白在分化红细胞和转基因小鼠中的表达，来研究转录因子和相关辅助因子在染色质可及性、珠蛋白位点构象变化和转录复合物募集的调节中的作用。生物素化的转录因子将使用链亲和素包被磁珠从细胞中分离出来，并进行质谱鉴定相关蛋白，染色质免疫沉淀（ChIP）鉴定相关DNA序列，结合ChIP和染色体构象捕获（3C）测定，鉴定在LCR和启动子非常接近的珠蛋白位点结构中起作用的蛋白质。

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.