Molecular Biology of Human Erythrocyte Alpha Spectrin

描述(申请人提供)：这项建议的长期目标是阐明红细胞膜骨架的关键成分--1-血影蛋白正常和异常表达的分子机制。血影蛋白的异常与遗传性溶血性贫血有关，有时是严重的。这个建议的第一个目的是识别和表征顺式序列、反式因子和表观遗传状态，包括染色质结构，跨越调节其在红系和非红系细胞中表达的？-Spectrin基因座。这些研究提出了一种假设，即血影蛋白和其他红细胞膜蛋白基因中的共同调控信号控制着它们的组织特异性表达。整合RNA转录组成、基因组结构、RNA聚合酶II结合、转录因子和调控蛋白结合以及组蛋白结构将提供红细胞膜基因结构、功能和调控的详细知识，并使我们能够识别控制红系细胞表达的共同调控信号。这一目标的研究将高通量基因组技术与基因表达的功能研究相结合。可利用的技术包括染色质免疫沉淀实验、随后的阵列杂交(CHIP-CHIP)或全基因组测序(CHIP-SEQ)、高通量DNase I超敏位点定位和基因表达的功能研究。从这些研究中获得的结果将有助于研究1-血影蛋白在红细胞生成、膜生物发生和遗传性红细胞疾病中的作用。这项建议的第二个目的是鉴定在血影蛋白连锁遗传性溶血性贫血患者中干扰血影蛋白基因调控和/或表达的突变，并表征这些突变对血影蛋白基因结构、功能和表达的影响。这些研究表明，β-血影蛋白的缺陷发生在功能重要的区域，它们的阐明将为正常和突变红细胞中β-血影蛋白基因的结构、功能和调控提供重要信息。将对扩增的患者基因组DNA进行核苷酸序列分析，以确定遗传性溶血性贫血病例中与β-血影蛋白定性和定量缺陷相关的遗传缺陷。在基因调控的功能研究中，将对已知的和新发现的？-Spectrin基因中的顺式调控元件进行询问和表征。总之，这些研究的结果将提供关于红系和非红系细胞中血影蛋白的结构、功能和调节的重要信息，并对红细胞血影蛋白连锁疾病的发病机制提供更多的见解。 与公共卫生相关：由于红细胞膜或内层的异常，许多人患有贫血或低血球计数。有些人需要手术或输血来治疗贫血。这个应用程序研究导致贫血的红细胞衬里的异常是什么、为什么以及如何引起的。

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the protein 1-spectrin, a critical component of the erythrocyte membrane skeleton. Abnormalities of ?-spectrin are associated with inherited hemolytic anemia, which is sometimes severe. The first aim of this proposal is to identify and characterize the cis-sequences, trans-factors, and epigenetic state, including chromatin architecture, across the ?-spectrin gene locus that regulate its expression in erythroid and nonerythroid cells. These studies address the hypothesis that common regulatory signatures in ?-spectrin and other erythrocyte membrane protein genes control their tissue-specific expression. Integration of mRNA transcript composition, genomic organization, RNA polymerase II binding, transcription factor and regulatory protein binding, and histone architecture will provide detailed knowledge of erythrocyte membrane gene structure, function, and regulation and allow us to identify a common regulatory signature that controls expression in erythroid cells. The studies in this aim combine high throughput genomic technologies with functional studies of gene expression. Techniques to be utilized include chromatin immunoprecipitation experiments followed by array hybridization (ChIP-chip) or whole genome sequencing (ChIP-seq), high throughput DNase I hypersensitive site mapping, and functional studies of gene expression. Results obtained from these studies will allow study of the role of 1-spectrin in erythropoiesis, membrane biogenesis, and inherited erythrocyte disorders. The second aim of this proposal is the identification of mutations that perturb ?- spectrin spectrin gene regulation and/or expression in patients with spectrin-linked inherited hemolytic anemias and characterization of the effect of these mutations on ?-spectrin gene structure, function, and expression. These studies address the hypothesis that defects of ?-spectrin occur in regions of functional importance and their elucidation will provide important information about the structure, function, and regulation of the ?-spectrin gene in normal and mutant erythrocytes. Nucleotide sequence analysis of amplified patient genomic DNA will be performed to identify genetic defects in cases of inherited hemolytic anemia associated with qualitative and quantitative defects of ?-spectrin. Previously known and newly identified cis-regulatory elements in the ?- spectrin gene will be interrogated and characterized in functional studies of gene regulation. Together, results from these studies will provide important information on the structure, function, and regulation of spectrin in erythroid and nonerythroid cells and shed additional insight into the pathogenesis of spectrin-linked disorders of the erythrocyte. PUBLIC HEALTH RELEVANCE: Many people suffer from anemia, or low blood count, due to abnormalities in the membranes, or lining, of their red blood cells. Some people require surgery or blood transfusions to treat the anemia. This application studies the what, why, and how of abnormalities of the lining of the red blood cell that cause the anemia.