Global Predictions and Tests of Erythroid Regulation

红细胞调节的全球预测和测试

• 批准号: 7351852
• 负责人: ROSS C HARDISON
• 金额: $ 44.28万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7351852
• 关键词: Acute Erythroblastic Leukemia; Binding; Binding Sites; Bioinformatics; Biology; Cells; Chemicals; Chromosomes; Computer Analysis; Condition; DNA; DNA Sequence; DNA-Binding Proteins; Data; Development; Effectiveness; Elements; Enhancers; Erythroblasts; Erythroid; Foundations; Future; Gene Expression; Genes; Genetic; Genetic Recombination; Genome; Genomics; Globin; Green Fluorescent Proteins; Hematopoiesis; Hemoglobinopathies; Human; Human Genome; Laboratories; Lead; Location; Longitudinal Studies; Luciferases; Measures; Molecular; Mus; Nucleic Acid Regulatory Sequences; Polymerase Chain Reaction; Proteins; Regulation; Regulatory Element; Reporter; Reporter Genes; Research Personnel; Reverse Transcription; Score; Sequence Alignment; Structure; System; Test Result; Testing; Therapeutic; Tissues; Validation; Work; base; beta Globin; chromatin immunoprecipitation; cohort; experimental analysis; feeding; human GATA1 protein; improved; in vivo; insight; mammalian genome; new technology; northern hybridization; novel; programs; promoter; restoration; tool; transcription factor

DESCRIPTION (provided by applicant): New technologies to better define the structure and expression of mammalian genomes offer exciting opportunities for understanding the genetic control of tissue development. Recent studies in our (two separate) laboratories used cell-based, molecular and bioinformatic approaches to generate potentially important insights into the structure, function and expression of erythroid genes. Now we aim to combine our preliminary data and expertise to conduct a genome-wide search for erythroid regulatory modules, i.e. the DNA sequences and proteins needed for erythroid regulation. This project will also serve as a testing ground for new genome-wide computational predictions of regulatory elements. We propose to define cohorts of co-regulated genes in induced murine erythroleukemia (MEL) cells and in Gata-1-null erythroid precursors (G1E cells) induced to differentiate by restoration of GATA-1 function. We will search for likely regulatory regions in noncoding genomic sequences encompassing these genes by using programs that analyze whole-genome human-mouse alignments to predict functional and regulatory sequences, plus searches for clusters of erythroid transcription factor binding sites. Strong candidates for regulatory sequences will be tested in erythroid expression cassettes that will be integrated into a targeted location in MEL cell and G1E cell chromosomes. We will measure their effects on expression using reporters such as green fluorescent protein and luciferase. After analyzing these results for features of the sequences and alignments that best correlate with demonstrable enhancer activity, we will incorporate those features into our programs that seek to discriminate regulatory sequences from other sequences. Reiterative cycles of analysis and experimental validation will lead to more effective predictions of erythroid regulatory sequences. Our comprehensive analysis of cis regulatory elements should lead to improved understanding of how gene expression is controlled and coordinated during erythroid maturation, a topic of relevance to the biology of hematopoiesis and to therapeutic strategies for hemoglobinopathies. More broadly, the experimental and computational approaches generated by our work should provide valuable tools for analyzing the control of gene expression in other tissues during normal development and various pathological states.

描述（由申请人提供）： 更好地定义哺乳动物基因组的结构和表达的新技术为理解组织发育的遗传控制提供了令人兴奋的机会。我们（两个独立的）实验室最近的研究使用基于细胞的分子和生物信息学方法来对红细胞基因的结构、功能和表达产生潜在的重要见解。现在，我们的目标是结合我们的初步数据和专业知识，对红细胞调节模块进行全基因组搜索，即红细胞调节所需的DNA序列和蛋白质。该项目还将作为新的全基因组调控元件计算预测的试验场。我们建议定义诱导鼠红白血病 (MEL) 细胞和通过恢复 GATA-1 功能诱导分化的 Gata-1 缺失红系前体细胞 (G1E 细胞) 中的共同调控基因组。我们将通过使用分析全基因组人-小鼠比对的程序来搜索包含这些基因的非编码基因组序列中可能的调控区域，以预测功能和调控序列，并搜索红细胞转录因子结合位点簇。强有力的候选调控序列将在红系表达盒中进行测试，该表达盒将整合到 MEL 细胞和 G1E 细胞染色体的目标位置。我们将使用绿色荧光蛋白和荧光素酶等报告基因来测量它们对表达的影响。在分析这些结果以找出与可证明的增强子活性最相关的序列特征和比对后，我们将把这些特征合并到我们的程序中，以寻求区分调控序列和其他序列。重复的分析和实验验证循环将导致对红细胞调节序列的更有效的预测。我们对顺式调控元件的全面分析应该有助于更好地理解红细胞成熟过程中基因表达如何控制和协调，这是一个与造血生物学和血红蛋白病治疗策略相关的主题。更广泛地说，我们的工作产生的实验和计算方法应该为分析正常发育和各种病理状态期间其他组织中基因表达的控制提供有价值的工具。