Global Predictions and Tests of Erythroid Regulation

红细胞调节的全球预测和测试

基本信息

批准号：
7185079
负责人：
ROSS C HARDISON
金额：
$ 44.09万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
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/7185079
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

项目摘要

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了解组织发育的遗传控制提供了令人兴奋的机会。在我们（两个独立的）实验室中的最新研究使用了基于细胞的，分子和生物信息学方法来产生对红细胞基因的结构，功能和表达的潜在重要见解。现在，我们旨在将我们的初步数据和专业知识结合起来，以进行整个基因组调节模块，即红细胞调节所需的DNA序列和蛋白质。该项目还将成为调节元素全基因组计算预测的新计算预测的测试基础。我们建议定义诱导的鼠红血球血症（MEL）细胞以及通过恢复GATA-1功能诱导的分化的GATA-1-无效红细胞前体（G1E细胞）中的共同调节基因的队列。我们将通过使用分析整个基因组人小鼠比对来预测功能和调节序列的程序，在非编码基因组序列中寻找可能的调节区域，并搜索搜索红细胞转录因子的结合位点的簇。强大的调节序列候选者将在红细胞表达盒中测试，这些盒将整合到MEL细胞和G1E细胞染色体中的靶向位置。我们将使用绿色荧光蛋白和荧光素酶等记者来衡量它们对表达的影响。在分析了与可证明的增强剂活性最有关系的序列和对齐的特征的结果之后，我们将将这些特征纳入我们的程序中，以试图将调节序列与其他序列区分开。分析和实验验证的重复循环将导致红斑调节序列的更有效预测。我们对CIS调节元件的全面分析应提高人们对基因表达如何控制和协调在红系成熟期间的理解，这是与造血性生物学相关的话题，以及对血红蛋白病的治疗策略。从更广泛的角度来看，我们的工作产生的实验和计算方法应提供有价值的工具，以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。