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.
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