Generation And Analysis Of Target Genes Libraries Of The Erythropoietic Transcription Factor Gata-1.

Abstract GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes. In erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes. In order to elucidate further the role of GATA-1 we applied an in vivo tagging methodology for the specific, quantitative biotinylation of this factor in mammalian cells (de Boer et al., 2003). We applied this method for identification of novel target genes of GATA-1 by performing pull-downs of crosslinked chromatin using streptavidin. We have also performed chromatin immunoprecipitations, where crosslinked chromatin was immunoprecipitated with antibodies against GATA-1, thus enriching for sequences bound in vivo by this factor in the immunoprecipitated DNA. Libraries of in vivo bound DNA targets were generated and a number of clones were sequenced. In order to facilitate the bioinformatics analysis of these libraries we generated TF Target Mapper (Transcription Factors Target Mapper). This is a BLAST search tool that allows rapid extraction of annotated information on genes around each hit and combines sequence cleaning/filtering, pattern searching and comparisons of the output list of genes or gene ontology IDs with user implemented lists. This tool was successfully applied to analyze sequences bound in vivo by the transcription factor GATA-1 and efficiently extracted information on genes around ChIPed sequences, thus identifying known and potentially novel GATA-1 gene targets. Using TF Target Mapper, 95 sequences were processed and annotated information on 372 genes 50kb upstream and downstream of each hit was extracted in 27 minutes. Among these genes, known targets of GATA-1, such as α-globin and ζ-globin, were readily identified by comparing to a list of known GATA-1 targets. This work is anticipated to provide a genome wide map of GATA-1 target genes in vivo. The identification of target genes and elucidation of their functions in hematopoiesis will allow the construction of complex transcriptional pathways that control lineage commitment and differentiation decisions.