Abstract
Transcription factor Gfi1b is required for erythroid and megakaryocytic differentiation. Gfi1b is over-expressed in patients and cell lines of erythroid and megakaryocytic leukemias. This indicates that this gene must be tightly regulated to achieve normal erythropoiesis and megakaryopoiesis. The mechanism of it is largely unknown. We have searched for conserved noncoding sequences from multiple evolutionary diverse species (multispecies conserved sequences, MCSs) between Gfi1b neighboring genes and localized several elements containing multiple highly conserved erythroid specific transcription factor binding sites. These MCSs localize with erythroid specific DNasa1 hypersensitive sites. Chromatin immunoprecipitation (ChIP) assays demonstrated GATA1/SCL/E2A/Ldb1/LMO2 pentameric complex and NF-E2, together with Gfi1b, binding in human and mouse cell lines and primary cells representing different stages of erythropoiesis and megakaryopoiesis. Analysis of histone modifications and cofactors binding in relation with Gfi1b expression and protein level gives clues about how activating and repressive complexes attach to Gfi1b promoter and potential long distance regulatory elements maintaining an equilibrium that changes with gene downregulation.
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