Purification and characterization of GATA-1 related transcription factor complexes and target genes by in vivo biotinylation tagging

The overall aim of this project is to understand the development and differentiation of erythroid cells on the basis of key transcription factor (TF) functions in hematopoiesis. Specific TFs serve to execute lineage decision, commitment and differentiation programs in hematopoiesis by regulating the expression of target genes. The ultimate goal of hematopoiesis is the continuous replenishment of the many different hematopoietic cell types in a constantly ongoing process. Genetic evidence indicates that the functions of key TFs such as TAL-1, GATA-1 and EKLF represent essential early-to-late developmental decisions in hematopoiesis (reviewed in ref. 1). Loss of GATA-1 in mice results in arrest at the pro-erythroblast stage and apoptosis2 while GATA-1 overexpression results in a block at the final stage of differentiation due to an absence of cell cycle arrest3. These data show a requirement for GATA-1 for differentiation past the pro-erythroblast stage as well as a role for GATA-1 protein levels in cell cycle regulation and terminal erythroid cell differentiation. GATA-1 interacts with TAL-1, the latter fulfilling an early requirement in the development of the hematopoietic system since lack of TAL-1 in mice leads to a complete absence of blood4. TAL-1 also interacts with the widely expressed factor Ldb15, which is the mammalian homolog of the Drosophila Chip gene thought to mediate long-range interactions between distant regulatory elements and target promoters6. Lack of EKLF in mice results in lethal anemia at the fetal liver stage due to a deficiency in b globin synthesis and failure in the production of normal red blood cells7-9. Binding sites for both GATA-1 and EKLF appear clustered together at many sites in the b globin locus (e.g. ref. 10), as well as in many other erythroid genes, thus suggesting that the two factors act in concert in regulating erythroid-specific transcription. The evidence outlined above is indicative of a network of overlapping and distinct transcription factor functions in erythropoiesis. However, despite a wealth of information regarding these factors, it is still not clear what role which particular complex plays in gene regulation (i.e. chromatin remodeling/modification and/or transcriptional activation/repression). It is evident that these factors act as multiprotein complexes, but only some interactions have been characterized (with little functional evidence). For example, GATA-1 forms several different complexes with potentially different subnuclear localization and function(s) (section 4b and ref. 11) but the role of these complexes is unknown. Beginning with GATA-1, we propose to gain insight into the function of these factors in hematopoiesis by taking a combined biochemical, cellular and genetic approach to characterize the protein complexes formed by GATA-1 and interacting proteins and to identify the genes targeted by these complexes in hematopoiesis. This aim translates into the following key objectives:i. the isolation and characterization of GATA-1 related transcription factor protein complexes from erythroid and/or precursor cellsii. the identification of erythroid gene targets for GATA-1 and interacting factors, e.g. TAL-1, EKLFiii. the functional characterization of newly identified protein partners and gene targets of hematopoietic transcription factors, primarily by genetic means (i.e.knockouts, RNAi).