Transcriptional Regulation of a Distinct GATA-1 Isoform during Selection of the Mast and Erythroid Lineages.

Current models of hematopoiesis suggest that in early, pluripotent progenitor cells, lineage-specific transcription factors are expressed at low levels. During differentiation, subsets of these transcription factors become dominantly expressed in a lineage-restricted fashion. Understanding how transcription factors are expressed in distinct cell-types is central to defining the regulatory events that occur during lineage selection.

GATA-1 is an essential transcriptional regulator for the erythroid and megakaryocyte lineages, while it is absent in neutrophils and monocytes. PU.1, on the other hand, is a critical transcription factor for neutrophils and monocytes, but it is not abundantly expressed in erythroid cells. Although these two factors have been shown to be antagonistic in monocytic and erythroid cells, both GATA-1 and PU.1 are required for the normal development of the mast lineage (Migliaccio et al., 2003, Walsh et al., 2002). Here we show that mast cells express a unique mRNA isoform of GATA-1 that is distinct from the major erythroid/megakaryocyte isoform. It is related, but not identical to the Ib transcript that has been described as a minor expressed form in erythroid cells (Tsai et al., 1991) and as a major expressed form in RNA isolated from CFU-GM primary myeloid cultures (Seshasayee et al., 2000). This GATA-1 mast cell isoform (GATA-1_mast) differs from the erythroid/megakaryocyte isoform by a unique, untranslated first exon that is alternatively spliced onto the downstream coding exons. In mast cells, GATA-1_mast is expressed from a promoter separate from that utilized in megakaryocytic and erythroid cells. Comparative analysis of genomic sequence of the GATA-1 locus in this region reveals modules of extensive phylogenetic conservation in mammals, including stretches containing both highly conserved PU.1 and GATA binding sites. We have performed chromatin immunoprecipitation studies with GATA-1 antibodies and have defined multiple regions of in vivo binding within the GATA-1 locus in erythroid cells. Addtional studies are underway utilizing the Scanning ChIP procedure (Zeller et al., 2001) to determine in vivo GATA-1, GATA-2, and PU.1 binding sites of these factors to the GATA-1 locus in mast cells. In order to determine whether PU.1 positively regulates the expression of the mast cell GATA-1 isoform, we have examined GATA-1_mast expression in PU.1 −/ − cells. PU.1 −/ − fetal liver cells cannot differentiate into mast cells in vitro; reintroduction of PU.1 expression restores mast cell differentiation. We show that PU.1 −/ − cells are deficient in expression of the GATA-1 mast cell mRNA isoform, and reintroduction of PU.1 into the PU.1 deficient cells markedly up-regulates the expression of GATA-1_mast.

Our findings demonstrate that PU.1 positively regulates a distinct GATA-1 isoform during mast cell differentiation. We propose a model in which GATA factors cooperate with PU.1 to direct cell-specific isoforms of transcriptional regulators during hematopoietic development.