Characterization of a Megakaryocyte-Specific Enhancer of the Key Hemopoietic Transcription Factor GATA1.

Abstract

Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well-studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the transcription factor GATA1, that is required for normal maturation of these two lineages. Megakaryocyte- and erythroid-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, we previously showed that the 3′ 179 base pairs (bp) of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show that mHS-3.5 binds key hemopoietic transcription factors in vivo (GATA1, SCL/TAL-1) and is required to maintain histone acetylation in the mGata1 locus in primary megakaryocytes. When deletional constructs containing mHS-3.5 were used to direct GATA1-LacZ reporter gene expression in transgenic mice, a 25 bp element within the 3′ 179bp in mHS-3.5, was critical for megakaryocyte expression. In vitro three uncharacterized DNA-binding activities A, B and C bind to the core of the 25 bp element, and these binding sites are conserved through evolution. Of these, only activity B is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals that activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows that sequence corresponding to the binding site for activity B is a previously unrecognised motif present in the cis-elements of other megakaryocyte-specific genes. In summary, we have identified a motif and a DNA-binding activity that are likely to be important in directing a megakaryocyte gene expression program distinct from that in red cells.