The induction of EKLF during the development of the erythroid lineage

Erythroid Kruppel-like factor (EKLF) is a red blood cell-specific transcription factor that is required for the expression of the adult beta-globin gene during definitive erythropoiesis in mammalian development. While the role of EKLF during globin gene activation in the fetal liver has been well characterized, recent studies suggest that EKLF may also fulfill important functions at an earlier point in embryogenesis. EKLF is initially expressed within the extraembryonic mesoderm of the yolk sac, the site of primitive hematopoiesis from which the first circulating blood cells of the embryonic vasculature arise. EKLF mRNA is detected in the yolk sac shortly after primitive hematopoietic progenitors appear, and mouse embryonic stem cell in vitro differentiation assays have shown its induction to be dependent on Bmp4/Smad signaling. Cis-regulatory regions of the EKLF gene contain Gata factor binding sites, which are required for transcriptional activity of EKLF reporter genes in transgenic mouse models.; In order to gain a better understanding of the mechanisms that underlie the initial activation of the EKLF gene in response to Bmp4, we chose to identify cis-regulatory elements within the EKLF locus and test their contribution to transcriptional control. To this end we undertook a comparative phylogenetic sequence analysis of the EKLF locus and developed a reporter gene assay that monitors the activity of EKLF transcription during the in vitro differentiation of mouse embryonic stem cells as a model of yolk sac hematopoiesis. We show that EKLF transcription is activated in progenitors prior to erythroid commitment and that this activity depends on previously known Gata factor binding sites as well as on newly identified Smad binding elements within the EKLF locus. In addition, we demonstrate that Gata2 occupies regulatory sites within the EKLF locus in vivo prior to its displacement by Gata1 during the differentiation of erythroid cells. In parallel, we identify the proto-oncogene Dek as a potential regulator of EKLF transcription. We propose a model by which Gata2 and Bmp4/Smad signaling synergize to activate EKLF expression prior to the onset of erythroid differentiation during hematopoiesis, suggesting that EKLF might play a role in establishing commitment to the erythroid lineage.