Otx1 Enhances Erythroid Differenciation through Activation of the SCL Gene.

Members of the paired class of homeobox proteins are critical determinants of left-right asymmetry and establish the antero-posterior axis, suggesting that they could also be involved in asymmetric determination within the hematopoietic system. We have previously shown that mice lacking Otx1, a bicoid homeodomain-containing gene, exhibit an impairment of the erythroid compartment, associated with decreased SCL levels. In the present study, we show that Otx1 is coexpressed with SCL in yolk sac during embryonic development; in differentiating embryonic stem cells, Otx1 is upregulated with SCL in both primitive and definitive erythroid colonies, while Otx expression is absent in cardiomyocytes and skeletomyocytes. To address the role of Otx1 in hematopoiesis, we overexpressed Otx1 in primary hematopoietic cells using the MSCV retrovirus. The gain of Otx1 function gives rise to a 6-fold increase in endogenous SCL levels together with an increase in TER119-positive erythroid cells. Strikingly, the generation of CD11b-positive myeloid cells was almost abrogated by ectopic Otx1 expression, suggesting that Otx1 favours the erythroid lineage at the expense of the myeloid lineage. Furthermore, we took several approaches to provide molecular and functional evidence that SCL is a direct transcriptional target of Otx1. Indeed, Otx1 synergizes with GATA-1 to activate transcription from the SCL proximal promoter and this activity is dependent on the proximal GATA site of the SCL promoter. Next, we show by chromatin immunoprecipitation that Otx1 and GATA-1 occupy the SCL proximal promoter in vivo in erythroid cells. At the molecular level, we show that Otx1 physically interacts with GATA-1 in erythroid cells, and the homeodomain of Otx1 is sufficient for this interaction. Finally, a gain of function of SCL rescues the erythroid deficiency of Otx1^−/− mice, consistent with the model in which SCL operates downstream of Otx1. Taken together, our observations indicate that Otx1, GATA-1 and SCL are involved in the same genetic pathway to specify the erythroid fate during hematopoiesis.