The PAX-SIX-EYA-DACH Network regulates GATA factors and drives human erythropoiesis

Abstract

Erythropoiesis is orchestrated by the coordinated action of multiple transcription factors. The master erythropoietic-regulator GATA1 is itself modulated via interactions with multiple co-regulatory factors. Though the PAX-SIX-EYA-DACH network (PSEDN) of conserved transcription factors has been well characterized in human organogenesis, a role for PSEDN members in hematopoietic systems has only recently been recognized (Liu et al., Nature 2019). Here we studied the PSEDN member SIX and discovered its ability to drive erythropoiesis of human hematopoietic cells. Overexpression of SIX1 or SIX2 in human TF1 erythroleukemia or primary CD34+ hematopoietic stem-progenitor cells (HSPCs) stimulated the generation of erythroid cells, as measured by flow cytometry, qPCR, and Western blot. Conversely, SIX1 knockout in TF1 cells or primary HSPCs reduced erythroid cell generation in response to erythropoietin. By gene set enrichment analysis of RNA-seq data, SIX1/SIX2 overexpression stimulated heme metabolism genes as well as genes known to be regulated by GATA1. SIX1/SIX2 overexpression reduced GATA2 and increased GATA1 expression, resembling GATA switching downstream of EPO signaling. To determine whether GATA1 was necessary for SIX1 to stimulate erythropoiesis, we generated GATA1-knockout cells using CRISPR/Cas9. In contrast to control cells, SIX1 OE in GATA1-knockout cells failed to stimulate erythropoiesis, indicating that SIX1 stimulation of erythropoiesis requires GATA1. To gain further insight into the mechanism by which SIX1/SIX2 stimulates erythropoiesis we generated a BirA*-SIX fusion protein to determine its interactome. Streptavidin-enrichment of biotinylated proteins in BirA*-SIX overexpression lysates revealed GATA1 and FOG1 as proximal interactors of BirA*-SIX. When co-expressed in HEK293T cells GATA1 and SIX1 were found to coimmunoprecipitate, suggesting the two proteins can physically interact in a complex. We demonstrated the functional consequence of the SIX1/SIX2 and GATA1 using a GATA1-dependent luciferase reporter construct. Cells in which SIX1/SIX2 and GATA1 were co-expressed exhibited significantly higher levels of luciferase expression compared to cells expressing only GATA1, suggesting SIX1 could stimulate GATA1-dependent transcription. Together our results suggest that SIX1 can stimulate erythropoiesis via multiple mechanisms. This thesis provides the first demonstration of a role for the PSEDN in erythropoiesis and reveal physical and functional interactions between two central developmental transcriptional networks (GATA/FOG and PSEDN).