Serine/Threonine Phosphorylation of RUNX1 Promotes Megakaryocytic Fate in Megakaryocyte-Erythroid Progenitors (HEL Cell Line Bulk mRNA-seq)
The differentiation of primary human megakaryocyte-erythroid progenitors (MEP) into  megakaryocytic (Mk) or erythroid (E) progenitors is critical for maintaining the blood  system, yet the mechanisms that regulate this fate specification remain unclear. Here,  we analyzed RNA-seq data and found that RUNX1 is a key regulator of differential gene  expression in MEP fate specification. Through viral transduction of primary MEP with  RUNX1, we demonstrated that overexpression of RUNX1 promotes Mk over E  specification, whereas pan-RUNX inhibition promotes E fate specification over Mk. We  found that although the levels of total RUNX1 do not differ between MkP and ErP, MkP  have higher levels of RUNX1 phosphorylated serine residues, and that serine/threonine  phosphorylation of RUNX1 dramatically increases its effectiveness. We used human  erythroleukemia (HEL) cell lines with expression of HA-tagged WT RUNX1,  phosphomimetic (RUNX1-4D) and non-phosphorylatable (RUNX1-4A) mutants to model  their effects on MEP. Although the chromatin association was not informative, the three  forms of RUNX1 caused differential expression of 2,625 genes. Approximately 40% of  these differentially expressed genes (DEGs) showed an increase with both RUNX1-WT  and RUNX1-4D while another 40% showed a decrease with both RUNX1-WT and  RUNX1-4D. We compared these DEGs with DEGs from primary human MEP, Mk  progenitors (MkP), and E progenitors (ErP), and found that the genes upregulated by  WT and RUNX1-4D in HEL cells overlapped significantly with the genes upregulated in  MkP vs. MEP, whereas genes downregulated by WT and RUNX1-4D in HEL cells  overlapped significantly with the genes downregulated in MkP vs. MEP. These results  suggest that phosphorylation of RUNX1 enhances RUNX1's function to activate or  repress transcription of target genes that are up/downregulated during MEP fate  specification. Overall design: Total RNA was extracted from HEL cell lines expressing HA-tagged WT RUNX1, RUNX1-4A, RUNX1-4D, along with an empty vector control HEL cell line. RNA was prepared from two biological replicates of each cell line.