GADD45b plays an essential role in the G-CSF triggered granulocytic differentiation of human hematopoietic cells

The mechanism of maturation arrest of granulopoiesis in congenital neutropenia (CN) is not fully elucidated. We found that GADD45b is induced by G-CSF in healthy HSPCs, but not in CN cells. We inhibited GADD45b expression in CD34+ cells and iPSCs by CRISPR/Cas9 RNP and found markedly diminished granulocytic differentiation in GADD45b knockout iPSCs and CD34+ cells. Transduction of CN patient HSPCs with GADD45B cDNA restored granulocytic differentiation. In silico analysis of GADD45B promoter, reporter gene and ChIP assays revealed CEBPA-mediated activation of GADD45b. GADD45b is regulating active DNA demethylation by promoting the recruiting of DNA demethylation machineries to specific genomic loci. Therefore, we investigated whether G-CSF-triggered granulocytic differentiation of HSPCs requires regulation of active gene demethylation by GADD45b using RNA sequencing and the Infinium methylation EPIC array of WT or GADD45b-edited CD34+ HSPCs treated or not with G-CSF. G-CSF treatment of HSPCs resulted in the robust changes of DNA methylation that was markedly reduced in GADD45b KO cells. We identified several signaling pathways that are regulated by GADD45b-mediated demethylation.