The PNT Domain of ERG Regulates Hematopoietic Stemness and is Required for the Maintenance of ERG-Induced Leukemia

The ETS transcription factor ERG has a crucial role in hematopoiesis. It is essential for hematopoietic stem cell maintenance and megakaryopoiesis. Increased expression of ERG is associated with poor prognosis in acute myeloid leukemia. Previous studies by us and others established ERG as an oncogene in human leukemia. The PNT domain of ERG is a conserved domain that, in some other ETS factors, is required for protein-protein interactions, but its function for ERG is unknown. To examine whether the PNT domain of ERG is essential for hematopoietic stem and progenitor cells (HSPC) transformation, murine fetal liver-derived-HSPC were transduced with human WT and deleted PNT (DelPNT) domain ERG and cultured in methylcellulose. In re-plating assays, the PNT domain was not essential for HSPC self-renewal yet, DelPNT-ERG-transduced cells demonstrated increased proliferation capacity. Flow cytometry analysis showed decreased expression of hematopoietic stem markers for DelPNT-ERG-transduced cells. Consistent with these findings, RNA sequencing revealed enrichment of genes associated with myeloid differentiation and loss of hematopoietic stem cell signature in HSPC transduced with DelPNT-ERG. Transduction-transplantation assays in C57B6 mice suggested that the PNT domain is not essential for leukemia development. Nevertheless, time to leukemia development was significantly delayed for delPNT-ERG-transduced mice. Gene expression analysis showed enrichment for cell cycle and Myc target genes in leukemia induced by WT-ERG as compared to DelPNT-ERG. The proximity map of ERG-interacting protein suggested that the PNT domain is essential for ERG association with the SWI-SNF complex, which was previously shown to mediate the viability and proliferation of leukemia cells, at least in part by the maintenance of Myc expression. To further validate this finding, we conducted ChIP-sequencing on myeloid progenitor cell line and revealed decreased intensities of histone modifications associated with an active enhancer at the Myc locus in cells transduced with DelPNT-ERG as compared to WT-ERG. These results suggest that in pre-leukemic conditions, the PNT domain has a role in preserving the stem cell phenotype associated with ERG, thereby preventing premature differentiation. Furthermore, ERG interaction with the SWI-SNF complex was significantly dependent on the PNT domain and may be essential for leukemia maintenance.