JAK2 is necessary and sufficient for interferon-gamma-induced transcription of the gene encoding gp91PHOX.

During the inflammatory response, interferon-gamma (IFN-gamma) increases transcription of the gene encoding gp91PHOX, a respiratory burst oxidase component. This gene (referred to as the CYBB gene) is transcribed in phagocytic cells differentiated beyond the promyelocyte stage, and transcription continues until cell death. Previous investigations identified a positive regulatory element in the proximal CYBB promoter referred to as the hematopoiesis-associated factor 1 (HAF1)-cis element. This element is activated by a multiprotein complex, which includes the IFN consensus sequence-binding protein (ICSBP). Interaction of this complex with the HAF1-cis element requires ICSBP tyrosine phosphorylation, which is induced by IFN-gamma stimulation of phagocytic cells. Previous studies also identified a negative cis element in the CYBB promoter. This element is repressed by the homeodomain protein HoxA10. HoxA10 tyrosine phosphorylation, which occurs in response to IFN-gamma, decreases HoxA10 DNA binding and therefore repression of CYBB transcription. In these studies, we determine Janus tyrosine kinase 2 (JAK2) activation is necessary and sufficient for IFN-gamma-induced CYBB transcription in phagocytic cells and also for ICSBP and HoxA10 tyrosine phosphorylation. Consistent with these results, we find JAK2 activation is sufficient to induce ICSBP interaction with the HAF1 element and abolish HoxA10 binding to the CYBBrepressor element. Therefore, these findings provide direct demonstration of JAK2 dependence of IFN-gamma-induced CYBB transcription. In addition, these results identify a mechanism mediating this effect.

SHP1 protein-tyrosine phosphatase regulates HoxA10 DNA binding and transcriptional repression activity in undifferentiated myeloid cells.

The homeodomain protein HoxA10 interacts with negative cis elements to repress gene transcription in undifferentiated myeloid cells. The CYBB and NCF2 genes, which encode the gp91(PHOX) and p67(PHOX) proteins, are two such HoxA10 target genes. During interferon gamma-induced myeloid differentiation, tyrosine phosphorylation decreases HoxA10 DNA binding affinity and transcriptional repression. Therefore, decreased HoxA10 repression contributes to increased CYBB and NCF2 transcription in differentiating myeloid cells. The current studies investigate modulation of HoxA10 repression activity during myelopoiesis. We determine that phosphorylation of tyrosine residues in the conserved homeodomain decreases HoxA10-DNA binding. We also determine that interaction of the homeodomain phosphotyrosine residues with an adjacent domain in the HoxA10 protein is necessary for decreased DNA binding affinity. Since SHP1 protein-tyrosine phosphatase antagonizes myeloid differentiation and decreases CYBB and NCF2 transcription, we investigated the influence of SHP1-protein-tyrosine phosphatase (PTP) on HoxA10 tyrosine phosphorylation. We find that SHP1-PTP activity increases HoxA10 target gene repression in undifferentiated myeloid cells. Consistent with this, SHP1-PTP interacts with HoxA10 and decreases homeodomain-tyrosine phosphorylation. These investigations suggest that SHP1-PTP activity, in undifferentiated myeloid cells, influences HoxA10 repression of myeloid-specific genes. Therefore, increased HoxA10 repression of myeloid gene transcription is a molecular mechanism for SHP1 inhibition of myeloid differentiation.