Abstract
STAT5B is a vital transcription factor for lymphocytes. Here, the function of two STAT5B mutations from human T-cell leukemias: one substituting tyrosine 665 with phenylalanine (STAT5B
) and the other with histidine (STAT5B
), was interrogated. In silico modeling predicted divergent energetic effects on homodimerization with a range of pathogenicity. In primary T cells in vitro, STAT5B
showed gain-of-function, whereas STAT5B
demonstrated loss-of-function. Introducing the mutation into the mouse genome illustrated that the gain-of-function
mutation resulted in accumulation of CD8
effector and memory and CD4
regulatory T cells, altering CD8
/CD4
ratios. In contrast, STAT5B
"knock-in" mice showed diminished CD8
effector and memory and CD4
regulatory T cells. In contrast to WT STAT5B, the STAT5B
variant displayed greater STAT5 phosphorylation, DNA binding, and transcriptional activity after cytokine activation, whereas the STAT5B
variant resembled a null. The work exemplifies how joining in silico and in vivo studies of single nucleotides deepens our understanding of disease-associated variants, revealing structural determinants of altered function, defining mechanistic roles, and, specifically here, identifying a gain-of-function variant that does not directly induce hematopoietic malignancy.