Stat5A and 5B transcription factors play a major role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological neoplasia and solid tumors. However, their mechanisms of action remain poorly understood and their molecular partners still need to be identified. Since Stat5A and 5B mainly differ in their carboxy-terminal transactivation domains, we sought to identify molecules that specifically bind to this domain, using a bacteria-match two-hybrid assay to screen a human Jurkat T cells library. This approach enabled us to isolate hTid1, a DNA-J protein homologous to a drosophila tumor suppressor, that interacts with Stat5B but not Stat5A. Specific interaction between Stat5B and hTid1 was confirmed by immunoprecipitation using various mouse and human lymphoïd cell lines and Cos-7 cells transfected with plasmids encoding both molecules. Using various deleted mutants, we showed that the cysteine-repeats containing region (CRR) of hTid1 was required for this interaction. Moreover, we found that the complex formed by hTid1 and STAT5B was dissociated upon cytokines- (IL-3, IL-7) or Tel-Jak2-induced tyrosine phosphorylation of STAT5B. We also brought evidence for a down-regulation of STAT5B transcriptional expression and activity by hTid1 in human hematopoietic cells. Indeed, overexpression of hTid1 but not of its CRR deleted form in Ba/F3 cells, resulted in a sharp reduction of Stat5B expression, a process that probably involves a proteasome-dependent degradation of Stat5B. By contrast, treatment of Ba/F3 cells with siRNA of hTid1 induced an increase of Stat5B expression. Finally, Cos-7 cells cotransfected with the prolactin receptor and Stat5B expression vectors and with a Stat5 specific luciferase constructs enabled us to demonstrate that overexpression of hTid1 inhibits of Stat5B transcription activity in a dose dependent manner. In conclusion, our findings define hTid1 as a novel negative regulator of Stat5B.

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