The transcription factor c-Myb is a key regulator of stem- and progenitor cells in the bone marrow, which has the task of forming most of the blood cells. However, maturation, activation and some proliferation of lymphoid cells occur in lymphoid organs like thymus. c-Myb is expressed throughout T-cell development in the thymus, and is essential at several stages during this development. The main objective of this project was to identify novel interaction partners of c-Myb, in an attempt to learn more about c-Myb function in general. In addition, we decided to specifically investigate if c-Myb can regulate RNA polymerase II pause-release through interaction with P-TEFb, which is composed of CDK9/CycT.
We performed a yeast two-hybrid screening with full length human c-Myb screened against a human thymus cDNA library. This resulted in the identification of 44 potential new interaction partners of c-Myb, where nine were found interesting. Four of the putative interaction partners were verified in remating experiments, and further analysis showed that their C-terminal domains had been "fished". Some false positive clones were found to code for scrambled proteins translated from the 5'UTR- and 3'UTR of their corresponding cDNAs. Interestingly, one of the clones encoding SUMO1 exhibited a c-Myb independent activation of reporter genes. Other clones encoded proteins that probably interact with SUMOylated c-Myb. Preliminary pulldown experiments were performed in the presence of deSUMOylation inhibitor N-ethylmaleimide to validate this assumption, but with no final conclusion.
The second part of the project was to attempt to confirm an interaction between c-Myb and the kinase CDK9 previously identified by immunoprecipitation and mass spectrometry. We could not detect an interaction in a yeast two-hybrid mating assay. However, a GST-pulldown detected a weak interaction between c-Myb and CDK9. Future studies are necessary to establish these novel interaction partners of c-Myb.