Selective inhibition of c-Myb DNA-binding by RNA polymers.

BACKGROUND: The transcription factor c-Myb is expressed in hematopoietic progenitor cells and other rapidly proliferating tissues, regulating genes important for proliferation, differentiation and survival. The DNA-binding domain (DBD) of c-Myb contains three tandemly arranged imperfect repeats, designated Myb domain R1, R2 and R3. The three-dimensional structure of the DBD shows that only the second and third Myb domains are directly involved in sequence-specific DNA-binding, while the R1 repeat does not contact DNA and only marginally affects DNA-binding properties. No structural information is available on the N-terminal 30 residues. Since deletion of the N-terminal region including R1 plays an important role in oncogenic activation of c-Myb, we asked whether this region confers properties beyond DNA-binding to the neighbouring c-Myb DBD. RESULTS: Analysis of a putative RNA-binding function of c-Myb DBD revealed that poly(G) preferentially inhibited c-Myb DNA-binding. A strong sequence-selectivity was observed when different RNA polymers were compared. Most interesting, the poly(G) sensitivity was significantly larger for a protein containing the N-terminus and the R1-repeat than for the minimal DNA-binding domain. CONCLUSION: Preferential inhibition of c-Myb DNA binding by poly(G) RNA suggests that c-Myb is able to interact with RNA in a sequence-selective manner. While R2 and R3, but not R1, are necessary for DNA-binding, R1 seems to have a distinct role in enhancing the RNA-sensitivity of c-Myb.

Transactivation properties of c-Myb are critically dependent on two SUMO-1 acceptor sites that are conjugated in a PIASy enhanced manner.

The transcription factor v-Myb is a potent inducer of myeloid leukemias, and its cellular homologue c-Myb plays a crucial role in the regulation of hematopoiesis. Recently, Bies and coworkers (Bies, J., Markus, J. & Wolff, L. (2002) J. Biol. Chem, 277, 8999-9009) presented evidence that murine c-Myb can be sumoylated under overexpression conditions in COS7 cells when cotransfected with FLAG-tagged SUMO-1. Here we provide independent evidence that human c-Myb is also subject to SUMO-1 conjugation under more physiological conditions as revealed by coimmunoprecipitation analysis of Jurkat cells and transfected CV-1 cells. Analysis in an in vitro conjugation system showed that modification of the two sites K503 and K527 is interdependent. A two-hybrid screening revealed that the SUMO-1 conjugase Ubc9 is one of a few major Myb-interacting proteins. The moderate basal level of sumoylation was greatly enhanced by cotransfection of PIASy, an E3 ligase for SUMO-1. The functional consequence of abolishing sumoylation was enhanced activation both of a transiently transfected reporter gene and of a resident Myb-target gene. When single and double mutants were compared, we found a clear correlation between reduction in sumoylation and increase in transcriptional activation. Enhancing sumoylation by contransfection of PIASy had a negative effect on both Myb-induced and basal level reporter activation. Furthermore, PIASy caused a shift in nuclear distribution of c-Myb towards the insoluble matrix fraction. We propose that the negative influence on transactivation properties by the negative regulatory domain region of c-Myb depends on the sumoylation sites located here.