AbstractsBiology & Animal Science

Analysis of a point mutation (D152V) in the transcription factor c- Myb and its effect on the interaction of c-Myb with Histone H3 and DNA

by Pimthanya Wanichawan Drågen

Institution: University of Oslo
Year: 1000
Keywords: mutasjoner punktmutasjoner D152V c-Myb; VDP::473
Record ID: 1280236
Full text PDF: https://www.duo.uio.no/handle/10852/11519


Hematopoiesis is the process by which the hematopoietic stem cells differentiate into a variety of specialized cells (mature blood cells). The development of the definitive hematopoietic cell lineages is regulated by a number of transcription factors, such as the DNA binding transcription factor, c-Myb. This factor is encoded by the c-myb proto-oncogene and loss of function of the c-myb gene results in embryonic lethality due to a failure to develop fetal liver hematopoiesis. The c-myb gene is highly expressed in immature, proliferative hematopoietic cells and its expression level declines as the immature hematopoietic cells differentiate. Ectopic expression of c-Myb inhibits differentiation of hematopoietic precursor cells. Recently, several mutants acting as knockdown alleles of c-myb was found to affect lineage commitment and differentiation by perturbing differentiation of erythoid precursors but allowing megakaryocytopoiesis. One of these mutations was a substitution of valine for aspartic acid at residue 152 in the DNA-binding domain of c-Myb. This mutant was isolated based on its property to rescue platelet defects in thrombopenic c-mpl-/- mice by producing supraphysiological expansion of megakaryocyte and platelet production. However, the molecular mechanism by which this mutation alters c-Myb function was not identified. The aim of this work was to examine the molecular mechanisms of the D152V mutation. Since the alteration lies in the DNA-binding domain (DBD) of c-Myb, it was natural to investigate changes in DNA-binding properties. In addition, the ability to associate with histone H3 was also studied. The DBD of c-Myb is composed of three tandem repeats each being similar to the chromatin interacting SANT-domain found in chromatin regulatory proteins (the Swi3, Ada2, TFIIIB, NcoR, and ISWI proteins). Together these repeats are responsible for the ability of c-Myb to bind to DNA in a sequence-specific fashion. Interestingly, the two last SANT-related repeats (R2R3) of c-Myb was recently found to interact also with the N-terminal of histone H3 (Mo et al., 2005). This was proposed to position the H3-tail for acetylation and represents a novel chromatin function of c-Myb-DBD. When the molecular mechanism altered by the D152V mutant was to be studied, we addressed both putative changes in DNA-binding properties, as well as changes in its interaction with histone H3. The latter was to see whether the D152V mutation disrupted the Myb-H3 interaction, which might be an alternative explanation of the the reduction of c-Myb activity revealed in the previous study (Carpinelli et al. 2004). GST-H3 fusion proteins were made and used to elucidate histone H3 interaction with the minimal DNA-binding domain (R2R3) of c-Myb wild type and D152V mutant. The results showed that the binding of R2R3-c-MybD152V to histone H3 was weaker than that detected in R2R3-c-Myb wild type. On the other hand, investigation of its effect on DNA-binding properties revealed unexpectedly an increase in DNA-binding activity of c-MybD152V.…