Hide metadata

dc.identifier.citationDrågen, Pimthanya Wanichawan. 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. Masteroppgave, University of Oslo, 2007en_US
dc.description.abstractHematopoiesis 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. A possible explanation may be that the substitution of the valine for an aspartic acid causes a removal of one negatively charge on the c-Myb protein surface, resulting in an increased DNA-binding of c-MybD152V compared to wild type. In conclusion, the results presented in this thesis indicate that the interaction of c-Myb with histone H3 is reduced by the D152V mutation, which may contribute to the reduction of c-Myb activity. To pursue these findings further, endogenous gene activation assays and detection of c-Myb-histone H3 interaction at the chromosomal level would be of interest.nor
dc.subjectmutasjoner punktmutasjoner D152V c-Myben_US
dc.titleAnalysis of a point mutation (D152V) in the transcription factor c- Myb and its effect on the interaction of c-Myb with Histone H3 and DNAen_US
dc.typeMaster thesisen_US
dc.creator.authorDrågen, Pimthanya Wanichawanen_US
dc.identifier.bibliographiccitationinfo:ofi/fmt:kev:mtx:ctx&ctx_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft.au=Drågen, Pimthanya Wanichawan&rft.title=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&rft.inst=University of Oslo&rft.date=2007&rft.degree=Masteroppgaveen_US
dc.contributor.supervisorOdd Stokke Gabrielsen/Elen Brendeford/Vilborg Matreen_US

Files in this item


No file.

Appears in the following Collection

Hide metadata