: Beeke Wienert
: v-Myb proteins and their oncogenic potential: A study on how two point mutations affect the interaction of v-Myb with other proteins
: Diplomica Verlag GmbH
: 9783842832916
: 1
: CHF 31.70
:
: Genetik, Gentechnik
: English
: 118
: kein Kopierschutz/DRM
: PC/MAC/eReader/Tablet
: PDF
The oncogene v-myb of the retroviruses AMV (avian myeloblastosis virus) and E26 (avian leukaemia virus) encodes a transcription factor (v-Myb) which is a truncated homolog of its cellular progenitor c-Myb. c-Myb plays an essential role in the development of haematopoietic cells and is known to be a regulator for many target genes. v-Myb AMV is responsible for the transformation of myelomonocytic cells and for arresting them in an immature stage, presumably because of a deregulation of the expression of specific target genes. In addition to the truncation of the coding region, a number of amino acid substitutions are responsible for the high oncogenicity of v-Myb AMV. Due to the amino acid substitutions, v-Myb AMV and v-Myb E26 differ in their target gene spectrum. The chicken mim-1 gene is activated by v-Myb E26 and c-Myb but not by v-Myb AMV. The gene consists of two cis-regulatory regions, a Myb responsive promoter and a cell-specific Myb-inducible enhancer. Recently, it has been proven that two amino acid substitutions in a hydrophobic patch in the transactivation domain of v-Myb AMV are sufficient to disrupt its ability to stimulate the enhancer. This work focuses on the consequences of these amino acid substitutions by investigating protein-protein interactions of the hydrophobic region of v-Myb AMV in comparison to v Myb E26. Previous experiments identified GRP78 as an interaction partner of v Myb. In this study, a cytosolic variant of GRP78, GRP78va, was confirmed to interact with both v-Myb proteins. It was shown that its interaction site is limited to a very small region of v-Myb preceding the hydrophobic patch. Additionally, it was shown that GRP78va associated with all other members of the Myb-family and also with C/EBPß and HIPK2, suggesting a non-sequence-specific binding of GRP78va. Furthermore, reporter gene experiments demonstrated a repressing effect of GRP78va on the transactivation potential of v-Myb E26. In addition, GST pull down assays and co-immunoprecipitation experiments were used to precipitate endogenous proteins that could represent potential interaction partners of v-Myb. SDS-PAGE analysis revealed candidate bands, but mass spectrometry analysis failed to identify any proteins relevant for interaction with v Myb. Two other proteins were tested for their interaction with the hydrophobic patch of v-Myb. Co-immunoprecipitation experiments confirmed that C/EBPß interacts with the hydrophobic region of v-Myb and that the amino acid substitutions seem to affect the interaction in a negative way. Furthermore, PRMT4 was identified as an interaction partner of v-Myb. Mapping experiments showed the interaction to be mediated by the hydrophobic region. The point mutations in v-Myb AMV appear to positively influence the affinity for PRMT4. The fact that a SUMO binding motif is located in the same region might suggest a potential involvement of SUMO in the interaction of PRMT4 and v-Myb.

Beeke Eienert, M.Sc., was born in Bremen in 1985. After finishing a Bachelor's degree in Biology and Chemistry at the Carl-von-Ossietzky Universität Oldenburg, she completed her Master's degree in Biowissenschaften in 2011. During a research stay in Australia, the author focused on researching transcription factors. She continued to focus on these in her Master's thesis and plans to further work within this research area in her doctoral dissertation at the UNSW, Sydney.
v-Myb proteins and their oncogenic potential1
Table of contents3
Abbreviations7
1 Abstract11
2 Introduction13
2.1 The myb gene family of transcription factors13
2.2 The haematopoeietic system18
2.3 The mim-1 gene as a model for gene regulation by v-Myb21
2.4 Aim of the study24
3 Material25
3.1 Chemicals25
3.2 Kits26
3.3 Devices and instruments26
3.4 Enzymes27
3.5 Antibodies28
3.6 Plasmids29
3.7 Oligonucleotides34
3.8 Bacterial strains34
3.9 Media and agar plates35
3.10 Cell culture and materials35
3.11 Cell lines36
3.12 Cell culture media36
3.13 Buffers and solutions37
4 Methods42
4.1 Preparation of competent bacteria42
4.2 Transformation of competent bacteria42
4.3 Plasmid DNA isolation42
4.4 Quantification of nucleic acids43
4.5 Modification of DNA by enzymes44
4.6 Agarose gel electrophoresis44
4.7 DNA fragment extraction45
4.8 Ligation45
4.9 Polymerase chain reaction (PCR)46
4.10 Passage and cultivation of cells47
4.11 Transient transfection by calcium phosphate co-precipitation47
4.12 Transient transfection by lipofection with MetafectenePro48
4.13 Bacterial GST-fusion protein expression and purification49
4.14 Protein extraction from eukaryotic cells49
4.15 SDS PAGE50
4.16 Gel staining50
4.17 Western blot and immuno detection51
4.18 GST pull-down assay52
4.19 GFP/ YFP trap52
4.20 Co-immunoprecipitation53
4.21 Reporter gene assay53
5 Results55
5.1 Introduction of different constructs of v-Myb57
5.2 Analysis of the interaction of the hydrophobic region of v-Myb with unidentified binding partners60
5.3 Analysis of the interaction of v-Myb with GRP7867
5.4 Analysis of the interaction between C/EBPß and the hydrophobic region of v-Myb77
5.5 Analysis of the interaction of PRMT4/CARM1 with v-Myb79
6 Discussion85
6.1 Endogenous pull-down experiments detected potential interaction partners of the hydrophobic region of v-Myb85
6.2 GRP78va interacts with both mutants of v-Myb86
6.3 C/EBPß interacts with the hydrophobic region of v-Myb89
6.4 PRMT4 as a newly identified interaction partner90
6.5 Future perspectives93
7 Appendix95
7.1 Table of figures95
7.2 References97
7.3 Clone charts106