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 arrests them in an immature stage, presumably by deregulating the expression of specific target genes. In addition to truncation of the coding region a number of amino acid (aa) substitutions are responsible for the high oncogenicity of v-Myb AMV. Due to the aa substitutions v-Myb AMV and v-Myb E26 differ in their target gene spectrum. Surprisingly, the chicken mim-1 gene can be activated by v-Myb E26 and c-Myb but not by v-Myb AMV. Recently it was shown that two aa substitutions in a hydrophobic patch in the transactivation domain of v-Myb AMV are sufficient to disrupt its ability to stimulate the Myb-responsive enhancer in mim-1.
This thesis focused on the consequences of these aa substitutions at the level of protein-protein interactions particularly investigating the hydrophobic regions of v-Myb AMV and v Myb E26. 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 small region of v-Myb preceding the hydrophobic patch. Additionally, it was shown that GRP78va also associated with other members of the Myb-family. Furthermore, reporter gene experiments demonstrated a repressing effect of GRP78va on the transactivation potential of v-Myb E26.
Two other proteins were tested for their interaction with the hydrophobic patch of v-Myb. Co-immunoprecipitation experiments confirmed that CCAAT-enhancer-binding protein β (C/EBPβ) interacts with the hydrophobic region of both v-Myb variants and that the aa substitutions in v-Myb AMV seem to weaken the interaction between the proteins. Furthermore, protein arginine methyltransferase 4 (PRMT4) was identified as an interaction partner of v-Myb. Mapping experiments showed that the interaction is mediated by the hydrophobic region. The point mutations in v-Myb AMV appear to positively influence the affinity for PRMT4 in comparison to v-Myb E26. 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.
Table of Contents
1. Abstract
2. Introduction
2.1 The myb gene family of transcription factors
2.1.1 Viral oncogenes of AMV and E26
2.1.2 Co-operating factors of c-Myb and v-Myb
2.2 The haematopoietic system
2.2.1 c-Myb is an important regulator of haematopoiesis
2.2.2 v-Myb with transforming abilities in haematopoiesis
2.3 The mim-1 gene as a model for gene regulation by v-Myb
2.3.1 v-Myb AMV is defective in activating the mim-1 enhancer
2.3.2 Two amino acid substitutions in the TAD of v-Myb AMV are sufficient to reduce its ability to stimulate the mim-1 enhancer
2.4 Aim of the study
3. Material
3.1 Chemicals
3.2 Kits
3.3 Devices and instruments
3.4 Enzymes
3.5 Antibodies
3.6 Plasmids
3.6.1 Prokaryotic expression vectors
3.6.2 Eukaryotic expression vectors
3.7 Oligonucleotides
3.8 Bacterial strains
3.9 Media and agar plates
3.10 Cell culture materials
3.11 Cell lines
3.12 Cell culture media
3.13 Buffers and solutions
4. Methods
Molecular biological techniques
4.1 Preparation of competent bacteria
4.2 Transformation of competent bacteria
4.3 Plasmid DNA isolation
4.4 Quantification of nucleic acids
4.5 Modification of DNA by enzymes
4.6 Agarose gel electrophoresis
4.7 DNA fragment extraction
4.8 Ligation
4.9 Polymerase chain reaction (PCR)
Cell culture techniques
4.10 Passage and cultivation of cells
4.11 Transient transfection by calcium phosphate co-precipitation
4.12 Transient transfection by lipofection with Metafectene®Pro
Protein biochemical techniques
4.13 Bacterial GST-fusion protein expression and purification
4.14 Protein extraction from eukaryotic cells
4.15 SDS PAGE
4.16 Gel staining
4.17 Western blot and immuno detection
4.18 GST pull-down assay
4.19 GFP/YFP trap
4.20 Co-immunoprecipitation
4.21 Reporter gene assay
5. Results
5.1 Introduction of different constructs of v-Myb
5.2 Analysis of the interaction of the hydrophobic region of v-Myb with unidentified binding partners
5.2.1 Endogenous GST pull-down experiments revealed Glucose regulated Protein 78 (GRP78) as an interaction partner of v-Myb
5.2.2 YFP trap experiments unveiled interesting protein bands of potential Myb-interacting proteins in SDS-PAGE
5.3 Analysis of the interaction of v-Myb with GRP78
5.3.1 Thapsigargin induces ER-stress and leads to expression of GRP78va
5.3.2 GRP78va interacts with v-Myb EP, v-Myb E26 and other proteins in co-transfection experiments
5.3.3 Influence of GRP78va on the transactivation potential of v-Myb E26
5.4 Analysis of the interaction between C/EBPβ and the hydrophobic region of v-Myb
5.5 Analysis of the interaction of PRMT4/CARM1 with v-Myb
5.5.1 PRMT4 interacts with the hydrophobic region of v-Myb
6. Discussion
6.1 Endogenous pull-down experiments detected potential interaction partners of the hydrophobic region of v-Myb
6.2 GRP78va interacts with both mutants of v-Myb
6.2.1 The specificity of the interaction with GRP78va
6.2.2 GRP78va reduces the transcriptional activity of v-Myb E26
6.3 C/EBPβ interacts with the hydrophobic region of v-Myb
6.4 PRMT4 as a newly identified interaction partner
6.4.1 The interaction site for PRMT4 is located in the hydrophobic region
6.4.2 Amino acid substitutions in the TAD of v-Myb AMV seem to affect the interaction with PRMT4
6.5 Future perspectives
7. Appendix
7.1 Table of figures
7.2 References
7.3 Clone charts
Research Objectives and Core Topics
The primary aim of this research is to elucidate the molecular interactions between the viral transcription factor v-Myb and potential regulatory partners, specifically focusing on the hydrophobic region within its transactivation domain. The study addresses why certain amino acid substitutions in this region disrupt the protein's ability to remodel chromatin at the mim-1 enhancer and investigates whether these mutations alter the binding affinity for known and newly identified transcriptional regulators.
- Functional characterization of v-Myb variants (AMV and E26) in comparison to cellular c-Myb.
- Identification of interaction partners for the v-Myb hydrophobic region using pull-down and YFP trap assays.
- Investigation of GRP78va, C/EBPβ, and PRMT4 as potential binding partners and their influence on transcriptional activity.
- Analysis of how oncogenic mutations affect protein-protein binding and chromatin remodeling capabilities.
Excerpt from the Book
2.4 Aim of the study
As described above, two conserved amino acid substitutions located in the hydrophobic region of the transactivation domain of v-Myb AMV (V267I and V270I) are sufficient to disrupt the chromatin remodelling activity of v-Myb at the mim-1 enhancer. One possible explanation for this observation is that these amino acid substitutions prevent the interaction of Myb with another transcriptional regulator.
The aim of this study is to identify potential interaction partners that bind to the hydrophobic region within the transactivation domain of the viral Myb protein. This work will also focus on proteins that are already known to interact with this domain and it will be investigated if their affinity for v-Myb differs due to the two amino acid substitutions in the hydrophobic region.
Chapter Summaries
1. Abstract: Provides an overview of the oncogenic v-Myb protein, its comparison to c-Myb, and the identification of GRP78, C/EBPβ, and PRMT4 as interaction partners.
2. Introduction: Details the Myb gene family, the haematopoietic system, and the regulatory role of v-Myb in leucamemic transformation and gene activation.
3. Material: Lists all chemicals, kits, laboratory equipment, bacterial strains, plasmids, and buffers utilized throughout the experimental procedures.
4. Methods: Describes the comprehensive molecular and cellular biology techniques employed, including DNA manipulation, protein expression, and interaction assays.
5. Results: Presents the findings regarding the interaction profiles of different v-Myb constructs with GRP78, C/EBPβ, and PRMT4, including the impact of these interactions on transactivation.
6. Discussion: Interprets the experimental results, examining the significance of the identified interactions and proposing models for how these associations influence the oncogenic potential of v-Myb.
7. Appendix: Contains the list of figures, comprehensive bibliography, and detailed clone charts for the vectors used.
Keywords
v-Myb, c-Myb, haematopoiesis, mim-1, transactivation domain, GRP78, C/EBPβ, PRMT4, oncogenesis, protein-protein interaction, chromatin remodeling, transcriptional regulation, molecular biology, YFP trap, GST pull-down
Frequently Asked Questions
What is the central focus of this research?
The research focuses on the molecular mechanisms of the viral transcription factor v-Myb, specifically how mutations in its hydrophobic region affect protein interactions and transcriptional regulation in leukaemic cells.
Which key transcription factor variants are studied?
The study compares the oncogenic v-Myb variants AMV and E26 against the cellular proto-oncogene c-Myb.
What is the primary research question?
The study investigates whether the loss of chromatin remodeling activity in v-Myb AMV is caused by the disruption of interactions with specific co-regulators due to key amino acid substitutions.
Which scientific methods are primarily utilized?
The study employs a range of techniques including GST pull-down assays, YFP trap experiments, co-immunoprecipitation, Western blot analysis, and reporter gene assays.
What does the main part of the work cover?
The main body of the work covers the methodological approach for identifying protein partners and the experimental results detailing the binding of GRP78, C/EBPβ, and PRMT4 to v-Myb variants.
What defines the core character of this work?
The work is characterized by its focus on viral oncogenesis, transcriptional regulation, and the identification of specific protein binding partners that modulate v-Myb activity.
How does GRP78va influence v-Myb activity?
Experiments show that GRP78va acts as a repressor, reducing the transactivation potential of v-Myb E26 by approximately half.
What role does PRMT4 play in relation to v-Myb?
PRMT4 was identified as a new interaction partner that binds to the hydrophobic region of v-Myb, with affinity appearing to be enhanced by the specific amino acid substitutions found in v-Myb AMV.
- Quote paper
- Beeke Wienert (Author), 2011, v-Myb proteins and their oncogenic potential: A study on how two point mutations affect the interaction of v-Myb with other proteins, Munich, GRIN Verlag, https://www.hausarbeiten.de/document/194449
