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 Devicesandinstruments
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 andagar plates
3.10 Cellculture materials
3.11 Cell lines
3.12 Cellculture media
3.13 Buffers andsolutions
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 Transienttransfection bylipofection 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
(GRP78) as an interaction partner of v-Myb
5.2.2 YFP trap experiments unveiled interesting protein bands of potential Mybinteracting 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 cotransfection experiments
5.3.3 Influence of GRP78va on the transactivation potential of v-Myb E26
5.4 Analysis of the interaction between C/EBPß 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/EBPß 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 Tableoffigures
7.2 References
7.3 Clonecharts
Abbreviations
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1 Abstract
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 arresting them in an immature stage, presumably because of deregulation the expression of specific target genes. In addition to 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 cell-specific Myb-inducible enhancer. Recently it was shown 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 focused 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 nonsequence-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 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.
2 Introduction
Leukaemia is the most common type of cancer in children and young adults (33 %) between the age of 0-20 and the leading cause of death by disease. The Leukaemia Research Foundation published the alarming fact of 43 000 estimated new cases of leukaemia in 2010. According to the numbers of deaths caused by leukaemia in 2010 alone, it becomes clear that in spite of growing research in this field it is still regarded as a lethal disease.
Generally, leukaemia is a malignant disease of the blood and blood-forming organs and is characterized by the uncontrolled proliferation of white blood cells. The balance between apoptosis and cell formation is disturbed so that haematopoietic cells proliferate to immature blasts replacing differentiated cells in the bone marrow. There are different forms of leukaemia which are classified according to the course of disease as acute or chronic, and concerning the type of blood cells affected as lymphoblastic or myeloid.
The exact causes of the different types of leukaemia still remain obscure although studies discovered risk factors like smoking, viruses, mutagens and genetic failures. All types of leukaemia result from failure in the regulation of differentiation and controlled proliferation of blood cells. Fundamental research concerning the regulation of haematopoiesis and understanding the molecular mechanisms behind these processes might possibly lead to innovative methods of treatment.
Differentiation of blood cells as well as their proliferation is controlled by a well attuned subset of genes that express essential growth factors. These genes are regulated by proteins that have either a positive or a negative influence on the transcription, so called transcription factors.
2.1 The myb gene family of transcription factors
The first identified member of the myb gene family of transcription factors is the viral oncogene v-myb carried by the retrovirus avian myeloblastosis virus (AMV) (Hall, 1941). AMV induces monoblastic leukaemia in chickens blocking the differentiation of immature cells (Ness et al., 1989). Twenty years later a second virus, avian erythroblastosis virus E26 was discovered (Ivanov, 1964). Birds infected with the E26 virus quickly develop acute myeloblastic or erythroblastic leukaemia (Radke et al., 1982). The oncogenic sequence that both v-myb variants share is actually a copy of the cellular form of myb (c-myb) and was obtained through reverse transcription of truncated myb cDNAs (Lavu and Reddy, 1986; Weinstein et al., 1986). This finding entailed further screening of cDNA libraries revealing cDNA clones of two closely related members of the myb family, A-myb and B-myb. They encode nuclear transcription factors and are highly homologous to c-myb in three regions (Figure 2.1) (Nomura et al., 1988).
The Myb proteins harbour an N-terminally located DNA binding domain which is highly conserved in all three members of the Myb protein family in vertebrates (Peters et al., 1987). This suggests the importance of the DNA/Myb interaction for the proper function of the transcription factors. They all share the ability to specifically recognize the same nucleotide sequence 5'-PyAAC(G/T)G-3' (Biedenkapp et al., 1988), which is known as the Myb binding site (MBS). Variation in the last two nucleotides results in lower affinity binding, but genuine targets mostly have multiple MBSs. Additionally it was shown that in some cases transcriptional activation by Myb is not dependent on DNA binding (Foos et al., 1993; Kanei- Ishii et al., 1994). The schematic structures of A-, B- and c-Myb are depicted in Figure 2.1.
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Figure 2.1: Schematic structures of the Myb proteins.
The transcription factors of the Myb protein family are depicted schematically. The DNA binding region is located at the N-terminus, the transactivating region centrally and the negative regulatory domain at the C- terminus. Dark areas mark regions that are highly conserved between all Myb proteins and light grey areas indicate highly conserved regions in the species human, mouse and chicken for each protein. Repeats R1, R2 and R3 in the DNA binding domain are depicted (adapted from (Rushton et al., 2003)).
All family members share a highly conserved DNA-binding domain at the N-terminus which is composed of three tandem repeats (R1, R2 and R3) of 51 to 52 amino acids. Each repeat contains three conserved tryptophans and is structured in three a-helices of which the last two form a helix-turn-helix related motif (Ogata et al., 1992; Zargarian et al., 1999). The minimal sequence-specific DNA binding domain consists of R2 and R3, whereas R1 seems to have a stabilizing effect on the protein/DNA-complex (Tanikawa et al., 1993). c-Myb is highly expressed in immature haematopoietic cells but is down regulated when it comes to terminal differentiation. This confirms its role as a master regulator in haematopoietic cell development (Duprey and Boettiger, 1985). Apparently c-Myb controls the balance between apoptosis, differentiation and proliferation of haematopoietic progenitor cells (Kumar et al., 2003; Oh and Reddy, 1999). c-Myb is also expressed in certain non-haematopoietic cells where its function is less clear. The discovery of the related proteins A- and B-Myb sharing highly conserved regions has led to the assumption that they might be functional homologs of c-Myb in non-haematopoietic cells. It was shown that A-Myb acts as a regulator in the developing central nervous system and mammary gland, B- cell differentiation and spermatogenesis (Trauth et al., 1994). Lack of A-Myb leads to arrest of spermatogenesis and defective development of the other compartments (Toscani et al., 1997). B-Myb expression is ubiquitous in all proliferating cells and appears to play a role in cell cycle regulation. B-Myb expression is maximal especially during the G1/S boundary of the cell cycle and is regulated by the transcription factor E2F (Lam et al., 1992; Lam and Watson, 1993). Knocking out this gene revealed a role for B-Myb in early development as mice lacking B-Myb died at an early embryonic stage. These findings indicate that each Myb protein plays a distinct role in development and all of them are crucial for proper growth of vertebrates (Tanaka et al., 1999). However, due to their related DNA-binding specificities and their similar roles in transcriptional regulation it is likely that the different Myb transcription factors might share some target genes (Ramsay and Gonda, 2008).
Most of the c-Myb target genes are positively regulated but a few are repressed by c-Myb. At the present moment over 80 genes are known to be regulated by Myb but even more are still awaiting validation (Ramsay and Gonda, 2008). The target genes can be classified in three functional groups: genes required for the maintenance of basic cellular functions, so called 'housekeeping' genes, like MAT2A and GSTM1; genes with specific functions in differentiated cell lineages such as mim-1, tom-1, gbx-1, lysozyme and ELA2 and genes linked to oncogenicity, which can be further classified as genes connected to proliferation (e.g. myc and kit), differentiation (e.g. GATA3) or survival (e.g. BCL2 and GRP78) (Ramsay and Gonda, 2008).
2.1.1 Viral oncogenes ofAMV and E26
The oncogenes carried by AMV (avian myeloblastosis virus) and Е2б (avian leukaemia virus) are truncated and mutated versions of the proto-oncogene c-myb.
v-myb AMV maintains parts of a typical retroviral structure, including an intact gag gene, a defective poi gene and a truncated env gene. The protein (48 kDa) is derived from a subgenomically spliced mRNA and lacks 71 amino acids at the N-terminus and 199 amino acids at the C-terminus (Rosson and Reddy, 1986). Truncation of envelope protein (Env) and deletions in the Pol protein in v-Myb AMV result in failure of the virus to replicate itself causing a dependence on helper viruses (Baluda and Reddy, 1994). In addition, v-Myb AMV contains 10 amino acid substitutions in the region derived from c-Myb thereby enhancing the oncogenic potential ofAMV (Dini et al., 1995; Introna et al., 1990).
E26 virus lacks most of the gag gene and does not contain a poi or env gene. Instead, it harbours the part of c-myb and also the sequence of an additional cellular gene, c-ets, resulting in a Gag-Myb-Ets fusion protein of 135 kDa (Leprince et al., 1983). Both the Myb part and the Ets part contribute to the oncogenicity of the protein. Comparing the structures of the viral Myb proteins to c-Myb reveals that v-Myb E26 shares a smaller region with c- Myb but, on the contrary, contains only one amino acid substitution (Figure 2.2).
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Figure 2.2: Schematic structure of v-Myb AMV and E26 in comparison to c-Myb.
Depicted are the schematic structures of c-Myb, v-Myb AMV and v-Myb E26. The repeats in the DNA binding domain (DBD) and the hydrophobic and acidic region of the transactivation domain (TAD) are illustrated. NRD: negative regulatory domain. Arrows mark amino acid substitutions in comparison to c-Myb.
Both viral proteins lack two thirds of repeat R1 and a part of the negative regulatory domain. Besides the point mutations these deletions are considered to be essential for the oncogenic potential of v-Myb (Dini et al., 1995).
2.1.2 Co-operating factors of c-Myb and v-Myb
The complexity of cell specific gene regulation implies that it is dependent on a network of multiple well attuned transcription factors. It has been shown that haematopoietic transcription factors associate with multi-protein-complexes independently from DNA binding and are recruited to cell specific enhancer and promoter regions of the target genes (Sieweke and Graf, 1998). Hence, v-Myb and c-Myb also co-operate with other transcription factors to fulfil their function as gene regulators.
One of the most discussed interactions of c- and v-Myb is the association with the CCAAT/enhancer binding protein ß (C/EBPß) via the DNA binding domain of Myb (Burk et al., 1993). C/EBPß plays an important role in the activation of certain Myb target genes, like the chicken mim-1 gene (see 2.3). Another C/EBP family member, C/EBPa, is known to interact with c-Myb and helps to activate the lysozyme gene (Ivanova et al., 2007). C/EBPa is also renowned to regulate the expression of tom-1 gene by associating with both v-Myb and c- Myb (Burk et al., 1997).
Similarly, the proteins p300 and CREB-binding protein (CBP) are cofactors that are involved in the regulation of DNA-binding transcription factors and are considered as functional homologs possessing acetyltransferase activity (Ogryzko et al., 1996). Transcription factors like c- and v-Myb as well as histones are acetylated by p300/CBP suggesting a role for p300/CBP in chromatin remodelling. Acetylation of histones by p300/CBP leads to chromatin opening and thereby to binding of transcription factors. c-Myb and v-Myb interact with p300/CBP via their transactivation domain what increases their transcriptional activity (Ness, 1999).
c- and v-Myb are also known to interact with several other cofactors like Ets-1, Ets-2 and PU.1 (Dudek et al., 1992; Oelgeschlager et al., 1996). Interestingly, GATA-1 and c-Myb act as antagonists in red blood cell differentiation, as GATA-1 binds to a binding site in the c-myb promoter repressing c-myb expression (Bartunek et al., 2003).
2.2 The haematopoietic system
Approximately 5 litres of blood circulate through an adult body transporting cells and solutes to the different organs. The blood flow ensures the transport of oxygen and carbon dioxide, the regulation of the body temperature and plays an important role in the immune response. Blood mainly consists of plasma and different types of cells that are classified in erythrocytes, leukocytes and platelets (Schmidt, 2007).
All blood cells arise from common pluripotent haematopoietic stem cells (HSC) and the development of different blood cell types from this HSC is called haematopoiesis (Figure 2.3).
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Figure 2.3: Haematopoiesis.
Development of haematopoietic cells arising from a pluripotent haematopoietic stem cell. See text for details. The level of c-Myb expression is indicated by grey wedges and circular arrows indicate the cell types in which c- Myb presence is required for self-renewal and expansion of the cell population (Ramsay and Gonda, 2008).
HSCs have the ability to proliferate for self-renewal and to further differentiate into myeloid or lymphoid stem cells. Lymphoid stem cells evolve into lymphoblasts and then differentiate into T or B lymphocytes. B lymphocytes can differentiate further into plasma cells. Myeloid stem cells generate during development either myeloblasts (that turn into basophils, neutrophils, eosinophils and macrophages), mast cells or megacaryocyte/erythroid progenitor cells. The latter ones develop into platelets via megakaryocytes or red blood cells via erythroblasts (Ramsay and Gonda, 2008).
2.2.1 c-Myb is an important regulator of haematopoiesis
The development of the haematopoietic system depends on many different factors. The fate of the progenitor cells is decided by coordinated expression or repression of transcription and growth factors, which regulate genes specific for cell differentiation (Orkin, 2000). c-Myb is one of these transcription factors playing an important role in haematopoietic lineage determination. c-Myb knock-out mice showed an interesting phenotype: up to day 13 of embryonic development they still seemed to grow normally, but at day 15 they became severely anaemic and died of anoxia. This led to the conclusion that c-Myb is not essential for embryonic but for adult-type erythropoiesis. Development of other haematopoietic lineages was similarly diminished (Mucenski et al., 1991).
Further studies revealed that the highest levels of c-Myb were found in cell lines representing immature or progenitor-like cells (Thompson and Ramsay, 1995). Overexpression of c-Myb leads to repression of normal differentiation and supports leukaemic transformation (Ganter and Lipsick, 1999; Introna and Golay, 1999). These observations suggest that one key function of c-Myb is to maintain the proliferative state of haematopoietic progenitor cells. This correlates to the levels of c-Myb expression which are highest in immature stem cells. During differentiation c-Myb expression is down regulated (Figure 2.3).
2.2.2 v-Myb with transforming abilities in haematopoiesis
Studies showed that newly hatched chickens infected with AMV developed rapidly fatal monoblastic leukaemia (Beard, 1963). This was further supported by cell transformation assays using chicken tissues rich in haematopoietic cells like embryonic yolk sac, spleen, bone marrow or peripheral blood (Baluda and Goetz, 1961). Normally, haematopoietic cells develop from progenitor cells to mature cell types in two weeks, but when they were incubated with AMV, large amounts of cells were observed which were indistinguishable from monoblasts found in peripheral blood of animals suffering from leukaemia. These assays confirmed that AMV increases the amount of monoblasts by inducing rapid division of progenitor cells that are otherwise committed to differentiate into monocytes and macrophages (McNagny and Graf, 1996). Interestingly, terminally differentiated macrophages are also transformed into monoblasts when infected with AMV suggesting that v-Myb AMV is also able to reverse differentiated phenotypes (Durban and Boettiger, 1981; Lipsickand Wang, 1999).
Chickens infected with E26 virus developed leukaemia which appeared to consist mainly of immature erythroblasts, but cell transformation assays revealed that E26 virus transformed both erythroid and myeloid cells (Radke et al., 1982). In contrast to AMV, E26 virus is also able to stimulate mitogenesis in primary fibroblasts (Ravel-Chapuis et al., 1991).
Effects on haematopoiesis of both retroviruses are depicted in Figure 2.4.
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Figure 2.4: Effect of v-Myb AMV and E26 on haematopoiesis.
Normal pathways of haematopoietic cells are indicated with arrows. The effect of the retroviruses AMV and E26 is visualized with thick horizontal red bars indicating the blocks in differentiation. The large triangle at the right indicates that c-Myb expression diminishes as haematopoietic cells develop and is generally absent in mature cells. (HSC haematopoietic stem cell, CMP common myeloid progenitor, CLP common lymphoid precursor, MEG megakaryocyte, GMP granulocytic (G) and monocytic (M) precursor cell, E erythroblast) (altered from (Lipsick and Wang, 1999).
It is still not clear how the oncogenic transformation by v-Myb exactly occurs. There are two major considerations, firstly the cell-specificity of v-Myb and secondly the context between transcriptional regulation and oncogenic potential (Lipsickand Wang, 1999).
Studies suggested that the haematopoietic specificity of AMV and E26 might be due to similarity to c-Myb in acting as a regulator for proliferation and differentiation of blood and lymphoid cells (Smarda and Lipsick, 1994; Todokoro et al., 1988). However, c-Myb is also expressed in other tissues and other truncated viruses do not show the same cell specificity as AMV and E26 but affect various tissues. This leads to the conclusion that the narrow cell specificity is also due to the point mutations present in v-Myb AMV and E26 (Lipsick and Wang, 1999).
Furthermore, Dini et al. and Engelke et al. discovered in 1995 that the highly leukaemogenic v-Myb AMV protein is a relatively weak transcriptional activator and that accumulated amino acid substitutions increase the transforming ability but decrease its transcriptional activity. These findings indicated that transcriptional activation and oncogenic transformation potentials do not always correlate. This is also supported by investigation of the mim-1 gene which is activated by c-Myb and v-Myb E26 but not by v-Myb AMV (Ness et al., 1989).
2.3 The mim-1 gene as a model for gene regulation by v-Myb
In 1989 screening experiments revealed a cellular gene, mim-1 (myb-induced myeloid protein-1), that is directly regulated by v-Myb. The mim-1 gene is activated by c- and v-Myb E26 and expressed only in the myelomonocytic lineage but not in other haematopoietic or non-haematopoietic cells (Ness et al., 1989). Only a subset of c-Myb expressing cells express the mim-1 gene suggesting that more tissue-specific factors are involved in the regulation as c-Myb alone is not sufficient (Burk et al., 1993). The mim-1 promoter does not only contain three Myb-binding sites, but it was shown that it also possesses binding sites for CCAAT/enhancer binding proteins (Burk et al., 1993) and that both types of binding sites are required for full activity of the promoter (Ness et al., 1993). Mapping of DNase l-hypersensitive sites (DHS) as a strategy to localize cis-acting regulatory elements could identify a powerful cell-specific enhancer in the mim-1 gene 2 kb upstream of the promoter (Chayka et al., 2005). The same work showed that a major function of v-Myb is apparently to activate the enhancer and to enable cooperation with the promoter by the help of C/EBPß and p300.
Little is known about the protein encoded by the mim-1 gene. It appears to have a molecular size of 35 kDa and its primary structure is similar to the chemotactic protein P33 except for one amino acid difference. There is evidence that Mim-1 might be the precursor protein for P33, which is an endogenous target protein for arginine-specific ADP-ribosyltransferase (Yamada et al., 1992). The Mim-1 protein is not only detectable in cells transformed by v- Myb but is also present in granules of normal promyelocytes and in fewer amounts in matured granulocytes in chickens (Ness et al., 1989).
2.3.1 v-Myb AMV is defective in activating the mim-1 enhancer
mim-1 is highly expressed in myeloid cells transformed by the retrovirus E26 whereas myelomonocytic cells transformed by AMV lack the granulocyte-specific Mim-1 protein and resemble monoblasts (Introna et al., 1990). Interestingly, v-Myb AMV is not able to stimulate expression ofthe mim-1 gene in vivo but was shown to activate the mim-1 promoterjust like v-Myb E26 in transient transfection experiments using artificial reporter gene constructs. These results suggested that there could be further cis-acting regulatory elements that are involved in the activation of the mim-1 gene. A Myb-responsive cell-specific enhancer was identified in addition to the mim-1 promoter. It co-operates with the promoter when synergistically activated by Myb and C/EBPß and thereby regulates the gene (Chayka et al., 2005). This synergistic activation of mim-1 appears to occur in two steps. First C/EBPß initiates chromatin opening at the enhancer region without activating the gene. Only when Myb is present mim-1 transcription occurs because Myb is able to open the chromatin at the promoter. C/EBPß cannot trigger chromatin opening at the promoter what might explain the failure of C/EBPß to induce mim-1 expression alone (Plachetka et al., 2008).
Furthermore it was reported that Myb-dependent activation of the gene involves extensive remodelling of the enhancer chromatin revealing that the so called 'oncogenic' amino acid substitutions in v-Myb AMV have disrupted the ability of the protein to remodel the enhancer chromatin that is necessary to activate the enhancer (Wilczek et al., 2009). These results explain the observations of v-Myb AMV being unable to induce the expression of mim-1 in myeloid cells although it is able to stimulate the promoter in reporter gene experiments.
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
C. Wilczek investigated in her work the effect of the oncogenic amino acid substitutions of v-Myb AMV with regard to their role in the mim-1 enhancer remodelling (Wilczek, 2008).
It was shown that while v-Myb E26 is competent in remodelling the chromosomal structure at the mim-1 enhancer and in repositioning a nucleosome in the enhancer region v-Myb AMV is defective in that function.
Further experiments showed that the amino acid substitutions in the DNA-binding domain and in the transactivation domain of v-Myb AMV are responsible for the failure to remodel the enhancer chromatin. Interestingly, it was found that two conserved amino acid substitutions (V267I and V270I) in the hydrophobic region located in the TAD of v-Myb AMV are sufficient to abolish the chromatin remodelling activity at the mim-1 enhancer. The sequence around the hydrophobic region of v-Myb AMV in comparison to other Myb proteins is depicted in Figure 2.5. * I
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Figure 2.5: Amino acid sequence around the hydrophobic region of v-Myb AMV in comparison to other Myb proteins.
Amino acid sequences of different Myb proteins from different species are depicted. The hydrophobic region is highlighted in black. Amino acid substitutions of v-Myb AMV in comparison to c-Myb are marked with arrows. The p300/CBP binding region is marked with a black bar (Wilczek et al., 2009).
The hydrophobic region of c-Myb and v-Myb is highlighted in black. It can be seen that this region is highly conserved among the c-Myb proteins of different species. The amino acids relevant for chromatin remodelling are mutated in v-Myb AMV (V267I and V270I).
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.
3 Material
3.1 Chemicals
The standard chemicals were purchased from the following companies: Acros Organics, New Jersey, USA; Amersham Biosciences, Freiburg, D; AppliChem, Darmstadt, D; BD (including Difco), New Jersey, USA; Biomol, Hamburg, D; Carl Roth GmbH & Co KG, Karlsruhe, D; Invitrogen GmbH including Gibco BRL), Karlsruhe, D; J. T. Baker, Deventer, NL; Merck KGaA, Darmstadt, D; MP (former ICN Biomedicals), Irvine, USA; Roche, Mannheim, D and Sigma- Aldrich (including Fluka), München, D.
Special chemicals were obtained from the following companies:
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3.2 Kits
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Nucleobond Kit PC 500 Macherey-Nagel Düren, D
3.3 Devices and instruments
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3.4 Enzymes
Restriction enzymes were purchased from New England Biolabs (NEB), Frankfurt a.M., D other enzymes were obtained from the following companies:
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3.5 Antibodies
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3.6 Plasmids
3.6.1 Prokaryotic expression vectors
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3.6.2 Eukaryotic expression vectors
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3.7 Oligonucleotides
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3.8 Bacterial strains
E.coli ToplOF' were used for transformation and multiplying of DNA. E.coli BL2l (DE3) were used for protein overexpression. The genotypes of the strains are as follows:
ToplOF': F' [laclq, TnlO (TetR)] mcrA A(mrr-hsdRMS-mcrBC) ф80 locZAMl5 AZlacX74 deoR recAl araDl39 D(ara-leu)7697 galU galK rspL(StrR) endAl nupG
BL2l (DE3): F-, dcm, ompThsdS(rB-, mB-), gal (DE3)ab
[...]
- 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.grin.com/document/194449
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