Odd Stokke Gabrielsen

• Fan, Qiong; Nørgaard, Rikke Christine; Grytten, Ivar; Ness, Cecilie Maria; Lucas, Christin; Vekterud, Kristin; Soedling, Helen; Matthews, Jason; Lemma, Roza Berhanu; Gabrielsen, Odd Stokke; Bindesbøll, Christian; Ulven, Stine Marie; Nebb, Hilde Irene; Grønning-Wang, Line Mariann & Sæther, Thomas (2020). LXRα Regulates ChREBPα Transactivity in a Target Gene-Specific Manner through an Agonist-Modulated LBD-LID Interaction. Cells.  ISSN 2073-4409.  9(5) . doi: 10.3390/cells9051214 Vis sammendrag

  The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in trans. The two factors interact via their key activation domains; the low glucose inhibitory domain (LID) of ChREBPα and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXRα in responding to nutritional cues that was overlooked due to LXR lipogenesis-promoting function.

• Fan, Qiong; Nørgaard, Rikke Christine; Grytten, Ivar; Ness, Cecilie Maria; Lucas, Christin; Vekterud, Kristin; Soedling, Helen; Matthews, Jason; Lemma, Roza Berhanu; Gabrielsen, Odd Stokke; Bindesbøll, Christian; Ulven, Stine Marie; Nebb, Hilde Irene; Grønning-Wang, Line Mariann & Sæther, Thomas (2019). Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction. BioRxiv.  ISSN 0362-4331. . doi: 10.1101/2019.12.20.869974 Vis sammendrag

  The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in liver. We have previously shown that LXR regulates ChREBP transcription and activity; however, the underlying mechanisms are unclear. In the current study, we demonstrate that LXRα and ChREBPα interact physically, and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα, and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP, but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in trans. The two factors interact via their key activation domains; ChREBPα’s low glucose inhibitory domain (LID) and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXRα that was overlooked due to LXR lipogenesis-promoting function.

• Fuglerud, Bettina Maria; Ledsaak, Marit; Rogne, Marie; Eskeland, Ragnhild & Gabrielsen, Odd Stokke (2018). The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation. Epigenetics & Chromatin.  ISSN 1756-8935.  11(1) . doi: 10.1186/s13072-018-0208-y Fulltekst i vitenarkiv.
• Rodriguez- Castañeda, Fernando; Lemma, Roza Berhanu; Cuervo Torre, Ignacio; Bengtsen, Mads; Moen, Lisa Marie; Ledsaak, Marit; Eskeland, Ragnhild & Gabrielsen, Odd Stokke (2018). The SUMO protease SENP1 and the chromatin remodeller CHD3 interact and jointly affect chromatin accessibility and gene expression. Journal of Biological Chemistry.  ISSN 0021-9258.  293(40), s 15439- 15454 . doi: 10.1074/jbc.RA118.002844 Fulltekst i vitenarkiv. Vis sammendrag

  The small ubiquitin-like modifier (SUMO) post-translationally modifies lysine residues of transcription factors and co-regulators and thereby contributes to an important layer of control of the activities of these transcriptional regulators. Likewise, deSUMOylation of these factors by the sentrin-specific proteases (SENPs) also plays a role in gene regulation, but whether SENPs functionally interact with other regulatory factors that control gene expression is unclear. In the present work, we focused on SENP1, specifically, on its role in activation of gene expression investigated through analysis of the SENP1 interactome, which revealed that SENP1 physically interacts with the chromatin remodeler chromodomain helicase DNA-binding protein 3 (CHD3). Using several additional methods, including GST pull-down and co-immunoprecipitation assays, we validated and mapped this interaction, and using CRISPR-Cas9–generated CHD3- and SENP1-KO cells (in the haploid HAP1 cell line), we investigated whether these two proteins are functionally linked in regulating chromatin remodeling and gene expression. Genome-wide ATAC-Seq analysis of the CHD3- and SENP1-KO cells revealed a large degree of overlap in differential chromatin openness between these two mutant cell lines. Moreover, motif analysis and comparison with ChIP-Seq profiles in K562 cells pointed to an association of CHD3 and SENP1 with CCCTC-binding factor (CTCF) and SUMOylated chromatin–associated factors. Lastly, genome-wide RNA-Seq also indicated that these two proteins co-regulate the expression of several genes. We propose that the functional link between chromatin remodeling by CHD3 and deSUMOylation by SENP1 uncovered here provides another level of control of gene expression.

• Bengtsen, Mads; Sørensen, Linda Therese; Aabel, Linn Ingeborg; Ledsaak, Marit; Matre, Vilborg & Gabrielsen, Odd Stokke (2017). The adaptor protein ARA55 and the nuclear kinase HIPK1 assist c-Myb in recruiting p300 to chromatin. Biochimica et Biophysica Acta. Gene Regulatory Mechanisms.  ISSN 1874-9399.  1860(7), s 751- 760 . doi: 10.1016/j.bbagrm.2017.05.001 Fulltekst i vitenarkiv.
• Fuglerud, Bettina Maria; Lemma, Roza Berhanu; Wanichawan, Pimthanya; Sundaram, Arvind; Eskeland, Ragnhild & Gabrielsen, Odd Stokke (2017). A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function. Nucleic Acids Research.  ISSN 0305-1048.  45(13), s 7681- 7696 . doi: 10.1093/nar/gkx364 Fulltekst i vitenarkiv.
• Simovski, Boris; Vodak, Daniel; Gundersen, Sveinung; Domanska, Diana Ewa; Azab, Abdulrahman; Holden, Lars; Holden, Marit; Grytten, Ivar; Rand, Knut Dagestad; Drabløs, Finn; Johansen, Morten; Mora Ortiz, Antonio Carlos; Lund-Andersen, Christin; Fromm, Bastian; Eskeland, Ragnhild; Gabrielsen, Odd Stokke; Ferkingstad, Egil; Nakken, Sigve; Bengtsen, Mads; Nederbragt, Alexander Johan; Thorarensen, Hildur Sif; Akse, Johannes Andreas; Glad, Ingrid Kristine; Hovig, Johannes Eivind & Sandve, Geir Kjetil (2017). GSuite HyperBrowser: integrative analysis of dataset collections across the genome and epigenome. GigaScience.  ISSN 2047-217X.  6(7), s 1- 12 . doi: 10.1093/gigascience/gix032 Fulltekst i vitenarkiv. Vis sammendrag

  Background: Recent large-scale undertakings such as ENCODE and Roadmap Epigenomics have generated experimental data mapped to the human reference genome (as genomic tracks) representing a variety of functional elements across a large number of cell types. Despite the high potential value of these publicly available data for a broad variety of investigations, little attention has been given to the analytical methodology necessary for their widespread utilisation. Findings: We here present a first principled treatment of the analysis of collections of genomic tracks. We have developed novel computational and statistical methodology to permit comparative and confirmatory analyses across multiple and disparate data sources. We delineate a set of generic questions that are useful across a broad range of investigations and discuss the implications of choosing different statistical measures and null models. Examples include contrasting analyses across different tissues or diseases. The methodology has been implemented in a comprehensive open-source software system, the GSuite HyperBrowser. To make the functionality accessible to biologists, and to facilitate reproducible analysis, we have also developed a web-based interface providing an expertly guided and customizable way of utilizing the methodology. With this system, many novel biological questions can flexibly be posed and rapidly answered. Conclusions: Through a combination of streamlined data acquisition, interoperable representation of dataset collections and customizable statistical analysis with guided setup and interpretation, the GSuite HyperBrowser represents a first comprehensive solution for integrative analysis of track collections across the genome and epigenome. The software is available at: https://hyperbrowser.uio.no

• Ledsaak, Marit; Bengtsen, Mads; Molværsmyr, Ann-Kristin; Fuglerud, Bettina Maria; Matre, Vilborg; Eskeland, Ragnhild & Gabrielsen, Odd Stokke (2016). PIAS1 binds p300 and behaves as a coactivator or corepressor of the transcription factor c-Myb dependent on SUMO-status. Biochimica et Biophysica Acta. Gene Regulatory Mechanisms.  ISSN 1874-9399.  1859(5), s 705- 718 . doi: 10.1016/j.bbagrm.2016.03.011
• Bengtsen, Mads; Klepper, Kjetil; Gundersen, Sveinung; Cuervo Torre, Ignacio; Drabløs, Finn; Hovig, Johannes Eivind; Sandve, Geir Kjetil F.; Gabrielsen, Odd Stokke & Eskeland, Ragnhild (2015). c-Myb Binding Sites in Haematopoietic Chromatin Landscapes. PLOS ONE.  ISSN 1932-6203.  10(7) . doi: 10.1371/journal.pone.0133280 Fulltekst i vitenarkiv. Vis sammendrag

  Strict control of tissue-specific gene expression plays a pivotal role during lineage commit- ment. The transcription factor c-Myb has an essential role in adult haematopoiesis and func- tions as an oncogene when rearranged in human cancers. Here we have exploited digital genomic footprinting analysis to obtain a global picture of c-Myb occupancy in the genome of six different haematopoietic cell-types. We have biologically validated several c-Myb foot- prints using c-Myb knockdown data, reporter assays and DamID analysis. We show that our predicted conserved c-Myb footprints are highly dependent on the haematopoietic cell type, but that there is a group of gene targets common to all cell-types analysed. Further- more, we find that c-Myb footprints co-localise with active histone mark H3K4me3 and are significantly enriched at exons. We analysed co-localisation of c-Myb footprints with 104 chromatin regulatory factors in K562 cells, and identified nine proteins that are enriched together with c-Myb footprints on genes positively regulated by c-Myb and one protein enriched on negatively regulated genes. Our data suggest that c-Myb is a transcription fac- tor with multifaceted target regulation depending on cell type.

• Mora Ortiz, Antonio Carlos; Sandve, Geir Kjetil; Gabrielsen, Odd Stokke & Eskeland, Ragnhild (2015). In the loop: promoter-enhancer interactions and bioinformatics. Briefings in Bioinformatics.  ISSN 1467-5463. . doi: 10.1093/bib/bbv097 Vis sammendrag

  Enhancer–promoter regulation is a fundamental mechanism underlying differential transcriptional regulation. Spatial chromatin organization brings remote enhancers in contact with target promoters in cis to regulate gene expression. There is considerable evidence for promoter–enhancer interactions (PEIs). In the recent years, genome-wide analyses have identified signatures and mapped novel enhancers; however, being able to precisely identify their target gene(s) requires massive biological and bioinformatics efforts. In this review, we give a short overview of the chromatin landscape and transcriptional regulation. We discuss some key concepts and problems related to chromatin interaction detection technologies, and emerging knowledge from genome-wide chromatin interaction data sets. Then, we critically review different types of bioinformatics analysis methods and tools related to representation and visualization of PEI data, raw data processing and PEI prediction. Lastly, we provide specific examples of how PEIs have been used to elucidate a functional role of non-coding single-nucleotide polymorphisms. The topic is at the forefront of epigenetic research, and by highlighting some future bioinformatics challenges in the field, this review provides a comprehensive background for future PEI studies.

• Alm-Kristiansen, Anne Hege; Lorenzo, Petra Isabel O.; Molværsmyr, Ann-Kristin; Matre, Vilborg; Ledsaak, Marit; Sæther, Thomas & Gabrielsen, Odd Stokke (2011). PIAS1 interacts with FLASH and enhances its co-activation of c-Myb. Molecular Cancer.  ISSN 1476-4598.  10 . doi: 10.1186/1476-4598-10-21
• Lorenzo, Petra Isabel O.; Brendeford, Elen Margrethe; Gilfillan, Siv; Gavrilov, Alexey A; Ledsaak, Marit; Razin, Sergey V; Eskeland, Ragnhild; Sæther, Thomas & Gabrielsen, Odd Stokke (2011). Identification of c-Myb target genes in K562 cells reveals a role for c-Myb as a master regulator. Genes & cancer.  ISSN 1947-6019.  2(8), s 805- 817 . doi: 10.1177/1947601911428224 Vis sammendrag

  The c-Myb transcription factor is an important regulator of hematopoietic cell development. c-Myb is expressed in immature hematopoietic cells and plays a direct role in lineage fate selection, cell cycle progression, and differentiation of myeloid as well as B- and T-lymphoid progenitor cells. As a DNA-binding transcription factor, c-Myb regulates specific gene programs through activation of target genes. Still, our understanding of these programs is incomplete. Here, we report a set of novel c-Myb target genes, identified using a combined approach: specific c-Myb knockdown by 2 different siRNAs and subsequent global expression profiling, combined with the confirmation of direct binding of c-Myb to the target promoters by ChIP assays. The combination of these 2 approaches, as well as additional validation such as cloning and testing the promoters in reporter assays, confirmed that MYADM, LMO2, GATA2, STAT5A, and IKZF1 are target genes of c-Myb. Additional studies, using chromosome conformation capture, demonstrated that c-Myb target genes may directly interact with each other, indicating that these genes may be coordinately regulated. Of the 5 novel target genes identified, 3 are transcription factors, and one is a transcriptional co-regulator, supporting a role of c-Myb as a master regulator controlling the expression of other transcriptional regulators in the hematopoietic system.

• Sæther, Thomas; Pattabiraman, D.R.; Alm-Kristiansen, Anne Hege; Vogt- Kielland, Linn Troye; Gonda, T.J. & Gabrielsen, Odd Stokke (2011). A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability. Oncogene.  ISSN 0950-9232.  30(2), s 212- 222 . doi: 10.1038/onc.2010.397
• Molværsmyr, Ann-Kristin; Sæther, Thomas; Gilfillan, Siv; Lorenzo, Petra Isabel O.; Kvaløy, Heidi; Matre, Vilborg & Gabrielsen, Odd Stokke (2010). A SUMO-regulated activation function controls synergy of c-Myb through a repressor-activator switch leading to differential p300 recruitment. Nucleic Acids Research.  ISSN 0305-1048.  38(15), s 4970- 4984 . doi: 10.1093/nar/gkq245
• Matre, Vilborg; Nordgård, Oddmund; Alm-Kristiansen, Anne Hege; Ledsaak, Marit & Gabrielsen, Odd Stokke (2009). HIPK1 interacts with c-Myb and modulates its activity through phosphorylation. Biochemical and Biophysical Research Communications - BBRC.  ISSN 0006-291X.  388(1), s 150- 154 . doi: 10.1016/j.bbrc.2009.07.139
• Alm-Kristiansen, Anne Hege; Norman, IL; Matre, Vilborg & Gabrielsen, Odd Stokke (2009). SUMO modification regulates the transcriptional activity of FLASH. Biochemical and Biophysical Research Communications - BBRC.  ISSN 0006-291X.  387(3), s 494- 499 . doi: 10.1016/j.bbrc.2009.07.053
• Alm-Kristiansen, Anne Hege; Sæther, Thomas; Matre, Vilborg; Otnæss, Siv Kolstø; Dahle, Øyvind & Gabrielsen, Odd Stokke (2008). FLASH acts as a co-activator of the transcription factor c-Myb and localizes to active RNA polymerase II foci. Oncogene.  ISSN 0950-9232.  27, s 4644- 4656 . doi: 10.1038/onc.2008.105
• Orvain, Christophe; Matre, Vilborg & Gabrielsen, Odd Stokke (2008). The transcription factor c-Myb affects pre-mRNA splicing. Biochemical and Biophysical Research Communications - BBRC.  ISSN 0006-291X.  372, s 309- 313 . doi: 10.1016/j.bbrc.2008.05.054
• Rønneberg, Jo Anders; Tost, Jörg; Solvang, Hiroko K.; Alnaes, GIG; Alnæs, Grethe Irene Grenaker; Johansen, Fredrik E.; Brendeford, Ellen M.; Yakhini, Zohar; Gut, Ivo G.; Lonning, PE; Lønning, Per Eystein; Børresen-Dale, Anne-Lise; Gabrielsen, Odd Stokke & Kristensen, Vessela Nedelcheva (2008). GSTP1 promoter haplotypes affect DNA methylation levels and promoter activity in breast carcinomas. Cancer Research.  ISSN 0008-5472.  68(14), s 5562- 5571 . doi: 10.1158/0008-5472.CAN-07-5828
• Berge, Tone; Matre, Vilborg; Brendeford, Elen Margrethe; Sæther, Thomas; Lüscher, Bernhard & Gabrielsen, Odd Stokke (2007). Revisiting a selection of target genes for the hematopoietic transcription factor c-Myb using chromatin immunoprecipitaion and c-Myb knockdown. Blood Cells, Molecules & Diseases.  ISSN 1079-9796.  39(3), s 278- 286 . doi: 10.1016/j.bcmd.2007.05.007
• Sæther, Thomas; Berge, T; Ledsaak, Marit; Matre, Vilborg; Alm-Kristiansen, Anne Hege; Dahle, O; Aubry, F & Gabrielsen, Odd Stokke (2007). The chromatin remodeling factor Mi-2 alpha acts as a novel co-activator for human c-Myb. Journal of Biological Chemistry.  ISSN 0021-9258.  282 . doi: 10.1074/jbc.M700755200
• Vad, Randi; Nafstad, E; Dahl, LA & Gabrielsen, Odd Stokke (2005). Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone. Journal of Biotechnology.  ISSN 0168-1656.  116
• Crowder, C; Dahle, Øyvind; Davis, RE; Gabrielsen, Odd Stokke & Rudikoff, S (2005). PML mediates IFN-alpha-induced apoptosis in myeloma by regulating TRAIL induction. Blood.  ISSN 0006-4971.  105
• Dahle, Øyvind; Bakke, Oddmund & Gabrielsen, Odd Stokke (2004). c-Myb associates with PML in nuclear bodies in hematopoietic cells. Experimental Cell Research.  ISSN 0014-4827.  297(1), s 118- 126 Vis sammendrag

  The c-Myb transcription factor plays a central role in the regulation of cell growth and differentiation of hematopoietic cells. Being the product of a proto-oncogene, one would expect c-Myb function to be modulated by signal transduction pathways, but our knowledge on such regulation of c-Myb is rather limited. Recently, we and others showed that c-Myb is subjected to sumoylation and that this posttranslational modification has considerable effect on c-Myb's activity. Interestingly, many proteins subjected to SUMO-1 conjugation associate with the promyelocytic leukemia (PML) protein and localize to PML nuclear bodies (PML NBs). Although the precise molecular function of PML NBs still remains to be defined, they seem to play a role in regulation of gene expression and are linked to specific cellular signaling. We show here that c-Myb localizes to PML NBs and that c-Myb interacts with PML as judged by immunofluorescence microcopy and co-immunoprecipitation experiments. Enforced expression of PML IV was shown to enhance c-Myb-dependent reporter activation. Our results imply a role for PML and possibly other components of PML NBs in regulating c-Myb's activity. This novel link between c-Myb and PML, two gene products being implicated in leukemic disorders, suggests that previously unknown mechanisms for regulating c-Myb's activity involving PML may exist.

• Dahle, Øyvind; Bakke, Oddmund & Gabrielsen, Odd Stokke (2004). c-Myb associates with PML in nuclear bodies in hematopoietic cells. Experimental Cell Research.  ISSN 0014-4827.  297, s 118- 126
• Nordgård, Oddmund; Andersen, Tor Øyvind & Gabrielsen, Odd Stokke (2004). Selective inhibition of c-Myb DNA-binding by RNA polymers. BMC Biochemistry.  ISSN 1471-2091.  5(15) . doi: 10.1186/1471-2091-5-15 Vis sammendrag

  The transcription factor c-Myb is expressed in hematopoietic progenitor cells and other rapidly proliferating tissues, regulating genes important for proliferation, differentiation and survival. The DNA-binding domain (DBD) of c-Myb contains three tandemly arranged imperfect repeats, designated Myb domain R1, R2 and R3. The three-dimensional structure of the DBD shows that only the second and third Myb domains are directly involved in sequence-specific DNA-binding, while the R1 repeat does not contact DNA and only marginally affects DNA-binding properties. No structural information is available on the N-terminal 30 residues. Since deletion of the N-terminal region including R1 plays an important role in oncogenic activation of c-Myb, we asked whether this region confers properties beyond DNA-binding to the neighbouring c-Myb DBD.

• Andersson, Kristin Brevik; Kowenz-Leutz, Elisabeth; Brendeford, Elen Margrethe; Tygsett, Ann-Helen Herwig; Leutz, Achim & Gabrielsen, Odd Stokke (2003). Phosphorylation dependent down-regulation of c-Myb DNA-binding is abrogated by a point mutation in the v-myb oncogene. Journal of Biological Chemistry.  ISSN 0021-9258.  278(6), s 3816- 3824
• Dahle, Øyvind; Andersen, Tor Øyvind; Nordgård, Oddmund; Matre, Vilborg; Del Sal, Giannino & Gabrielsen, Odd Stokke (2003). Transactivation properties of c-Myb are critically dependent on two SUMO-1 acceptor sites that are conjugated in a PIASy enhanced manner. European Journal of Biochemistry (EJB).  ISSN 0014-2956.  270(6), s 1338- 1348
• Matre, Vilborg; Høvring, Per Ivar; Fjeldheim, Åse Karine; Helgeland, Lars; Orvain, Christophe; Andersson, Kristin Brevik; Gautvik, Kaare M & Gabrielsen, Odd Stokke (2003). The human neuroendocrine thyrotropin-releasing hormone receptor promotor is activated by the haematopoietic transcription factor c-Myb. Biochemical Journal.  ISSN 0264-6021.  372, s 851-859
• Nordgård, Oddmund; Dahle, Øyvind; Andersen, Tor Øyvind & Gabrielsen, Odd Stokke (2001). JAB1/CSN5 interacts with the GAL4 DNA binding domain: a note of caution about two-hybrid interactions. Biochimie.  ISSN 0300-9084.  83(10), s 969- 971 Vis sammendrag

  The Jun activation domain binding protein 1 (JAB1) was first identified as an interaction partner and coactivator of c-Jun. Subsequently, it was found to be a subunit of the COP9 signalosome (CSN) and termed CSN subunit 5 (CSN5). This complex regulates light-mediated development in plants and plays an essential role in a variety of organisms. A striking feature of JAB1/CSN5 is its reported interaction with a wide range of proteins and its modulation of their activity or stability. We applied the yeast two-hybrid system to screen for proteins interacting with the DNA-binding domain of the transcription factor c-Myb and found JAB1/CSN5 among the double-positive clones. To our surprise JAB1/CSN5 was shown to interact with the DNA-binding domain of GAL4 alone and had to be rejected as a false positive in the GAL4-based two-hybrid system. This finding emphasizes the necessity of particular caution when JAB1/CSN5 is found in two-hybrid screenings.

• Berge, Tone; Bergholtz, Stine Louise; Andersson, Kristin Brevik & Gabrielsen, Odd Stokke (2001). A novel yeast system for in vivo selection of recognition sequences: defining an optimal c-Myb-responsive element. Nucleic Acids Research.  ISSN 0305-1048.  29(20 (E99)) Vis sammendrag

  Yeast (Saccharomyces cerevisiae) has proved to be a highly valuable tool in a range of screening methods. We present in this work the design and use of a novel yeast effector-reporter system for selection of sequences recognised by DNA-binding proteins in vivo. A dual HIS3-lacZ reporter under the control of a single randomised response element facilitates both positive growth selection of binding sequences and subsequent quantification of the strength of the selected sequence. A galactose-inducible effector allows discrimination between reporter activation caused by the protein under study and activation due to endogenous factors. The system mimics the physiological gene dosage relationship between transcription factor and target genes in vivo by using a low copy effector plasmid and a high copy reporter plasmid, favouring sequence selectivity. The utility of the novel yeast screening system was demonstrated by using it to refine the definition of an optimal recognition element for the c-Myb transcription factor (MRE). We present screening data supporting an extended MRE consensus closely mimicking known strong response elements and where a sequence of 11 nt influences activity. Novel features include a more strict sequence requirement in the second half-site of the MRE where a T-rich sequence is preferred in vivo.

• Bergholtz, Stine Louise; Andersen, Tor Øyvind; Andersson, Kristin Brevik; Borrebæk, Jørgen; Lüscher, Bernhard & Gabrielsen, Odd Stokke (2001). The highly conserved DNA-binding domains of A-, B- and c-Myb differ with respect to DNA-binding, phosphorylation and redox properties. Nucleic Acids Research.  ISSN 0305-1048.  29(17), s 3546- 3556 Vis sammendrag

  In the Myb family, as in other families of transcription factors sharing similar DNA-binding domains (DBDs), diversity of function is believed to rely mainly on the less conserved parts of the proteins and on their distinct patterns of expression. However, small conserved differences between DBDs of individual members could play a role in fine-tuning their function. We have compared the highly conserved DBDs of the three vertebrate Myb proteins (A-, B- and c-Myb) and found distinct functional differences. While A- and c-Myb behaved virtually identically in a variety of DNA-binding assays, B-Myb formed complexes of comparatively lower stability, rapidly dissociating under competitive conditions and showing less tolerance to binding site variations. The three protein domains also differed as substrates for protein kinases. Whereas PKA in theory should target the DBDs of A- and c-Myb, but not B-Myb, only c-Myb was phosphorylated by PKA. CK2 phosphorylated all three proteins, although on different sites in the N-terminal region. Finally, B-Myb was remarkably sensitive to cysteine-directed oxidation compared to the other Myb proteins. Our data suggest that the small differences that have evolved between individual Myb family members lead to clear differences in DBD properties even if their sequence recognition remains the same.

• Pinson, Benoit; Brendeford, Elen Margrethe; Gabrielsen, Odd Stokke & Daignan-Fornier, Bertrand (2001). Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors. Nucleic Acids Research.  ISSN 0305-1048.  29(2), s 527- 535 Vis sammendrag

  Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix-turn-helix-related motif with the cysteine packed in the hydrophobic core of the repeat.

• Pinson, Benoit; Gabrielsen, Odd Stokke & Daignan-Fornier, Bertrand (2000). Redox regulation of AMP synthesis in yeast: a role of the Bas1p and Bas2p transcription factors. Molecular Microbiology.  ISSN 0950-382X.  36(6), s 1460- 1469 Vis sammendrag

  Expression of yeast AMP synthesis genes (ADE genes) was severely affected when cells were grown under oxidative stress conditions. To get an insight into the molecular mechanisms of this new transcriptional regulation, the role of the Bas1p and Bas2p transcription factors, known to activate expression of the ADE genes, was investigated. In vitro, DNA-binding of Bas1p was sensitive to oxidation. However, this sensitivity could not account for the regulation of the ADE genes because we showed, using a BAS1-VP16 chimera, that Bas1p DNA-binding activity was not sensitive to oxidation in vivo. Consistently, a triple cysteine mutant of Bas1p (fully resistant to oxidation in vitro) was unable to restore transcription of the ADE genes under oxidative conditions. We then investigated the possibility that Bas2p could be the oxidative stress responsive factor. Interestingly, transcription of the PHO5 gene, which is dependent on Bas2p but not on Bas1p, was found to be severely impaired by oxidative stress. Nevertheless, a Bas2p cysteine-free mutant was not sufficient to confer resistance to oxidative stress. Finally, we found that a Bas1p-Bas2p fusion protein restored ADE gene expression under oxidative conditions, thus suggesting that redox sensitivity of ADE gene expression could be due to an impairment of Bas1p/Bas2p interaction. This hypothesis was further substantiated in a two hybrid experiment showing that Bas1p/Bas2p interaction is affected by oxidative stress.

• Pinson, Benoit; Kongsrud, Torill Line; Ording, Eli; Johansen, Lena; Daignan-Fornier, Bertrand & Gabrielsen, Odd Stokke (2000). Signaling through regulated transcription factor interaction: mapping of a regulatory interaction domain in the Myb-related Bas1p. Nucleic Acids Research.  ISSN 0305-1048.  28(23), s 4665- 4673 Vis sammendrag

  Gene activation in eukaryotes is inherently combinatorial depending on cooperation between different transcription factors. An example where this cooperation seems to be directly exploited for regulation is the Bas1p/Bas2p couple in yeast. Bas1p is a Myb-related transcription factor that acts together with the homeodomain-related Bas2p (Pho2p) to regulate purine and histidine biosynthesis genes in response to extracellular purine limitation. We show that fusion of the two factors abolished adenine repression, suggesting that what is regulated by adenine is the Bas1p-Bas2p interaction. Analysis of Bas1p deletions revealed a critical domain (Bas1p interaction and regulatory domain, BIRD) acting in two-hybrid assays as an adenine-dependent Bas1p-Bas2p interaction domain. BIRD had a dual function, as an internal repressor of a centrally located Bas1p transactivation domain on the ADE1 promoter and as a Bas2p-dependent activator on the HIS4 promoter. This promoter-dependent behavior reflected a differential binding to the two promoters in vivo. On ADE1 Bas1p bound the promoter efficiently by itself, but required adenine limitation and Bas2p interaction through BIRD for derepression. On HIS4 efficient promoter binding and derepression required both factors and adenine limitation. We propose a promoter-dependent model for adenine regulation in yeast based on controlled Bas1p-Bas2p interactions through BIRD and exploited differentially by the two promoters.

• Andersson, Kristin Brevik; Berge, Tone; Matre, Vilborg & Gabrielsen, Odd Stokke (1999). Sequence selectivity of c-Myb in vivo. Resolution of a DNA target specificity paradox. Journal of Biological Chemistry.  ISSN 0021-9258.  274(31), s 21986- 21994 Vis sammendrag

  We have investigated the basis for the striking difference between the broad DNA sequence selectivity of the c-Myb transcription factor minimal DNA-binding domain R(2)R(3) in vitro and the more restricted preference of a R(2)R(3)VP16 protein for Myb-specific recognition elements (MREs) in a Saccharomyces cerevisiae transactivation system. We show that sequence discrimination in yeast is highly dependent on the expression level of Myb effector protein. Full-length c-Myb and a C-terminally truncated protein (residues 1-360) were also included in the study. All of the tested Myb proteins displayed very similar DNA binding properties in electrophoretic mobility shift assays. Only minor differences between full-length c-Myb and truncated c-Myb(1-360) were observed. In transactivation studies in CV-1 cells, the MRE selectivity was highest at low expression levels of Myb effector proteins. However, the discrimination between MRE variants was rapidly lost with high input levels of effector plasmid. In c-Myb-expressing K-562 cells, the high degree of MRE selectivity was retained, thereby confirming the relevance of the results obtained in the yeast system. These data suggest that the MRE selectivity of c-Myb is an intrinsic property of only the R(2)R(3) domain itself and that the transactivation response of a specific MRE in vivo may be highly dependent on the expression level of the Myb protein in the cell.

• Kristensen, Vessela Nedelcheva; Haraldsen, E.K.; Anderson, K.B.; Lønning, Per Eystein; Erikstein, Bjørn; Kåresen, Rolf; Gabrielsen, Odd Stokke & Børresen-Dale, Anne-Lise (1999). CYP 17 and breast cancer risk; the polymorphism in the 5' flanking area of the gene does not influence binding to SP-1. Cancer Research.  ISSN 0008-5472.  59, s 2825- 2828
• Kristensen, Vessela Nedelcheva; Haraldsen, Ellen Christine; Andersson, Kristin Brevik; Lønning, Per Eystein; Erikstein, Bjørn; Kåresen, Rolf; Gabrielsen, Odd Stokke & Børresen-Dale, Anne-Lise (1999). CYP17 and breast cancer risk: the polymorphism in the 5' flanking area of the gene does not influence binding to Sp-1. Cancer Research.  ISSN 0008-5472.  59(12), s 2825- 2828 Vis sammendrag

  The ability of a motif of the CYP17 5' untranslated region, created by a polymorphic T to C substitution, to bind to the human transcription factor Sp-1 was investigated. No binding of any of the polymorphic alleles was observed in electromobility shift assay. No other sequence within +1 to +100 of each of the CYP17 alleles formed complex with the Sp-1 or enhanced binding to the polymorphic CACC box. Genotyping of 510 breast cancer patients and 201 controls revealed no difference in genotype frequencies. Age at onset, tumor grade, lymph node status and distant metastases, stage, and estrogen and progesterone receptor status were not associated with the CYP17 genotype.

• Zargarian, Loussinee; Tilly, Veronique Le; Jamin, Nadege; Chaffotte, A.; Gabrielsen, Odd Stokke; Toma, Flavio & Alpert, B. (1999). Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb. Biochemistry.  ISSN 0006-2960.  38(6), s 1921- 1929 Vis sammendrag

  The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex.The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.

• Brendeford, Elen Margrethe; Andersson, Kristin Brevik & Gabrielsen, Odd Stokke (1998). Nitric oxide (NO) disrupts specific DNA binding of the transcription factor c-Myb in vitro. FEBS Letters.  ISSN 0014-5793.  425(1), s 52- 56 Vis sammendrag

  In an attempt to elucidate signal transduction pathways which may modulate DNA binding of the transcription factor c-Myb, we investigated whether c-Myb could be a target for the signaling molecule nitric oxide (NO) in vitro. NO-generating agents severely inhibited specific DNA binding of the c-Myb minimal DNA-binding domain R2R3. This inhibition was readily reversible upon treatment with excess DTT. A redox-sensitive cysteine (C130) was required for this NO sensitivity. Moreover, a DNA-binding domain carrying two of the avian myeloblastosis virus (AMV)-specific mutations (L106H, V117D) appeared to be less sensitive to S-nitrosylation than the wild-type c-Myb. This difference in NO sensitivity may influence the regulation of wild type versus AMV v-Myb protein function.

• Pinson, Benoit; Sagot, Isabelle; Borne, Françoise; Gabrielsen, Odd Stokke & Daignan-Fornier, Bertrand (1998). Mutations in the yeast Myb-like protein Bas1p resulting in discrimination between promoters in vivo but not in vitro. Nucleic Acids Research.  ISSN 0305-1048.  26(17), s 3977- 3985 Vis sammendrag

  Bas1p is a yeast transcription factor that activates expression of purine and histidine biosynthesis genes in response to extracellular purine limitation. The N-terminal part of Bas1p contains an Myb-like DNA binding domain composed of three tryptophan-rich imperfect repeats. We show that mutating the conserved tryptophan residues in the DNA binding domain of Bas1p severely impairs in vivo activation of target genes and in vitro DNA binding of Bas1p. We also found that two mutations (H34L and W42A) in the first repeat make Bas1p discriminate between promoters in vivo . These two BAS1 mutants are able to activate expression of an HIS4-lacZ fusion but not that of ADE1-lacZ or ADE17-lacZ fusions. Surprisingly, these mutant proteins bind equally well to the three promoters in vitro , suggesting that the mutations affect the interaction of Bas1p with some promoter-specific factor(s) in vivo . By mutating a potential nucleotide binding site in the DNA binding domain of Bas1p, we also show that this motif does not play a major role in purine regulation of Bas1p activity. Finally, using a green fluorescence protein (GFP)-Bas1p fusion, we establish the strict nuclear localization of Bas1p and show that it is not affected by extracellular adenine.

• Brendeford, Elen Margrethe; Myrset, Astrid Hilde; Hegvold, Anne Bostad; Lundin, Maria & Gabrielsen, Odd Stokke (1997). Oncogenic point mutations induce altered conformation, redox sensitivity, and DNA binding in the minimal DNA binding domain of avian myeloblastosis virus v-Myb. Journal of Biological Chemistry.  ISSN 0021-9258.  272(7), s 4436- 4443 Vis sammendrag

  c-Myb is the founder member of a class of transcription factors with tryptophan-rich repeats responsible for DNA binding. Activated oncogenic forms of Myb are encoded by the avian retroviruses, avian myeloblastosis virus (AMV) and E26. AMV v-Myb encodes a truncated protein with 11 point mutations relative to c-Myb. The mutations in the DNA binding domain (DBD) were reported to impose distinct phenotypes of differentiation on transformed myeloid cells (Introna, M., Golay, J., Frampton, J., Nakano, T., Ness, S. A., and Graf, T. (1990) Cell 63, 1287-1297). The molecular mechanism operating has remained elusive since no change in sequence specificity has been found. We introduced AMV-specific point mutations in the minimal DBD of chicken c-Myb and studied their effect on structure and function of the purified protein. Fluorescence emission spectra and fluorescence quenching experiments showed that the AMV-specific point mutations had a significant effect on the conformation of the DBD, giving rise to a more compact structure, a change that was accompanied by a reduced sensitivity toward cysteine-specific alkylation and oxidation. The DNA binding properties were also altered by the AMV-specific point mutations, leading to protein-DNA complexes with highly reduced stability. This reduction in stability was, however, more severe with certain subtypes of binding sequences than with others. This differential behavior was also observed in an in vivo model system where DBD-VP16 fusions were coexpressed with various reporters. These findings imply that different subsets of Myb-responsive promoters may react differentially toward the AMV-specific mutations, a phenomenon that could contribute to the altered patterns of gene expression induced by the AMV v-Myb relative to wild type c-Myb.

• Brendeford, Elen Margrethe; Myrset, Astrid Hilde; Hegvold, Anne Bostad; Lundin, Maria & Gabrielsen, Odd Stokke (1997). Oncogenic point mutations induce altered conformation, redox-sensitivity and DNA-binding in the minimal DNA-binding domain of Avian Myeloblastosis Virus v-Myb. Journal of Biological Chemistry.  ISSN 0021-9258.  272(74436), s 4436- 4443
• Xue, Bin; Gabrielsen, Odd Stokke & Myrset, Astrid Hilde (1997). Capillary electrophoretic mobility shift assay (CEMSA) of a protein-DNA complex. Journal of Capillary Electrophoresis.  ISSN 1079-5383.  4(5), s 225- 231 Vis sammendrag

  The utility of capillary electrophoresis in the study of DNA-protein binding is demonstrated, using the minimal DNA binding domain of the onco-protein c-Myb (R2R3) and a specific target DNA sequence as a model system. The capillary electrophoresis method is based on simple UV detection at 260 nm with a linear polymer buffer and a coated capillary, and requires no labeling or derivatization of the DNA. A specific protein-DNA complex is observable as a retarded peak, which increases with increasing protein concentration with a corresponding reduction in the free DNA peak. With DNA and protein preparations of known concentrations, a test for sequence-specific binding can be completed within 10 min.

• Hegvold, Anne Bostad & Gabrielsen, Odd Stokke (1996). The importance of the linker connecting the repeats of the c-Myb oncoprotein may be due to a positioning function. Nucleic Acids Research.  ISSN 0305-1048.  24(20), s 3990- 3995 Vis sammendrag

  The DNA-binding domain of the oncoprotein c-Myb consists of three imperfect tryptophan-rich repeats, R1, R2 and R3. Each repeat forms an independent mini-domain with a helix-turn-helix related motif and they are connected by linkers containing highly conserved residues. The location of the linker between two DNA-binding units suggests a function analogous to a dimerisation motif with a critical role in positioning the recognition helices of each mini-domain. Mutational analysis of the minimal DNA-binding domain of chicken c-Myb (R2 and R3), revealed that besides the recognition helices of each repeat, the linker connecting them was of critical importance in maintaining specific DNA-binding. A comparison of several linker sequences from different Myb proteins revealed a highly conserved motif of four amino acids in the first half of the linker: LNPE (L138 to E141 in chicken c-Myb R2R3). Substitution of residues within this sequence led to reduced stability of protein-DNA complexes and even loss of DNA-binding. The two most affected mutants showed increased accessibility to proteases, and fluorescence emission spectra and quenching experiments revealed greater average exposure of tryptophans which suggests changes in conformation of the proteins. From the structure of R2R3 we propose that the LNPE motif provides two functions: anchorage to the first repeat (through L) and determination of the direction of the bridge to the next repeat (through P).

Se alle arbeider i Cristin

• Fan, Qiong; Nørgaard, Rikke Christine; Grytten, Ivar; Ness, Cecilie Maria; Lucas, Christin; Vekterud, Kristin; Södling, Helen; Matthews, Jason; Lemma, Roza Berhanu; Gabrielsen, Odd Stokke; Bindesbøll, Christian; Ulven, Stine Marie; Nebb, Hilde Irene; Grønning-Wang, Line Mariann & Sæther, Thomas (2019). LXRα interacts with the glucose-sensing transcription factor ChREBPα to regulate its transcriptional activity.. Vis sammendrag

  The cholesterol-sensing nuclear receptor Liver X Receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in the regulation of glucose and lipid metabolism. LXR does this job in part by regulating the expression of ChREBP. We have previously shown that LXR also regulates ChREBP activity. To get a better understanding of mechanisms at play, we asked if LXR and ChREBP interact physically. Interestingly, LXRα binds to ChREBPα, but not the shorter isoform ChREBPβ. Co-immunoprecipitation (CoIP) of different LXR and ChRBEP domains shows that it is ChREBPα’s low glucose inhibitory domain (LID), which is lacking in ChREBPβ, that interacts with the ligand-binding domain (LBD) of LXRα. In line with this, we see a surprisingly high co-occupancy of LXR and ChREBP on regulatory regions in the mouse genome when re-analysing two independently published chromatin immunoprecipitation-sequencing (ChIP-seq) datasets. Moreover, Functional studies show that LXRα is able to co-activate together with ChREBPα, but not ChREBPβ, and increase ChREBP-specific target gene expression in vitro and in vivo. Unexpectedly however, ligand-engaged LXR exhibits a repressive effect on the expression of the same genes in primary mouse hepatocytes, in contrast to what we observe with target genes that are common to LXR and ChREBP. Performing CoIP and ChIP on selected target genes, we demonstrate mechanistically that the repressive effect most likely is due to a weakened ChREBPα:LXRα interaction and reduced binding of ChREBP to chromatin. Altogether, the novel transcriptional complex comprising ChREBPα and LXRα adds to the intricate integration of nutrient signals in glucose and lipid metabolism.

• Fan, Qiong; Nørgaard, Rikke Christine; Grytten, Ivar; Ulven, Stine Marie; Lucas, Christin; Bindesbøll, Christian; Lemma, Roza Berhanu; Gabrielsen, Odd Stokke; Grønning-Wang, Line Mariann; Nebb, Hilde Irene & Sæther, Thomas (2018). LXRα interacts with the glucose-sensing transcription factor ChREBPα and increases its transcriptional activity.
• Mo, Ingrid; Eriksson, Anna; Brorson, Ina Skaara; Ledsaak, Marit; Harbo, Hanne Flinstad; Gabrielsen, Odd Stokke & Berge, Tone (2017). Identifying protein interaction partners for DEXI - encoded by the dexamethasone-induced gene, DEXI.
• Matre, Vilborg; Aabel, Linn Ingeborg; Ledsaak, Marit; Vollsund, Pernille; Sæther, Thomas & Gabrielsen, Odd Stokke (2013). PTM-crosstalk: SUMO1 acts as a docking domain for the nuclear kinase HIPK1 mediating SUMO-linked phosphorylation of c-Myb and other targets. Vis sammendrag

  Like histones, many transcription factors (TFs) are modified at multiple sites by a diverse set of posttranslational modifications (PTMs) such as phosphorylation, acetylation, methylation, and sumoylation. PTMs are believed to be linked ("TF code") through crosstalk. A classical scenario of PTM-crosstalk is that one specific PTM acts as a docking site for a modification enzyme introducing another PTM. SUMO (small ubiquitin-like modifier) conjugated to TFs is a reversible PTM that modulates the function and/or interactions of the TFs. Here we focus on how SUMO may induce other PTMs in TFs. Our model is the transcription factor c-Myb and the nuclear homeodomain-interacting protein kinases (HJPKl and HJPK2). We have previously identified HIPK1 as an interaction partner for c-Myb (Matre et al, 2009). The function of c-Myb is largely affected by SUMO-conjugation (Molværsmyr et al, 2010) and SUMO-binding (Sæther et al. 2011). Here, we have studied c-Myb PTM-crosstalk by investigating the link between SUMO-conjugation and phosphorylation. We conclude that SUMO conjugated to c-Myb directs the recruitment of HIPK1, making the sumoylated protein the preferred kinase substrate. The SUMO-binding property of HIPK1 is dependent on an active kinase domain. The SUMO-docking property extends to HIPK2 and CtBP1. This work reveals a novel mechanism for PTM-crosstalk on TFs.

• Matre, Vilborg; Aabel, Linn Ingeborg; Ledsaak, Marit; Vollsund, Pernille; Sæther, Thomas & Gabrielsen, Odd Stokke (2012). PTM-crosstalk: SUMO1 acts as a docking domain for the nuclear kinase HIPK1 mediating SUMO-linked phosphorylation of c-Myb and other targets.
• Molværsmyr, Ann-Kristin; Ledsaak, Marit; Alm-Kristiansen, Anne Hege; Lorenzo, Petra Isabel O.; Matre, Vilborg; Sæther, Thomas & Gabrielsen, Odd Stokke (2012). The SUMO E3 ligase PIAS1 interacts with c-Myb and enhances its transcriptional activity. Vis sammendrag

  PIAS1 belongs to the family of Protein Inhibitor of Activated STAT. In addition to their function as negative regulators of STAT signaling, PIAS proteins also act as SUMO E3 ligases, enhancing sumoylation of target proteins. This activity is dependent on a RING finger domain present in PIAS proteins. Even though sumoylation of trancriptional regulators often leads to inhibition of their activity, PIAS proteins are known to act as both negative and positive regulators of transcription, in both SUMO-dependent or independent manners. We have previously identified FLASH (FLICE associated huge protein) as a co-activator of the transcription factor c-Myb. To find additional FLASH-associated proteins, we performed a yeast two-hybrid screening with FLASH as bait, and identified the SUMO E3 ligase PIAS1 as an interacting partner. Functional assays revealed that PIAS1 enhances FLASH's activity and its ability to co-activate c-Myb in a RING finger-dependent manner. The three proteins, FLASH, PIAS1, and c-Myb, are all co-localized with active RNA polymerase II foci. As an E3 ligase we expect PIAS1 to sumoylate c-Myb and inhibit the transcriptional activity of c-Myb. Contrarily, we only see weak sumoylation of c-Myb and an enhancement of c-Myb transactivation. We are currently studying the involvement of p300 in the PIAS1 enhancement of c-Myb transcriptional activity. We suggest that the RING finger domain of PIAS1 have two functions, one being a SUMO E3 ligase and another recruiting the co-activator p300.

• Sæther, Thomas; Bengtsen, Mads; Molværsmyr, Ann-Kristin; Pattabiraman, D.R.; Alm-Kristiansen, Anne Hege; Ledsaak, Marit; Gonda, T.J. & Gabrielsen, Odd Stokke (2012). Regulating the regulator: SUMO functions as a master switch for the transcription factor c-Myb. Vis sammendrag

  The oncoprotein c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. This transcriptional activator plays a direct role in lineage fate selection, cell cycle progression, and differentiation of myeloid as well as B- and T-lymphoid progenitors. Recent studies have shown that c-Myb accomplishes this by controlling the expression of several important hematopoietic transcriptions factors, and in that way acts as a master regulator. c-Myb is modified by SUMO on two lysines in its C-terminal regulatory domain (CRD), and is able to bind SUMO via a SUMO-interacting motif (SIM) in the central transactivation domain. Both these SUMO-contacts are important in regulating the transactivation activity of c-Myb, and breaking these contacts makes c-Myb super active. We have found that the ability of c-Myb to synergize with other transcription factors on compound promoters is restricted by synergy control, linked to the SUMOylation of CRD. De-SUMOylation of c-Myb leads to a dramatic change in synergy behavior, which correlates with a SUMO-dependent differential recruitment of p300 and a corresponding local change in histone H3 and H4 acetylation. Using cell-based transformation assays, we have addressed the abilities of c-Myb SUMO binding and conjugation mutants to transform hematopoietic cells. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhances the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated confers a high proliferative ability and causes an effective differentiation block. This establishes SUMO binding as a mechanism responsible for keeping the transforming potential of the oncoprotein in check. Taken together this implies that SUMO functions as a master regulatory switch for c-Myb. Given that the SUMO-system may be perturbed in different cancers, c-Myb may be particularly vulnerable to such changes and thus mediate dramatic downstream effects.

• Sæther, Thomas; Molværsmyr, Ann-Kristin; Gilfillan, Siv; Alm-Kristiansen, Anne Hege; Aabel, Linn Ingeborg; Matre, Vilborg & Gabrielsen, Odd Stokke (2011). The hematopoietic transcription factor c-Myb functions as an epigenetic organizer. Vis sammendrag

  The c-Myb protein belongs to a group of early hematopoietic transcription factors that are important for progenitor generation and proliferation. These factors have been hypothesized to participate in establishing epigenetic patterns specific for hematopoietic genes. Consistent with its role as a regulator of stem and progenitor cells, a picture is now emerging where c-Myb appears to be directly involved in chromatin modulation. Several lines of evidence, both from our lab and others, point to a particular involvement of c-Myb in histone interaction and chromatin modification. Importantly, the DNA-binding domain of c-Myb not only binds DNA. Its SANT properties also enables it to bind to the N-terminal tail of histone H3 and facilitates histone acetylation by p300. We show that SUMOylation of c-Myb causes differential recruitment of p300 to chromatin leading to a local change in histone H3 and H4 acetylation, supporting the hypothesis of SUMO as a determinant of chromatin signatures. p300/CBP was the first chromatin modifying enzyme demonstrated to be recruited to regulatory gene elements by c-Myb. Today the catalogue also includes MLL, PIAS1, HIPK1, and Mi-2, all of which have enzymatic activity. Together with FLASH and PIAS1, c-Myb also co-localizes with active RNA polymerase II foci, resembling transcription factories. The aim of the present work in to define a specific Myb-induced epigenetic signature by correlating the recruitment of these co-factors with histone marks on Myb-regulated genomic elements.

• Rangberg, Anbjørg; Sæther, Thomas & Gabrielsen, Odd Stokke (2010). Design of a system for inducible SUMOylation of the transcription factor c-Myb. Vis sammendrag

  c-Myb is a transcription factor involved in proliferation and differentiation of hematopoietic progenitor cells. Its activity is regulated by post-translational modifications, including SUMOylation. SUMO (Small Ubiquitin-related Modifier) is covalently conjugated to two specific lysines in c-Myb located in its negative regulatory domain (K503 and K527). Its transactivating potential is severely reduced, even when only a small portion of Myb is SUMOylated. To study the dynamics of c-Myb SUMOylation we have designed a system for inducible SUMOylation inspired by the USDDS method. SUMO is conjugated to targets in a process involving three different enzymes, one of these being the SUMO-conjugating enzyme Ubc9. By bringing Ubc9 and c-Myb together in vivo the level of SUMOylated c-Myb increases. We have taken advantage of the inducible heterodimerization system from ARIAD (www.ariad.com/wt/tertiarypage/kits_info). In dimerization-dependent SUMOylation, c-Myb and Ubc9 are fused to the chemically activatable heterodimerization domains FKBP and FRB respectively. By addition of a small “dimerizer” (rapamycin derivate) the fusion proteins are brought together and SUMO-modification induced. We have established and validated this system in living cells, and are now addressing biologically relevant questions with regard to c-Myb and SUMO. Inducible chimeras might prove to be a valuable tool for the study of posttranslational modifications in general.

• Sæther, Thomas; Molværsmyr, Ann-Kristin; Lorenzo, Petra Isabel O.; Gilfillan, Siv; Kvaløy, Heidi; Matre, Vilborg & Gabrielsen, Odd Stokke (2010). A SUMO-regulated activation function (SRAF) controls synergy of c-Myb through a repressor-activator switch. Vis sammendrag

  Synergy between transcription factors is a well-known phenomenon. An interesting twist to the phenomenon of synergy was the identification of a short protein motif that restricted activator synergy and mediated “synergy control” (SC). Notably, SC motifs turned out to be sites of SUMO-conjugation, and sumoylation was shown to disrupt synergy. The transcription factor c-Myb plays a key role in controlling gene programs during proliferation and differentiation of hematopoietic cells. Several target genes are activated by c-Myb in synergy with other transcription factors, and c-Myb activity is regulated by sumoylation. In the present work we investigate the effect of sumoylation of c-Myb on its synergistic capacity. We find evidence for a strong SUMO-governed synergy control of the factor. Moreover, we make observations that point to a more general mechanism underlying the control of transcriptional synergy. In our mechanistic model the restriction of activating functions (AFs) through sumoylation is a central aspect.

• Sæther, Thomas; Pattabiraman, D.R.; Aabel, Linn Ingeborg; Matre, Vilborg; Gonda, T.J. & Gabrielsen, Odd Stokke (2010). A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability. Vis sammendrag

  c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb. Recently, our lab has identified several interaction partners of c-Myb. Curiously, all these partners are in one way or another linked to the SUMO-system. In this work, we address a novel SUMO-link of c-Myb, non-covalent binding of SUMO to c-Myb. Based on the consensus SUMO-interacting motif (SIM) we identified and examined putative SIMs in human c-Myb. The analysis shown below establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check.

• Sæther, Thomas; Molværsmyr, Ann-Kristin; Lorenzo, Petra Isabel O.; Kvaløy, Heidi; Matre, Vilborg & Gabrielsen, Odd Stokke (2009). A SUMO-regulated activation function (SRAF) controls synergy of c-Myb through a repressor-activator switch. Vis sammendrag

  Synergy between transcription factors operating together on complex promoters is a key aspect of gene activation. The ability of specific factors to synergize is restricted by sumoylation (synergy control). Focusing on the hematopoietic transcription factor c-Myb, we found evidence for a strong synergy control linked to SUMO-conjugation in its negative regulatory domain (NRD), while AMV v-Myb had escaped synergy control. Mechanistic studies revealed a SUMO-dependent switch in the function of NRD. When NRD is sumoylated, it reduces the activity of c-Myb. When sumoylation is abolished, the function of NRD switches into being activating, providing the factor with a second activation function (AF). Thus c-Myb harbours two AFs, one constitutively active and one in NRD being SUMO-regulated (SRAF). This double AF augments c-Myb synergy at compound natural promoters. A similar SUMO-dependent switch was observed in the C-terminal regulatory domain of p53. We therefore propose a general model for SUMO-mediated synergy control. In this model, SUMO controls synergy by determining the number and strength of AFs associated with a promoter through the bound transcription factors.

• Vollsund, Pernille; Bergholtz, Stine Louise; Sæther, Thomas; Aasland, Rein & Gabrielsen, Odd Stokke (2009). Understanding the phosphorylation-sumoylation cross talk in the transcription factor c-Myb. Vis sammendrag

  The transcription factor c-Myb is involved in proliferation and differentiation of hematopoietic cells. c-Myb is regulated by several post-translational modifications (PTMs), including phosphorylation and sumoylation. SUMO (small ubiquitin related modifier) modifies the target protein by covalent conjugation to specific lysines. Previous studies from our group described sumoylation of c-Myb at two sites (K503 and K527) in its negative regulatory domain (NRD) causing severely reduced activity1 . SUMO-conjugation mainly modulates the ability of c-Myb to synergize on complex promoters2. Various phosphorylations of c-Myb have also been reported3,4. Preliminary results indicated that sumoylation and phosphorylation might be closely linked, suggesting a PTM crosstalk. Hence, a series of mutants were made to establish if there is a dependency between these modifications or if they are independent. The preliminary data reported here are mainly based on analysis of migration shifts after phosphatase treatments.

• Sæther, Thomas; Molværsmyr, Ann-Kristin; Matre, Vilborg; Lorenzo, Petra Isabel O. & Gabrielsen, Odd Stokke (2008). SUMO-conjugation of the transcription factor c-Myb controls cooperative behaviour and induces an activator-to-repressor switch in the negative regulatory domain. Vis sammendrag

  The transcription factor c-Myb plays a key role in controlling gene programs during proliferation and differentiation of hematopoietic cells. Several target genes are activated by c-Myb in synergy with other transcription factors. It has been reported that the ability of specific factors to synergize is restricted by sumoylation (synergy control). Since c-Myb is being sumoylated, we investigated the effect of sumoylation of c-Myb on its synergistic capacity. By using a set of dedicated reporters to quantify its synergy behaviour, we found evidence for a strong synergy control linked to SUMO-conjugation in the negative regulatory domain (NRD) of c-Myb. Interestingly, the oncogenic version of c-Myb, AMV v-Myb, lacking most of the NRD including two SUMO-conjugation sites, appears to have escaped synergy control, as judged by the high synergy factor measured. The search for a mechanism revealed a SUMO-dependent switch in the function of NRD. When NRD is sumoylated, it acts negatively by reducing the activity of c-Myb. When sumoylation is abolished, NRD switches into a transactivation domain (TAD), providing the factor with a second TAD. This implies a new picture of c-Myb having two TADs, one centrally located and constitutively active and one in NRD being under SUMO-control. We observed that this increase in the number of TADs augments c-Myb synergy at compound natural promoters as predicted. Furthermore, we suggest that this SUMO-dependent switch mechanism is not specific for c-Myb, since we observed a similar switch when we analyzed the effect of sumoylation in the C-terminal regulatory domain of p53. We therefore propose a general model for SUMO-mediated synergy control in which SUMO restrains the number of active TADs and thus the available co-activator interaction surfaces. In this model, SUMO controls synergy by determining the number and strength of independent and active TADs associated with a promoter through the bound transcription factors.

• Molværsmyr, Ann-Kristin; Kvaløy, Heidi; Sæther, Thomas; Matre, Vilborg & Gabrielsen, Odd Stokke (2007). Controlling gene expression through SUMO-conjugation - the importance of synergy. Vis sammendrag

  Synergy control (SC) is a concept attributed to a short motif (PsiKxE) found in a number of nuclear regulators, acting to limit their transcriptional synergy. Disruption of this motif enhances synergistic activation at compound response elements, without altering the activity driven from a single site. Since SC motifs were found to be identical to sumoylation sites, the research has been focused on SUMO as a repressive modifier per se. We believe that the multiplicity aspect of the SC-motifs is not sufficiently appreciated in the current understanding of how SUMO controls transcriptional processes. We have found that SC is strongly operating on the oncoprotein c-Myb. By using reporters with variable numbers of MREs, we have observed that the level of synergy appears to be highly dependent on whether c-Myb can be sumoylated or not. Studies on v-Myb, the oncogenic version of c-Myb which cannot be sumoylated, imply that escape from SC might be part of an oncogenic strategy. Working with this model, we unexpectedly came across a conditional transactivation domain (TAD), embedded within c-Myb’s traditionally described negative regulatory domain (NRD). When tethering c-Myb NRD to a Gal4 responsive reporter, this domain switches from repressing to strongly activating transcription, when sumoylation is abrogated by mutating the SC motif. The mechanism and precise location of this switchable TAD are currently being studied. Furthermore, an exciting feature of the SUMO-switchable TAD construct is the possibility to monitor the efficiency or equilibrium of the SUMO conjugation/de-conjugation apparatus in the cell. This is now being tested.

• Sæther, Thomas; Berge, Tone; Ledsaak, Marit; Matre, Vilborg; Alm-Kristiansen, Anne Hege; Dahle, Øyvind; Aubry, Florence & Gabrielsen, Odd Stokke (2007). The chromatin remodelling factor Mi-2alpha acts as a novel co-activator for human c-Myb. Vis sammendrag

  The c-Myb protein belongs to a group of early hematopoietic transcription factors that are important for progenitor generation and proliferation. These factors have been hypothesized to participate in establishing chromatin patterns specific for hematopoietic genes. In a two-hybrid screening we identified the chromatin remodelling factor Mi-2alpha as an interaction partner for human c-Myb. The main interacting domains were mapped to the N-terminal region of Mi-2alpha and the DNA-binding domain of c-Myb. Surprisingly, functional analysis revealed that Mi-2alpha previously studied as a subunit in the NuRD co-repressor complex, enhanced c-Myb dependent reporter activation. Consistently, knock-down of endogenous Mi-2alpha in c-Myb expressing K562 cells led to down-regulation of the c-Myb target genes NMU and ADA. When wild-type and helicase-dead Mi-2alpha were compared, the Myb-Mi-2alpha co-activation appeared to be independent of the ATPase/DNA helicase activity of Mi-2alpha. The rationale for the unexpected co-activator function seems to lie in a dual function of Mi-2alpha, by which this factor is able to repress transcription in a helicase-dependent and activate in a helicase-independent fashion, as revealed by Gal4 tethering experiments. Interestingly, desumoylation of c-Myb potentiated the Myb-Mi-2alpha transactivational co-operation, as did cotransfection with p300.

• Alm-Kristiansen, Anne Hege; Sæther, Thomas; Ledsaak, Marit; Matre, Vilborg & Gabrielsen, Odd Stokke (2006). FLASH is a coregulator of the transcription factor c-Myb.
• Sæther, Thomas; Berge, Tone; Ledsaak, Marit; Dahle, Øyvind; Matre, Vilborg; Lüscher, Bernhard & Gabrielsen, Odd Stokke (2004). Human c-Myb interacts with the Mi-2alpha subunit of the NuRD complex. Vis sammendrag

  The c-Myb protein belongs to a group of ´early´ haematopoietic transcription factors that are important for progenitor generation and proliferation. These factors have been hypothesized to participate in establishing chromatin patterns specific for haematopoietic genes [Muller and Leutz (2001) Curr Opin Genet Dev 11, 167-174]. In a two-hybrid screen, we identified Mi-2alpha as an interaction partner for human c-Myb. Mi-2alpha is a member of the NuRD (Nucleosome Remodelling and histone Deacetylase) co-repressor complex and responsible for its chromatin remodelling activity. Using various interaction assays, we confirmed the association and mapped the main interacting domains to the C-terminal region of Mi-2alpha (aa 1653-2000) and the FAETL region (leucin-rich; aa. 352-417) of c-Myb. Myb shows affinity for the entire NuRD complex as revealed by analysis of other components of the complex; in transfected cells we detected association between c-Myb and HDAC1, and from Jurkat nuclear extracts we pulled down endogenous c-Myb using antibodies against MTA2 and RbAp46. Functional analysis revealed that while the C-terminal interaction domain in Mi-2alpha decreased c-Myb´s DNA binding and inhibited Myb-dependent reporter-activation, the full-length Mi-2alpha apparently enhanced c-Myb dependent reporter activation, a phenomenon that seems to be related to Mi-2alpha stabilizing the c-Myb protein. The link between SUMO-1 modification of c-Myb and Mi-2alpha interaction is currently being addressed. Overall, our results point to the direct involvement of c-Myb in the restructuring of chromatin through its interaction with the NuRD complex.

• Dahle, Øyvind; Bergeland, Trygve; Nordgård, Oddmund; Andersen, Tor Øyvind; Sal, Giannino Del & Gabrielsen, Odd Stokke (2001). SUMOylation of the human oncoprotein c-Myb and its implications. Vis sammendrag

  The c-Myb oncoprotein binds specific DNA-sequences and regulates target genes important for normal function of the hematopoietic system. It was first identified as v-myb encoded by leukemic viruses, which can induce myeloblastic leukemia in chicken and mice. The v-Myb protein is a truncated version of the normal c-Myb protein. To understand molecular and functional differences between c-Myb and v-Myb we set out to identify novel c-Myb interaction partners. Here we report the identification of hUbc9 as a interaction partner of human c-Myb in a GAL4-based two-hybrid screening using human bone marrow and K562 libraries. Ubc9 is an E2-homolougous enzyme and conjugates the ubiquitin-like protein SUMO-1 to specific protein targets (e.g. p53 and c-Jun). SUMOylation has been shown to protect proteins against ubiquitin-mediated degradation and is also important for the formation of subnuclear structures called PML nuclear bodies. In this work we show that human c-Myb is SUMOylated by Ubc9. Indirect immunofluorescence experiments show that c-Myb co-localizes with SUMO-1 and PML in nuclear bodies. We have identified two consensus sites of SUMOylation, which are deleted in v-Myb. Mutation of these sites eliminates SUMOylation of c-Myb, increases its activity dramatically and alters the subnuclear localization of c-Myb to some extent.

• Berge, Tone; Dittrich, Oliver; Gabrielsen, Odd Stokke & Lüscher, Bernhard (2001). Chromatin Immunoprecipitation (ChIP) analysis of the human adenosine deaminase (hADA) gene ¿ c-Myb and polymerase occupancy and histone acetylation status. Vis sammendrag

  The c-Myb protein is a transcription factor encoded by the c-myb proto-oncogene. A large body of evidence suggests that c-Myb is involved in regulating cell growth and differentiation in hematopoietic cells. As a transcription factor c-Myb exerts its function through recognition of specific response elements in the regulatory regions of its target genes. Much effort has been put into identifying relevant target genes for c-Myb action, and binding sites for c-Myb have been identified in an increasing number of gene promoters, including mim-1, neutrophil elastase (ELA2), cdc-2, c-myc, c-myb, bcl-2, TCR d and g chains, tom-1, CD4, ADA, lck and RAG-2. Using the Chromatin Immunoprecipitation (ChIP) technique, c-Myb binding to different proposed c-Myb target genes has been analysed in different cell lines. We found that c-Myb bound to intron 1 of the human adenosine deaminase (hADA) gene, containing a c-Myb binding motif. We also compared this binding to the Myb-occupancy observed on other proposed c-Myb targets. ChIP data of polymerase loading and histone acetylation of the hADA locus will be discussed in relation to c-Myb binding and hADA gene expression levels.

• Kongsrud, Torill Line; Pinson, Benoit; Daignan-Fornier, Bertrand & Gabrielsen, Odd Stokke (2001). The Myb-related Bas1p transcription factor ¿ dissection of domains involved in DNA-interaction in vitro and in vivo. Vis sammendrag

  Bas1p is a Myb-related TF that cooperates with the homeodomain-related Bas2p to regulate purine and histidine biosynthesis genes in yeast in response to extracellular adenine limitation. An adenine-dependent signal appears to somehow suppress the interaction between Bas1p and Bas2p and thereby inhibit expression of ADE genes. We identified a ¿BIRDomain¿ in Bas1p that is critical for both the adenine regulatory response and the interaction with Bas2p (1). In the present work we have further dissected functional domains of Bas1p involved in DNA-binding. When we mapped the DNA-binding domain of Bas1p using a HIS4-derived Bas1p-response element in vitro (2), the very N-terminal was not essential for binding. When the N-terminal deletion was assayed in vivo, it surprisingly abolished activation of an ADE1-lacZ reporter. A VP16-fusion approach implied that the lack of activation was caused by loss of DNA-binding in vivo. When elements from the ADE1 promoter was assayed for binding to wt and N-terminally truncated recombinant Bas1p, we found again the N-terminal to be dispensable for DNA-binding in vitro. This implies that the N-terminal of the Bas1p could be important for productive interaction with its binding sites in a chromatin context but not on naked DNA. Analysis of the phenomenon using chromatin-related disruption strains will be presented.

• Matre, Vilborg; Høvring, Per Ivar; Fjeldheim, Åse Karine; Orvain, Christophe; Andersson, Kristin Brevik; Gautvik, Kaare M & Gabrielsen, Odd Stokke (2001). Transactivation of the human TRH by c-Myb; a possible link beetween the neuroendocrine and the immune systems. Vis sammendrag

  Thyrotropin (TRH) receptor (TRHR) is a G protein-coupled receptor playing a crucial role in the anterior pituitary where it controls the synthesis and secretion of TSH and PRL. Its widespread presence not only in the CNS, but also in thymus indicates unknown functions of TRH, possibly a role in the immune system. We report that the human TRHR promoter contains several putative response elements for the haematopoetic transcription factor c-Myb an is highly Myb-responsive in transfection assays. DNA-binding analysis revealed one preferred Myb-binding site in intron 1. This element conferred Myb-dependent activatian of reporter plasmids in CV-1 cells. The Myb-dependent activatian of TRHR promoter was strongly suppressed by a dominant negative Myb-Engrailded fusion. Furthermore, RT-PCR analysis of rat tissues and cell lines showed that the TRHR gene is expressed both in thymocytes, bone marrow and the NK-cell line RNK-16. Also, specific TRH agonist binding to cell surface receptors was demonstrated in thymocytes and RNK-16 cells. Our findings suggest a novel link between the neuroendocrine and the immune systems at the transcriptional level.

• Orvain, Christophe & Gabrielsen, Odd Stokke (2001). Evidence that the transcription factor c-Myb is involved in pre-mRNA splicing.
• Dahle, Øyvind; Nordgård, Oddmund; Recordon, Patricia & Gabrielsen, Odd Stokke (2000). Two-hybrid screening for protein interaction partners of c-Myb. Vis sammendrag

  The myb gene family consists of three members A-, B- and c-myb, that are related through their high homology in the N-terminal DNA-binding domain (DBD). These genes encode transcription factors, which activate their target genes through a central transactivation domain. Although the three Myb proteins have overlapping DNA binding specificity, their transactivation potential varies with cell type and promoter context. Therefore they probably have different cell specific interaction partners. Myb proteins also have a C-terminal regulatory domain, that regulates their transactivation of the target genes. We have focused this work on c-Myb, which has a well established role in oncogenesis. Previous studies has shown that c-Myb cooperates with other transcription factors in transactivation of target promotors. In this work we have applied a GAL4-based two-hybrid system to identify proteins that interact with the DBD of c-Myb. At the present stage, we have performed three screenings with the DBD as bait, and 8 clones were found to activate both HIS3 and LacZ reporters. Three of these encoded JAB-1 (Jun Activation domain Binding protein) and the other five GEF-2 (gangliosid exchange factor). JAB-1 was shown to interact with the Gal4-DBD and is clearly a false positive. GEF-2 interacts specifically with c-Myb-DBD, but is so far poorly charachterized. We are now in the process of making a new bait, which spans a region in the C-terminal part of c-Myb.

• Nordgård, Oddmund; Dahle, Øyvind; Andersen, Tor Øyvind & Gabrielsen, Odd Stokke (2000). Is JAB1 an interaction partner of c-Myb?. Vis sammendrag

  The Jun activation domain binding protein 1 (JAB1) was first identified as a coactivator of c-Jun using the activation domain of c-Jun as a bait in a GAL4-based two-hybrid screening [1]. More recently, JAB1 were found to be part of the mammalian 450 kDa signalosome complex, which is similar to the plant COP9 complex involved in light-mediated signal transduction [2,3]. The 450 kDa signalosome phosphorylates I\kappa B and c-Jun and is structurally similar to the proteasome regulatory complex and the eIF3 translation-initiation factor complex. Two-hybrid screenings have recently identified JAB1 as an interaction partner of the cyclin-dependent-kinase inhibitorq protein p27^{Kip1} and the Bcl-3 oncoprotein [4,5]. The c-Myb oncoprotein is a transcription factor that regulates proliferation and differentiation in hematopoietic cells [6]. We searched for novel interaction partners of c-Myb using its DNA-binding domain (DBD) as bait in a GAL4-based yeast two-hybrid system. Screening a cDNA library from human bone marrow, we identified JAB1 as a putative c-Myb interactant. However, co-transformation of the yeast reporter strain with a vector encoding the JAB1-fusion protein and a vector encoding only the DNA-binding DNA-binding domain (DBD) of GAL4 activated the reporter genes without c-Myb. Thus, our studies indicate that JAB1 interacts with the GAL4-DBD alone and therefore has to be added to the already long list of false positive in the GAL4-based yeast two-hybrid system. The possibility that c-Myb interacts specifically with JAB1 was rejected.

• Berge, Tone; Andersson, Kristin Brevik & Gabrielsen, Odd Stokke (2000). An in vivo selection system based on yeast to identify optimal response-elements for DNA-binding transcription factors. Vis sammendrag

  The onco-protein c-Myb is a DNA-binding transcription factor regulating genes important for differentiation, proliferation and apoptosis of haematopoietic cells. It is a paradox that c-Myb on one hand regulates critical genes in specific cell types, but on the other hand binds a DNA-target sequence so loosely defined that Myb-responsive elements (MRE) must be present at an improbable frequency in the genome. One solution to this paradox is that Myb proteins cooperate with other transcription factors. Another contribution may be that MREs are more precisely defined in vivo than what is revealed from in vitro studies. We have recently shown that MRE-sequences that bind c-Myb equally well in vitro and give similar transactivation levels in transient transfected mammalian cells, result in very different transactivation responses in vivo in an effector-reporter system in yeast where more physiological protein levels can be obtained (Andersson et al., 1999). In this work we constructed a dual reporter plasmid where two genes, HIS3 and lacZ, are under control of the same promoter element. Myb-dependent activation of the reporter induces expression of a fusion protein with two active parts, imidazoleglycerol-phosphate dehydratase and b-galactosidase. The HIS3 gene product allows us to screen many transformants by growing them on selective medium, while a b-galactosidase activity assay gives us the degree of transactivation obtained. We have used this system with a partially randomised library to screen for optimal MRE¿s. Until now, 2000 clones are screened and out of these 130 contain reporter plasmids that are transactivated by c-Myb, a fraction of them again gives high b-galactosidase activity. We will describe this yeast selection system, present the different MRE¿s that were selected and from this deduce optimal Myb-response elements in vivo.

• Berge, Tone; Andersson, Kristin Brevik & Gabrielsen, Odd Stokke (2000). An in vivo selection system in yeast to identify optimal response elements for the c-Myb transcription factor. Vis sammendrag

  The onco-protein c-Myb is a DNA-binding transcription factor regulating genes important for differentiation, proliferation and apoptosis of haematopoietic cells. It is a paradox that c-Myb on one hand regulates critical genes in specific cell types, but on the other hand binds a DNA-target sequence so loosely defined that Myb-responsive elements (MRE) must be present at an improbable frequency in the genome. One solution to this paradox is that Myb proteins cooperate with other transcription factors. Another contribution may be that MREs are more precisely defined in vivo than what is revealed from in vitro studies. We have recently shown that MRE-sequences that bind c-Myb equally well in vitro and give similar transactivation levels in transient transfected mammalian cells, result in very different transactivation responses in vivo in an effector-reporter system in yeast where more physiological protein levels can be obtained (Andersson et al., 1999). In this work we constructed a dual reporter plasmid where two yeast genes, HIS3 and lacZ, are fused and under control of the same promoter element. We have used this system with a partially randomised MRE library (keeping the AAC core of MRE constant) to screen for optimal MREs. Of 2000 clones screened, 130 contained reporter plasmids that were transactivated by c-Myb, and of these, 70 gave b-galactosidase activity above 20 units. From sequencing of rescued reporters, we deduced an in vivo consensus sequence for MRE. This consensus conforms to the in vitro derived consensus, but shows that nucleotides in positions outside the earlier identified MRE seem to be important for binding of Myb to DNA in vivo. In addition, our method showed a strong selection for multiple Myb-core binding sites. In this screening, we also identified some novel MREs showing high Myb-dependent activation in vivo despite important deviations from the established MRE consensus. Studies of these novel sites are in progress.

• Brendeford, Elen Margrethe; Andersson, Kristin Brevik; Tygsett, Ann-Helen Herwig; Kowenz-Leutz, Elisabeth; Leutz, Achim & Gabrielsen, Odd Stokke (2000). A phosphorylation site in the DNA-binding domain of the transcription factor c-Myb that is lost in AMV v-Myb. Vis sammendrag

  The oncoprotein c-Myb is a sequence specific transcription factor, regulating genes important for proliferation and differentiation of hematopoietic cells. Its DNA-binding domain (DBD) consists of three tryptophan rich HTH-related repeats designated R1, R2 and R3. The c-Myb protein has been extensively characterized, but little information is available regarding regulation of c-Myb function by signal transduction mechanisms. Recently we have identified a kinase that is able to phosphorylate the DBD of c-Myb but not its oncogenic counterpart v-Myb from avian myeloblastosis virus (AMV). As expected for a phosphorylation in DBD, DNA-binding was affected by the kinase, but in a fashion that depended on the recognition site studied. Several assays have been used to study the functional consequence of this phosphorylation, including use of phospho-specific antibody to monitor the extent of phosphorylation in vivo. These observations suggests a possible connection between an oncogenic form of a transcription factor and a signal transduction mechanism, where the oncogenic form of the protein escape cellular control because it can no longer be phosphorylated by the kinase.

• Brendeford, Elen Margrethe; Andersson, Kristin Brevik; Tygsett, Ann-Helen Herwig; Kowenz-Leutz, Elisabeth; Leutz, Achim & Gabrielsen, Odd Stokke (2000). STUDY OF A PHOSPHORYLATION SITE IN THE DNA-BINDING DOMAIN OF THE TRANSCRIPTION FACTOR c-MYB WHICH IS ABSENT IN AMV v-MYB. Vis sammendrag

  The oncoprotein c-Myb is a sequence specific transcription factor, regulating genes important for proliferation and differentiation of hematopoietic cells. Its DNA-binding domain (DBD) consists of three tryptophan rich HTH-related repeats designated R1, R2 and R3. The c-Myb protein has been extensively characterized, but little information is available regarding regulation of c-Myb function by signal transduction mechanisms. Recently we have identified a kinase that is able to phosphorylate the DBD of c-Myb but not its oncogenic counterpart v-Myb from avian myeloblastosis virus (AMV). As expected for a phosphorylation in DBD, DNA-binding was affected by the kinase, but in a fashion that depended on the recognition site studied. Several assays have been used to study the functional consequence of this phosphorylation, including use of phospho-specific antibody to monitor the extent of phosphorylation in vivo. These observations suggests a possible connection between an oncogenic form of a transcription factor and a signal transduction mechanism, where the oncogenic form of the protein escape cellular control because it can no longer be phosphorylated by the kinase.

• Gabrielsen, Odd Stokke; Matre, Vilborg & Bergholtz, Stine Louise (2000). Protein-Oligonucleotide Interactions, In R. A. Meyers (ed.),  Encyclopedia of Analytical Chemistry.  John Wiley & Sons.  s 5997 - 6017 Vis sammendrag

  The specific binding of a protein to a nucleic acid is a first step in several central processes in a living cell. Sequence-specific protein-DNA interactions are crucial for the functional read-out of genetic information. Sequence recognition is the result of a concerted action of many weak chemical interactions of different types between the protein and its DNA target, including non-specific electrostatic interactions, hydrogen bonding and van der Waals contacts. The precise complementarity of shape between the two macromolecules facilitates specific chemical recognition to be established. The electrophoretic mobility shift assay (EMSA) and several variants of footprinting are simple electrophoretic methods developed to study protein-DNA interactions. Because the specificity is determined by the nucleic acid sequence, the same methods can be exploited for a wide range of proteins simply by changing the sequence of the nucleic acid. EMSA detects sequence-specific DNA-binding activity in a protein sample as a separate migrating band in a non-denaturating gel. A footprinting method provides more detailed information on the precise location of a bound protein along the DNA fragment through the removal of specific bands in a pattern of cleaved fragments separated by electrophoresis. Both methods are highly sensitive due to the use of radioactively labeled oligonucleotides and can be performed with protein samples of low purity. When combined these methods are capable of providing a picture of the protein-DNA complex with a great deal of molecular detail, only surpassed by the more demanding methods of crystallography and NMR.

• Pinson, Benoit; Kongsrud, Torill Line; Ording, Eli; Johansen, Lena; Daignan-Fornier, Bertrand & Gabrielsen, Odd Stokke (2000). The Myb-related yeast transcription factor Bas1p has a signal-responsive repression/activation-domain. Vis sammendrag

  We are interested in whether the Myb-related Bas1p could become a fertile model for c-Myb research. The study of a yeast factor has the advantage of allowing a combined genetic and biochemical approach. Bas1p is a Saccharomyces cerevisiae transcription factor that cooperates with the homeodomain-related Bas2p to regulate expression of purine (ADE genes) and histidine (HIS genes) biosynthesis genes. This expression responds to extracellular purine limitation, i.e. expression of Bas1p/Bas2p target genes are reduced in the presence of external adenine. Bas1p and Bas2p functions can easily be scored in vivo since mutations that abolish the function of one of these factors lead to histidine auxotrophy in a gcn4 genetic background. Our goal was to determine the molecular mechanism involved in this signal responsive regulation.

• Nordgård, Oddmund & Gabrielsen, Odd Stokke (1999). Macromolecular interactions of the c-Myb oncoprotein.
• Berge, Tone; Andersson, Kristin Brevik & Gabrielsen, Odd Stokke (1999). Improved model systems for studies of transcriptional control in vivo.
• Brendeford, Elen Margrethe; Andersson, Kristin Brevik; Pinson, Benoit; Daignan-Fornier, Bertrand & Gabrielsen, Odd Stokke (1999). REDOX REGULATION OF SPECIFIC DNA-BINDING OF THE TRANSCRIPTION FACTOR c-MYB.
• Pinson, Benoit; Gabrielsen, Odd Stokke & Daignan-Fornier, Bertrand (1999). Redox regulation of yeast biosynthesis genes.
• Olsen, O. N.; Bergholtz, Stine Louise; Granum, P.E. & Gabrielsen, Odd Stokke (1998). Studies on the molecular interactions of c-Myb by one -hybrid and pull-down strategies.
• Andersson, Kristin Brevik; Berge, Tone; Matre, Vilborg & Gabrielsen, Odd Stokke (1998). The activity of Myb MRE's in vivo is dependent on the R2R3 domain alone and the protein expression level.
• Berge, Tone; Andersson, Kristin Brevik & Gabrielsen, Odd Stokke (1998). Design of an in vitro selection system based on yeast to identify optimal response-elements for DNA-binding transcription factors.
• Gabrielsen, Odd Stokke (1998). Når gener blir syke-hva genteknologien har lært oss om kreft.
• Gabrielsen, Odd Stokke (1998). The Myb-related transcription factor BAS1 in yeast - a useful model system for c-Myb?.
• Kongsrud, Torill Line; Johansen, L.; Ording, Eli; Pinson, Benoit; Daignan-Fornier, Bertrand & Gabrielsen, Odd Stokke (1998). The Myb-related yeast transcription factor BAS1 contains a chameleon domain as an internal repressor and as an external activation domain.
• Kongsrud, Torill Line; Paramu, V.; Ording, Eli; Johansen, L. & Gabrielsen, Odd Stokke (1998). Transactivation studies of BAS1 and BAS2, two related transcription factors from yeast.
• Olsen, O. N. & Gabrielsen, Odd Stokke (1998). One hybrid screening for proteins binding the Myb recognititon.
• Andersson, Kristin Brevik & Gabrielsen, Odd Stokke (1997). Functional differences between Myb-binding sequences in mammalian cells.
• Berge, Tone; Andersson, Kristin Brevik & Gabrielsen, Odd Stokke (1997). Design of an in vivo selection system based on yeast to identify optimal respons-element for DNA-binding transcription factors.
• Brendeford, Elen Margrethe; Bergholtz, Stine Louise; Ording, Eli; Risøen, Per Arne; Matre, Vilborg & Gabrielsen, Odd Stokke (1997). C-Myb - Midifractions that affect DNA-binding.
• Gabrielsen, Odd Stokke (1997). The Myb-domain - a flexible DNA-binding domain with regulatory potential, used by trancripton factors from yeast ro humans.
• Gabrielsen, Odd Stokke (1997). The Myb-domain - a flexible DNA-binding domain with regulatory potential, used by transcription factors from yeast to humans.
• Gabrielsen, Odd Stokke (1997). The oncoprotein c-Myb - some lessons from studies in vitro, in yeast and in cell culture.
• Ording, Eli; Bergholtz, Stine Louise; Brendeford, Elen Margrethe; Jamin, Nadege & Gabrielsen, Odd Stokke (1997). Flexibility in the second half-sie sequence recognized by the c-Myb R2 domain - in vitro and in vivo analysis.
• Borrebæk, Jørgen; Henriksen, J. & Gabrielsen, Odd Stokke (1996). DNA-binding studies of the B-Myb transcription factor.
• Borrebæk, Jørgen; Ording, Eli & Gabrielsen, Odd Stokke (1996). B-Myb versus c-Myb. In vitro DNA-binding studies.
• Brendeford, Elen Margrethe; Myrset, Astrid Hilde; Hegvold, Anne Bostad; Lundin, Maria & Gabrielsen, Odd Stokke (1996). Altered conformation, DNA-binding and redox-sensitivity on the minimal DNA-binding domain of AMV v-Myb.
• Gabrielsen, Odd Stokke (1996). BAS1 - a Myb homologue in yeast.
• Gabrielsen, Odd Stokke (1996). c-Myb-transription factor and oncoprotein - some aspects of structure, DNA-binding and regulatory mechanisms.

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