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Fan, Qiong; Nørgaard, Rikke Christine; Grytten, Ivar; Ness, Cecilie Maria; Lucas, Christin & Vekterud, Kristin
[Vis alle 15 forfattere av denne artikkelen]
(2019).
LXRα interacts with the glucose-sensing transcription factor ChREBPα to regulate its transcriptional activity.
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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.
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Fan, Qiong; Nørgaard, Rikke Christine; Grytten, Ivar; Ulven, Stine Marie; Lucas, Christin & Bindesbøll, Christian
[Vis alle 11 forfattere av denne artikkelen]
(2018).
LXRα interacts with the glucose-sensing transcription factor ChREBPα and increases its transcriptional activity.
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Mo, Ingrid; Eriksson, Anna; Brorson, Ina Skaara; Ledsaak, Marit; Harbo, Hanne Flinstad & Gabrielsen, Odd Stokke
[Vis alle 7 forfattere av denne artikkelen]
(2017).
Identifying protein interaction partners for DEXI
- encoded by the dexamethasone-induced gene, DEXI
.
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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.
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Sæther, Thomas; Bengtsen, Mads; Molværsmyr, Ann-Kristin; Pattabiraman, D.R.; Alm-Kristiansen, Anne Hege & Ledsaak, Marit
[Vis alle 8 forfattere av denne artikkelen]
(2012).
Regulating the regulator: SUMO functions as a master switch for the transcription factor c-Myb.
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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.
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Molværsmyr, Ann-Kristin; Ledsaak, Marit; Alm-Kristiansen, Anne Hege; Lorenzo, Petra Isabel O.; Matre, Vilborg & Sæther, Thomas
[Vis alle 7 forfattere av denne artikkelen]
(2012).
The SUMO E3 ligase PIAS1 interacts with c-Myb and enhances its transcriptional activity.
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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.
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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.
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Sæther, Thomas; Molværsmyr, Ann-Kristin; Gilfillan, Siv ; Alm-Kristiansen, Anne Hege; Aabel, Linn Ingeborg & Matre, Vilborg
[Vis alle 7 forfattere av denne artikkelen]
(2011).
The hematopoietic transcription factor c-Myb functions as an epigenetic organizer.
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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.
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Sæther, Thomas; Molværsmyr, Ann-Kristin; Lorenzo, Petra Isabel O.; Gilfillan, Siv ; Kvaløy, Heidi & Matre, Vilborg
[Vis alle 7 forfattere av denne artikkelen]
(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.
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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.
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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.
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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.
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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.
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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.
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Sæther, Thomas; Berge, Tone; Ledsaak, Marit; Matre, Vilborg; Alm-Kristiansen, Anne Hege & Dahle, Øyvind
[Vis alle 8 forfattere av denne artikkelen]
(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.
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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.
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Alm-Kristiansen, Anne Hege; Sæther, Thomas; Ledsaak, Marit; Matre, Vilborg & Gabrielsen, Odd Stokke
(2006).
FLASH is a coregulator of the transcription factor c-Myb.
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Sæther, Thomas; Berge, Tone; Ledsaak, Marit; Dahle, Øyvind; Matre, Vilborg & Lüscher, Bernhard
[Vis alle 7 forfattere av denne artikkelen]
(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.
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Orvain, Christophe & Gabrielsen, Odd Stokke
(2001).
Evidence that the transcription factor c-Myb is involved in pre-mRNA splicing.
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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.
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Matre, Vilborg; Høvring, Per Ivar; Fjeldheim, Åse Karine; Orvain, Christophe; Andersson, Kristin Brevik & Gautvik, Kaare M
[Vis alle 7 forfattere av denne artikkelen]
(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.
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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.
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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.
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Gabrielsen, Odd Stokke; Matre, Vilborg & Bergholtz, Stine Louise
(2000).
Protein-Oligonucleotide Interactions.
I Meyers, R. A. (Red.),
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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Nordgård, Oddmund & Gabrielsen, Odd Stokke
(1999).
Macromolecular interactions of the c-Myb oncoprotein.
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Berge, Tone; Andersson, Kristin Brevik & Gabrielsen, Odd Stokke
(1999).
Improved model systems for studies of transcriptional control in vivo.
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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.
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Pinson, Benoit; Gabrielsen, Odd Stokke & Daignan-Fornier, Bertrand
(1999).
Redox regulation of yeast biosynthesis genes.
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Gabrielsen, Odd Stokke
(1998).
Når gener blir syke-hva genteknologien har lært oss om kreft.
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Gabrielsen, Odd Stokke
(1998).
The Myb-related transcription factor BAS1 in yeast - a useful model system for c-Myb?
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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.
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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.
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Olsen, O. N. & Gabrielsen, Odd Stokke
(1998).
One hybrid screening for proteins binding the Myb recognititon.
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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.
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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.
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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.
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Gabrielsen, Odd Stokke
(1997).
The Myb-domain - a flexible DNA-binding domain with regulatory potential, used by trancripton factors from yeast ro humans.
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Gabrielsen, Odd Stokke
(1997).
The Myb-domain - a flexible DNA-binding domain with regulatory potential, used by transcription factors from yeast to humans.
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Gabrielsen, Odd Stokke
(1997).
The oncoprotein c-Myb - some lessons from studies in vitro, in yeast and in cell culture.
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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.
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Brendeford, Elen Margrethe; Bergholtz, Stine Louise; Ording, Eli; Risøen, Per Arne; Matre, Vilborg & Gabrielsen, Odd Stokke
(1997).
C-Myb - Midifractions that affect DNA-binding.
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Andersson, Kristin Brevik & Gabrielsen, Odd Stokke
(1997).
Functional differences between Myb-binding sequences in mammalian cells.