Ann-Kristin Molværsmyr

• Søland, Tine Merete; Lipka, Aleksandra Katarzyna; Ruus, Ann-Kristin; Molværsmyr, Ann-Kristin; Galtung, Hilde & Haug, Trude Marie (2024). Extracellular vesicles from cancer cell lines of different origins drive the phenotype of normal oral fibroblasts in a CAF-like direction. Frontiers in Oncology. 14. doi: 10.3389/fonc.2024.1456346. Fulltekst i vitenarkiv Vis sammendrag

  Introduction: Normal oral fibroblasts (NOFs) are located in the connective tissue of the oral mucosa. The NOFs play an important role in wound healing, tumor progression, and metastasis. They are subjected to influence by external and internal stimuli, among them extracellular vesicles (EVs), that are considered as important players in cell to cell communication, especially in carcinogenesis and metastatic processes. During tumorigenesis, stromal NOFs may undergo activation into cancer-associated fibroblasts (CAFs) that modify their phenotype to provide pro-oncogenic signals that in turn facilitate tumor initiation, progression, and metastasis. The aim of the study was to reveal the effect of EVs derived from local (oral squamous cell carcinoma – OSCC) and distant (pancreatic adenocarcinoma – PDAC; malignant melanoma brain metastasis – MBM) cancer origin on NOFs and their possible change into a CAF-like direction. Methods: The effect of each of the cancer EV types on NOFs proliferation, viability, and migration was tested. Also, changes in gene expression of the well-established CAF biomarkers ACTA2, FAP, PDGFR, and two putative CAF biomarkers, the Ca2+- activated ion channels ANO1 and KCNMA, were studied. Results: Obtained results indicate that NOFs receive and process signals transmitted by EVs originating from both OSCC, PDAC, and MBM. The fibroblast response was dependent on EV origin and concentration, and duration of EV exposure. Conclusion: The present results indicate that the molecular cargo of the EVs direct NOFs towards a pro-tumorigenic phenotype.

• Ledsaak, Marit; Bengtsen, Mads; Molværsmyr, Ann-Kristin; Fuglerud, Bettina Maria; Matre, Vilborg & Eskeland, Ragnhild [Vis alle 7 forfattere av denne artikkelen] (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. Fulltekst i vitenarkiv
• Alm-Kristiansen, Anne Hege; Lorenzo, Petra Isabel O.; Molværsmyr, Ann-Kristin; Matre, Vilborg; Ledsaak, Marit & Sæther, Thomas [Vis alle 7 forfattere av denne artikkelen] (2011). PIAS1 interacts with FLASH and enhances its co-activation of c-Myb. Molecular Cancer. 10. doi: 10.1186/1476-4598-10-21. Fulltekst i vitenarkiv Vis sammendrag

  Background FLASH is a huge nuclear protein involved in various cellular functions such as apoptosis signalling, NF-κB activation, S-phase regulation, processing of histone pre-mRNAs, and co-regulation of transcription. Recently, we identified FLASH as a co-activator of the transcription factor c-Myb and found FLASH to be tightly associated with active transcription foci. As a huge multifunctional protein, FLASH is expected to have many interaction partners, some which may shed light on its function as a transcriptional regulator. Results To find additional FLASH-associated proteins, we performed a yeast two-hybrid (Y2H) screening with FLASH as bait and identified the SUMO E3 ligase PIAS1 as an interaction partner. The association appears to involve two distinct interaction surfaces in FLASH. We verified the interaction by Y2H-mating, GST pulldowns, co-IP and ChIP. FLASH and PIAS1 were found to co-localize in nuclear speckles. Functional assays revealed that PIAS1 enhances the intrinsic transcriptional activity of FLASH in a RING finger-dependent manner. Furthermore, PIAS1 also augments the specific activity of c-Myb, and cooperates with FLASH to further co-activate c-Myb. The three proteins, FLASH, PIAS1, and c-Myb, are all co-localized with active RNA polymerase II foci, resembling transcription factories. Conclusions We conclude that PIAS1 is a common partner for two cancer-related nuclear factors, c-Myb and FLASH. Our results point to a functional cooperation between FLASH and PIAS1 in the enhancement of c-Myb activity in active nuclear foci.

• Molværsmyr, Ann-Kristin; Sæther, Thomas; Gilfillan, Siv; Lorenzo, Petra Isabel O.; Kvaløy, Heidi & Matre, Vilborg [Vis alle 7 forfattere av denne artikkelen] (2010). A SUMO-regulated activation function controls synergy of c-Myb through a repressor-activator switch leading to differential p300 recruitment. Nucleic Acids Research (NAR). ISSN 0305-1048. 38(15), s. 4970–4984. doi: 10.1093/nar/gkq245. Fulltekst i vitenarkiv

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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. Fulltekst i vitenarkiv 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 [Vis alle 8 forfattere av denne artikkelen] (2012). Regulating the regulator: SUMO functions as a master switch for the transcription factor c-Myb. Fulltekst i vitenarkiv 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 [Vis alle 7 forfattere av denne artikkelen] (2011). The hematopoietic transcription factor c-Myb functions as an epigenetic organizer. Fulltekst i vitenarkiv 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.

• 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. Fulltekst i vitenarkiv 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; 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. Fulltekst i vitenarkiv 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.

• 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. Fulltekst i vitenarkiv 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. Fulltekst i vitenarkiv 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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