Marit Ledsaak

• Nguyen, Xuan Thang; Martiensen, Emily Louise Beecher; Abdelhalim, Mohamed; Tran, Trung; Ledsaak, Marit & Rogne, Marie [Show all 8 contributors for this article] (2026). Proteome-wide multi-omics profiling of osteosarcoma transcription factor networks. bioRxiv (preprint article). doi: 10.64898/2026.03.29.714917. Full text in Research Archive Show summary

  Osteosarcoma (OS) is an aggressive bone cancer that most commonly affects children and young adults. OS exhibits a high degree of genomic complexity, as well as cellular plasticity, and dynamic transcriptional regulation is suggested to contribute to treatment resistance and metastasis. Cell lines are well characterized as models to advance our knowledge on OS biology. HOS and U2OS cells have increased invasiveness and higher migratory ability compared with MG63. In this study, we employed a tandem array of consensus transcription factor response elements (catTFREs) proteomic approach to characterize transcription factor (TF) regulatory networks related to OS aggressiveness. We mapped 7,594 proteins and enriched 352 transcription factors and coregulators. When we integrated proteomics with cell line specific gene expression and chromatin accessibility we classified the proteins into different OS cell line dependent sub-clusters and identified TFs and coregulators common for all cell lines and specific for individual cell lines. We demonstrate that RUNX2, MYBL2 and HMGA2 are specifically enriched in HOS and U2OS and may be linked to the cell aggressiveness. ETV5, JUNB, NFIX and ZEB1 were among TFs specific to MG63. Our analysis provides a more comprehensive understanding of the transcriptional drivers that shape OS regulatory landscapes and may have future therapeutic implications.

• Rossini, Roberto; Oshaghi, Mohammadsaleh; Nekrasov, Maxim; Bellanger, Aurélie Nathalie Pascale; Domaschenz, Renae & Dijkwel, Yasmin [Show all 13 contributors for this article] (2025). Loss of multilevel 3D genome organization during breast cancer progression. Genome Research. ISSN 1088-9051. p. 1–18. doi: 10.1101/gr.280791.125. Full text in Research Archive Show summary

  Breast cancer entails intricate alterations in genome organization and expression. However, how three-dimensional (3D) chromatin structure changes in the progression from a normal to a breast cancer malignant state remains unknown. To address this, we have conducted an analysis combining Hi-C data with lamina-associated domains (LADs), epigenomic marks, and gene expression in an in vitro model of breast cancer progression. Our results reveal that although the fundamental properties of topologically associating domains (TADs) are overall maintained, significant changes occur in the organization of compartments and subcompartments. These changes are closely correlated with alterations in the expression of oncogenic genes. We also observe a restructuring of TAD–TAD interactions, coinciding with a loss of spatial compartmentalization and radial positioning of the 3D genome. Notably, we identify a previously unrecognized interchromosomal insertion event, wherein a locus on Chromosome 8 housing the MYC oncogene is inserted into a highly active subcompartment on Chromosome 10. This insertion is accompanied by the formation of de novo enhancer contacts and activation of MYC , illustrating how structural genomic variants can alter the 3D genome during oncogenesis. In summary, our findings provide evidence for the loss of genome organization at multiple scales during breast cancer progression, revealing novel relationships between genome 3D structure and oncogenic processes.

• Lemma, Roza Berhanu; Ledsaak, Marit; Fuglerud, Bettina Maria; Castañeda, Fernando Rodriguez-; Eskeland, Ragnhild & Gabrielsen, Odd Stokke (2023). MYB regulates the SUMO protease SENP1 and its novel interaction partner UXT, modulating MYB target genes and the SUMO landscape. Journal of Biological Chemistry. ISSN 0021-9258. 299(9). doi: 10.1016/j.jbc.2023.105062. Full text in Research Archive Show summary

  SUMOylation is a post-translational modification frequently found on nuclear proteins, including transcription factors (TFs) and coactivators. By controlling the activity of several TFs, SUMOylation may have far-reaching effects. MYB is an example of a developmental TF subjected to SUMO-mediated regulation, through both SUMO conjugation and SUMO binding. How SUMO affects MYB target genes is unknown. Here, we explored the global effect of reduced SUMOylation of MYB on its downstream gene programs. RNA-Seq in K562 cells after MYB knockdown and rescue with mutants having an altered SUMO status revealed a number of differentially regulated genes and distinct gene ontology term enrichments. Clearly, the SUMO status of MYB both quantitatively and qualitatively affects its regulome. The transcriptome data further revealed that MYB upregulates the SUMO protease SENP1, a key enzyme that removes SUMO conjugation from SUMOylated proteins. Given this role of SENP1 in the MYB regulome, we expanded the analysis, mapped interaction partners of SENP1, and identified UXT as a novel player affecting the SUMO system by acting as a repressor of SENP1. MYB inhibits the expression of UXT suggesting that MYB is able not only to control a specific gene program directly but also indirectly by affecting the SUMO landscape through SENP1 and UXT. These findings suggest an autoactivation loop whereby MYB, through enhancing SENP1 and reducing UXT, is itself being activated by a reduced level of repressive SUMOylation. We propose that overexpressed MYB, seen in multiple cancers, may drive this autoactivation loop and contribute to oncogenic activation of MYB.

• Lemma, Roza Berhanu; Fleischer, Thomas; Martinsen, Emily; Ledsaak, Marit; Kristensen, Vessela N. & Eskeland, Ragnhild [Show all 8 contributors for this article] (2022). Pioneer transcription factors are associated with the modulation of DNA methylation patterns across cancers. Epigenetics & Chromatin. 15. doi: 10.1186/s13072-022-00444-9. Full text in Research Archive Show summary

  Methylation of cytosines on DNA is a prominent modification associated with gene expression regulation. Aberrant DNA methylation patterns have recurrently been linked to dysregulation of the regulatory program in cancer cells. To shed light on the underlying molecular mechanism driving this process, we hypothesised that aberrant methylation patterns could be controlled by the binding of specific transcription factors (TFs) across cancer types. By combining DNA methylation arrays and gene expression data with TF binding sites (TFBSs), we explored the interplay between TF binding and DNA methylation in 19 cancer types. We performed emQTL (expression–methylation quantitative trait loci) analyses independently in each cancer type and identified 13 TFs whose expression levels are correlated with local DNA methylation patterns around their binding sites in at least 2 cancer types. The 13 TFs are mainly associated with local demethylation and are enriched for pioneer function, suggesting a specific role for these TFs in modulating chromatin structure and transcription in cancer patients. Furthermore, we confirmed that de novo methylation is precluded across cancers at CpGs lying in genomic regions enriched for TF binding signatures associated with SP1, CTCF, NRF1, GABPA, KLF9, and/or YY1. The modulation of DNA methylation associated with TF binding was observed at cis-regulatory regions controlling immune- and cancer-associated pathways, corroborating that the emQTL signals were derived from both cancer and tumor-infiltrating cells. As a case example, we experimentally confirmed that FOXA1 knock-down is associated with higher methylation in regions bound by FOXA1 in breast cancer MCF-7 cells. Finally, we reported physical interactions between FOXA1 with TET1 and TET2 both in an in vitro setup and in vivo at physiological levels in MCF-7 cells, adding further support for FOXA1 attracting TET1 and TET2 to induce local demethylation in cancer cells.

• Lemma, Roza Berhanu; Ledsaak, Marit; Fuglerud, Bettina Maria; Sandve, Geir Kjetil; Eskeland, Ragnhild & Gabrielsen, Odd Stokke (2021). Chromatin occupancy and target genes of the haematopoietic master transcription factor MYB. Scientific Reports. 11(9008). doi: 10.1038/s41598-021-88516-w. Full text in Research Archive Show summary

  The transcription factor MYB is a master regulator in haematopoietic progenitor cells and a pioneer factor affecting differentiation and proliferation of these cells. Leukaemic transformation may be promoted by high MYB levels. Despite much accumulated molecular knowledge of MYB, we still lack a comprehensive understanding of its target genes and its chromatin action. In the present work, we performed a ChIP-seq analysis of MYB in K562 cells accompanied by detailed bioinformatics analyses. We found that MYB occupies both promoters and enhancers. Five clusters (C1–C5) were found when we classified MYB peaks according to epigenetic profiles. C1 was enriched for promoters and C2 dominated by enhancers. C2-linked genes were connected to hematopoietic specific functions and had GATA factor motifs as second in frequency. C1 had in addition to MYB-motifs a significant frequency of ETS-related motifs. Combining ChIP-seq data with RNA-seq data allowed us to identify direct MYB target genes. We also compared ChIP-seq data with digital genomic footprinting. MYB is occupying nearly a third of the super-enhancers in K562. Finally, we concluded that MYB cooperates with a subset of the other highly expressed TFs in this cell line, as expected for a master regulator

• Næs, Guro; Storesund, Jan Ove; Udayakumar, Priyanga-Dina; Ledsaak, Marit & Gabrielsen, Odd Stokke (2020). Dissecting the transactivation domain (tAD) of the transcription factor c‐Myb to assess recent models of tAD function. FEBS Open Bio. 10(11), p. 2329–2342. doi: 10.1002/2211-5463.12978. Full text in Research Archive Show summary

  Transcription factors use a DNA‐binding domain to localize their action and a transactivation domain (tAD) to stimulate activation of the associated gene. Recent work has renewed interest in how tADs activate genes, which remains poorly understood. Key features in the new models are exposure of short linear motifs (SLMs) and liquid–liquid phase separation (LLPS). Inspired by the new models for tAD function, we decided to revisit the tAD of the haematopoietic transcription factor c‐Myb by performing a mutational analysis to see how these new models fit and potentially explain the tAD behaviour of this master regulator. We know that c‐Myb has an acidic tAD, which contains a well‐characterized SLM in the form of a LxxLL motif. By testing 12 alanine‐scanning mutants and three mutants with major reorganization of its tAD in two mammalian reporter systems, we found a pattern of effects very close to what would be expected from the SLM‐exposure model, with strong effects exerted by both acidic replacements and SLM mutation. When the same mutants were tested in a yeast system, the pattern of effects was dramatically different, with the SLM mutation exerting no effect, and tAD behaviour was much less affected by small alterations, as would be expected from a LLPS model. These observations are discussed in the light of the two new tAD models, and a two‐step hypothesis for transactivation, combining both models, is proposed.

• Castañeda, Fernando Rodriguez-; Lemma, Roza Berhanu; Torre, Ignacio Cuervo; Bengtsen, Mads; Moen, Lisa Marie & Ledsaak, Marit [Show all 8 contributors for this article] (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), p. 15439–15454. doi: 10.1074/jbc.RA118.002844. Full text in Research Archive Show summary

  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.

• 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. 11(1). doi: 10.1186/s13072-018-0208-y. Full text in Research Archive Show summary

  Background The concept of pioneer transcription factors is emerging as an essential part of the epigenetic regulation, taking place during cell development and differentiation. However, the precise molecular mechanism underlying pioneer factor activity remains poorly understood. We recently reported that the transcription factor c-Myb acts as a pioneer factor in haematopoiesis, and a point mutation in its DNA binding domain, D152V, is able to abrogate this function. Results Here, we show that specific histone modifications, including H3K27ac, prevent binding of c-Myb to histone tails, representing a novel effect of histone modifications, namely restricting binding of a pioneer factor to chromatin. Furthermore, we have taken advantage of the pioneer-defect D152V mutant to investigate mechanisms of c-Myb’s pioneer factor activity. We show that c-Myb-dependent transcriptional activation of a gene in inaccessible chromatin relies on c-Myb binding to histones, as well as on c-Myb interacting with the histone acetyltransferase p300. ChIP assays show that both wild type and the D152V mutant of c-Myb bind to a selected target gene at its promoter and enhancer, but only wild-type c-Myb causes opening and activation of the locus. Enhancement of histone acetylation restores activation of the same gene in the absence of c-Myb, suggesting that facilitating histone acetylation is a crucial part of the pioneer factor function of c-Myb. Conclusions We suggest a pioneer factor model in which c-Myb binds to regions of closed chromatin and then recruits histone acetyltransferases. By binding to histones, c-Myb facilitates histone acetylation, acting as a cofactor for p300 at c-Myb bound sites. The resulting H3K27ac leads to chromatin opening and detachment of c-Myb from the acetylated chromatin. We propose that the latter phenomenon, acetylation-induced chromatin dissociation, represents a mechanism for controlling the dynamics of pioneer factor binding to chromatin.

• 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), p. 751–760. doi: 10.1016/j.bbagrm.2017.05.001. Full text in Research Archive Show summary

  LIM-domain proteins, containing multiple cysteine-rich zinc finger-like motifs, have been shown to play diverse roles in several cellular processes. A common theme is that they mediate important protein-protein interactions that are key to their function. Androgen receptor-associated protein 55 (ARA55) belongs to this family of bridging proteins containing four C-terminal LIM domains. It has a dual role with functions both at focal adhesions and in the nucleus, apparently shuttling between the two compartments. In the present work, we have expanded our understanding of its nuclear functions by showing that it interacts with three nuclear regulators not previously linked to ARA55. We first identified ARA55 as a novel interaction partner of the nuclear kinase HIPK1 and found that ARA55, like HIPK1, also interacts with the transcription factor c-Myb. In search of a function for these associations, we observed that the coactivator p300 not only binds to c-Myb, but to ARA55 as well. When combined, c-Myb, p300, HIPK1 and ARA55 caused strong synergistic activation of a chromatinized reporter gene. In parallel, all partners, including p300, were efficiently recruited to chromatin at the c-Myb-bound promoter. Consistent with this cooperation, we found that c-Myb and ARA55 share a common set of target genes in an osteosarcoma cellular context. We propose that ARA55 and HIPK1 assist c-Myb in recruiting the coactivator and acetyltransferase p300 to chromatin.

• Ledsaak, Marit; Bengtsen, Mads; Molværsmyr, Ann-Kristin; Fuglerud, Bettina Maria; Matre, Vilborg & Eskeland, Ragnhild [Show all 7 contributors for this article] (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), p. 705–718. doi: 10.1016/j.bbagrm.2016.03.011. Full text in Research Archive
• Lorenzo, Petra Isabel O.; Brendeford, Elen Margrethe; Gilfillan, Siv; Gavrilov, Alexey A; Ledsaak, Marit & Razin, Sergey V [Show all 9 contributors for this article] (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), p. 805–817. doi: 10.1177/1947601911428224. Full text in Research Archive Show summary

  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.

• Alm-Kristiansen, Anne Hege; Lorenzo, Petra Isabel O.; Molværsmyr, Ann-Kristin; Matre, Vilborg; Ledsaak, Marit & Sæther, Thomas [Show all 7 contributors for this article] (2011). PIAS1 interacts with FLASH and enhances its co-activation of c-Myb. Molecular Cancer. 10. doi: 10.1186/1476-4598-10-21. Full text in Research Archive Show summary

  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.

• 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), p. 150–154. doi: 10.1016/j.bbrc.2009.07.139. Full text in Research Archive
• Sæther, Thomas; Berge, T; Ledsaak, Marit; Matre, Vilborg; Alm-Kristiansen, Anne Hege & Dahle, O [Show all 8 contributors for this article] (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. Full text in Research Archive

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• Mo, Ingrid; Eriksson, Anna; Brorson, Ina Skaara; Ledsaak, Marit; Harbo, Hanne Flinstad & Gabrielsen, Odd Stokke [Show all 7 contributors for this article] (2017). Identifying protein interaction partners for DEXI - encoded by the dexamethasone-induced gene, DEXI. Full text in Research Archive
• 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. Full text in Research Archive Show summary

  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.

• Molværsmyr, Ann-Kristin; Ledsaak, Marit; Alm-Kristiansen, Anne Hege; Lorenzo, Petra Isabel O.; Matre, Vilborg & Sæther, Thomas [Show all 7 contributors for this article] (2012). The SUMO E3 ligase PIAS1 interacts with c-Myb and enhances its transcriptional activity. Full text in Research Archive Show summary

  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 [Show all 8 contributors for this article] (2012). Regulating the regulator: SUMO functions as a master switch for the transcription factor c-Myb. Full text in Research Archive Show summary

  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.

• 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. Full text in Research Archive
• Sæther, Thomas; Berge, Tone; Ledsaak, Marit; Matre, Vilborg; Alm-Kristiansen, Anne Hege & Dahle, Øyvind [Show all 8 contributors for this article] (2007). The chromatin remodelling factor Mi-2alpha acts as a novel co-activator for human c-Myb. Full text in Research Archive Show summary

  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. Full text in Research Archive
• Sæther, Thomas; Berge, Tone; Ledsaak, Marit; Dahle, Øyvind; Matre, Vilborg & Lüscher, Bernhard [Show all 7 contributors for this article] (2004). Human c-Myb interacts with the Mi-2alpha subunit of the NuRD complex. Full text in Research Archive Show summary

  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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