Marit Ledsaak

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

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

• Bengtsen, Mads; Sørensen, Linda Therese; Aabel, Linn Ingeborg; Ledsaak, Marit; Matre, Vilborg & Gabrielsen, Odd Stokke (2017). The adaptor protein ARA55 and the nuclear kinase HIPK1 assist c-Myb in recruiting p300 to chromatin. Biochimica et Biophysica Acta. Gene Regulatory Mechanisms.  ISSN 1874-9399.  1860(7), s 751- 760 . doi: 10.1016/j.bbagrm.2017.05.001 Fulltekst i vitenarkiv.
• Ledsaak, Marit; Bengtsen, Mads; Molværsmyr, Ann-Kristin; Fuglerud, Bettina Maria; Matre, Vilborg; Eskeland, Ragnhild & Gabrielsen, Odd Stokke (2016). PIAS1 binds p300 and behaves as a coactivator or corepressor of the transcription factor c-Myb dependent on SUMO-status. Biochimica et Biophysica Acta. Gene Regulatory Mechanisms.  ISSN 1874-9399.  1859(5), s 705- 718 . doi: 10.1016/j.bbagrm.2016.03.011
• Alm-Kristiansen, Anne Hege; Lorenzo, Petra Isabel O.; Molværsmyr, Ann-Kristin; Matre, Vilborg; Ledsaak, Marit; Sæther, Thomas & Gabrielsen, Odd Stokke (2011). PIAS1 interacts with FLASH and enhances its co-activation of c-Myb. Molecular Cancer.  ISSN 1476-4598.  10 . doi: 10.1186/1476-4598-10-21
• Lorenzo, Petra Isabel O.; Brendeford, Elen Margrethe; Gilfillan, Siv; Gavrilov, Alexey A; Ledsaak, Marit; Razin, Sergey V; Eskeland, Ragnhild; Sæther, Thomas & Gabrielsen, Odd Stokke (2011). Identification of c-Myb target genes in K562 cells reveals a role for c-Myb as a master regulator. Genes & cancer.  ISSN 1947-6019.  2(8), s 805- 817 . doi: 10.1177/1947601911428224 Vis sammendrag

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

• Matre, Vilborg; Nordgård, Oddmund; Alm-Kristiansen, Anne Hege; Ledsaak, Marit & Gabrielsen, Odd Stokke (2009). HIPK1 interacts with c-Myb and modulates its activity through phosphorylation. Biochemical and Biophysical Research Communications - BBRC.  ISSN 0006-291X.  388(1), s 150- 154 . doi: 10.1016/j.bbrc.2009.07.139
• Sæther, Thomas; Berge, T; Ledsaak, Marit; Matre, Vilborg; Alm-Kristiansen, Anne Hege; Dahle, O; Aubry, F & Gabrielsen, Odd Stokke (2007). The chromatin remodeling factor Mi-2 alpha acts as a novel co-activator for human c-Myb. Journal of Biological Chemistry.  ISSN 0021-9258.  282 . doi: 10.1074/jbc.M700755200

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• Mo, Ingrid; Eriksson, Anna; Brorson, Ina Skaara; Ledsaak, Marit; Harbo, Hanne Flinstad; Gabrielsen, Odd Stokke & Berge, Tone (2017). Identifying protein interaction partners for DEXI - encoded by the dexamethasone-induced gene, DEXI.
• Matre, Vilborg; Aabel, Linn Ingeborg; Ledsaak, Marit; Vollsund, Pernille; Sæther, Thomas & Gabrielsen, Odd Stokke (2013). PTM-crosstalk: SUMO1 acts as a docking domain for the nuclear kinase HIPK1 mediating SUMO-linked phosphorylation of c-Myb and other targets. Vis sammendrag

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

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

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

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

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

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

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

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

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

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