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  • 1.
    Arabi, Azadeh
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Berkson, Rachel
    Wright, Anthony P.
    Södertörn University, School of Life Sciences.
    Regulation of the nucleolar structure by the oncoprotein c-Myc and proteasomesManuscript (preprint) (Other academic)
  • 2.
    Arabi, Azadeh
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Rustum, Cecilia
    Södertörn University, Avdelning Naturvetenskap. Stockholm University.
    Hallberg, Einar
    Södertörn University, Avdelning Naturvetenskap.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Accumulation of c-Myc and proteasomes at the nucleoli of cells containing elevated c-Myc protein levels2003In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 116, no 9, p. 1707-1717Article in journal (Refereed)
    Abstract [en]

    c-Myc is a predominately nuclear transcription factor that is a substrate for rapid turnover by the proteasome system. Cancer-related mutations in c-Myc lead to defects in its degradation and thereby contribute to the increase in its cellular level that is associated with the disease. Little is known about the mechanisms that target c-Myc to the proteasomes. By using a GFP fusion protein and live analysis we show that c-Myc shuttles between the nucleus and cytoplasm and thus it could be degraded in either compartment. Strikingly, at elevated levels of expression c-Myc accumulates at nucleoli in some cells, consistent with saturation of a nucleolus-associated degradation system in these cells. This idea is further supported by the observation that proteasome inhibitor treatment causes accumulation of c-Myc at the nucleoli of essentially all cells. Under these conditions c-Myc is relatively stably associated with the nucleolus, as would be expected if the nucleolus functions as a sequestration/degradation site for excess c-Myc. Furthermore, during elevated c-Myc expression or proteasome inhibition, nucleoli that are associated with c-Myc also accumulate proteasomes. c-Myc and proteasomes co-localise in intranucleolar regions distinct from the dense fibrillar component of the nucleolus. Based on these results we propose a model for c-Myc downregulation where c-Myc is sequestered at the nucleoli. Sequestration of c-Myc is accompanied by recruitment of proteasomes and may lead to subsequent degradation.

  • 3.
    Arabi, Azadeh
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wu, Siqin
    SLU.
    Ridderstråle, Karin
    SLU.
    Bierhoff, Holger
    German Cancer Research Center, Heidelberg, Germany.
    Shiue, Chiounan
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Fatyol, Karoly
    German Cancer Research Center, Heidelberg, Germany.
    Fahlén, Sara
    SLU.
    Hydbring, Per
    SLU.
    Söderberg, Ola
    Uppsala universitet.
    Grummt, Ingrid
    German Cancer Research Center, Heidelberg, Germany.
    Larsson, Lars-Gunnar
    SLU.
    Wright, Anthony P H
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription2005In: Nature Cell Biology, ISSN 1465-7392, E-ISSN 1476-4679, Vol. 7, no 3, p. 303-310Article in journal (Refereed)
    Abstract [en]

    The c-Myc oncoprotein regulates transcription of genes that are associated with cell growth, proliferation and apoptosis(1). c-Myc levels are modulated by ubiquitin/proteasome-mediated degradation(1). Proteasome inhibition leads to c-Myc accumulation within nucleoli(2), indicating that c-Myc might have a nucleolar function. Here we show that the proteins c-Myc and Max interact in nucleoli and are associated with ribosomal DNA. This association is increased upon activation of quiescent cells and is followed by recruitment of the Myc cofactor TRRAP, enhanced histone acetylation, recruitment of RNA polymerase I (Pol I), and activation of rDNA transcription. Using small interfering RNAs (siRNAs) against c-Myc and an inhibitor of Myc - Max interactions, we demonstrate that c-Myc is required for activating rDNA transcription in response to mitogenic signals. Furthermore, using the ligand-activated MycER ( ER, oestrogen receptor) system, we show that c-Myc can activate Pol I transcription in the absence of Pol II transcription. These results suggest that c-Myc coordinates the activity of all three nuclear RNA polymerases, and thereby plays a key role in regulating ribosome biogenesis and cell growth.

  • 4. Bergh, F T
    et al.
    Flinn, Elisabeth M
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Svaren, J
    Wright, Anthony P
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Horz, W
    Comparison of nucleosome remodeling by the yeast transcription factor Pho4 and the glucocorticoid receptor2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 12, p. 9035-9042Article in journal (Refereed)
    Abstract [en]

    Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively. In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR, Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity. However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter. Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site. In contrast, GR had little effect on the stability of the MMTV B nucleosome. A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome. Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure. Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation.

  • 5. Beskow, Anne
    et al.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences.
    Comparative analysis of regulatory transcription factors in Schizosaccharomyces pombe and budding yeasts2006In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 23, no 13, p. 929-935Article in journal (Refereed)
    Abstract [en]

    Regulatory transcription factors (rTFs), which bind specific DNA sequences in the regulatory regions of genes and subsequently activate or repress transcription, play a central role in programming genomic expression. The number of rTFs in a species might therefore reflect its functional complexity. For simple organisms like yeast, a relatively small number of rTFs might be expected that is fairly constant between yeast species. We show that the budding yeast, Saccharomyces cerevisiae, contains 201 rTfs, which is one of the largest rTF numbers found in yeast species for which genome sequences are available. This is a much higher number than the 129 rTFs found in the fission yeast, Schizosaccharomyces pombe, which is currently the yeast with the lowest number of rTFs. Comparative analysis of several different budding yeast species shows that most of the 'extra' rTFs found in S. cerevisiae were probably acquired as a result of a whole genome duplication (WGD) event that occurred in an ancestor of a subset of budding yeast species. However, we also show that budding yeast species that have not been affected by the WGD contain a greater number of rTFs than S. pombe (mean = 145). Thus, two or more mechanisms have led to the 60% increase in rTFs in S. cerevisiae compared to S. pombe. This difference may correlate with a more extensive functional divergence in budding yeasts compared to fission yeasts. The relatively small number of rTFs in S. pombe make this organism an attractive model for global studies of mechanisms that programme gene expression.

  • 6.
    Durand-Dubief, Mickael
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Sinha, Indranil
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Fagerström-Billai, Fredrik
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Bonilla, Carolina
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Anthony
    Södertörn University, School of Life Sciences. Karolinska Instiutet.
    Grunstein, Michael
    University of California, Los Angeles, CA, USA.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation and retrotransposon silencing2007In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 26, no 10, p. 2477-2488Article in journal (Refereed)
    Abstract [en]

    Expression profiling, ChiP-CHIP and phenotypic analysis were used to investigate the functional relationships of class III NAD(+)-dependent HDACs (Sirtuins) in fission yeast. We detected significant histone acetylation increases in Sirtuin mutants at their specific genomic binding targets and were thus able to identify an in vivo substrate preference for each Sirtuin. At heterochromatic loci, we demonstrate that although Hst2 is mainly cytoplasmic, a nuclear pool of Hst2 colocalizes with the other Sirtuins at silent regions (cen, mat, tel, rDNA), and that like the other Sirtuins, Hst2 is required for rDNA and centromeric silencing. Interestingly we found specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation. Hst2 directly represses genes involved in transport and membrane function, whereas Hst4 represses amino-acid biosynthesis genes and Tf2 retrotransposons. A specific role for Hst4 in Tf2 50 mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation.

  • 7.
    Fagerström-Billai, Fredrik
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Durand-Dubief, Mikael
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Individual Subunits of the Ssn6-Tup11/12 corepressor are selectively required for repression of different target genes2007In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 27, no 3, p. 1069-1082Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae Ssn6 and Tup1 proteins form a corepressor complex that is recruited to target genes by DNA-bound repressor proteins. Repression occurs via several mechanisms, including interaction with hypoacetylated N termini of histones, recruitment of histone deacetylases (HDACs), and interactions with the RNA polymerase II holoenzyme. The distantly related fission yeast, Schizosaccharomyces pombe, has two partially redundant Tup1-like proteins that are dispensable during normal growth. In contrast, we show that Ssn6 is an essential protein in S. pombe, suggesting a function that is independent of Tup11 and Tup12. Consistently, the group of genes that requires Ssn6 for their regulation overlaps but is distinct from the group of genes that depend on Tup11 or Tup12. Global chip-on-chip analysis shows that Ssn6 is almost invariably found in the same genomic locations as Tup11 and/or Tup12. All three corepressor subunits are generally bound to genes that are selectively regulated by Ssn6 or Tup11/12, and thus, the subunit specificity is probably manifested in the context of a corepressor complex containing all three subunits. The corepressor binds to both the intergenic and coding regions of genes, but differential localization of the corepressor within genes does not appear to account for the selective dependence of target genes on the Ssn6 or Tup11/12 subunits. Ssn6, Tup11, and Tup12 are preferentially found at genomic locations at which histones are deacetylated, primarily by the Clr6 class I HDAC. Clr6 is also important for the repression of corepressor target genes. Interestingly, a subset of corepressor target genes, including direct target genes affected by Ssn6 overexpression, is associated with the function of class II (CIr3) and III (Hst4 and Sir2) HDACs.

  • 8.
    Fagerström-Billai, Fredrik
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Anthony P H
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Functional comparison of the Tup11 and Tup12 transcriptional corepressors in fission yeast2005In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 25, no 2, p. 716-727Article in journal (Refereed)
    Abstract [en]

    Gene duplication is considered an important evolutionary mechanism. Unlike many characterized species, the fission yeast Schizosaccharomyces pombe contains two paralogous genes, tup11(+) and tup12(+), that encode transcriptional corepressors similar to the well-characterized budding yeast Tup1 protein. Previous reports have suggested that Tup11 and Tup12 proteins play redundant roles. Consistently, we show that the two Tup proteins can interact together when expressed at normal levels and that each can independently interact with the Ssn6 protein, as seen for Tup1 in budding yeast. However, tup11(-) and tup12(-) mutants have different phenotypes on media containing KCl and CaCl2. Consistent with the functional difference between tup11(-) and tup12- mutants, we identified a number of genes in genome-wide gene expression experiments that are differentially affected by mutations in the tup11(+) and tup12(+) genes. Many of these genes are differentially derepressed in tup11(-) mutants and are over-represented in genes that have previously been shown to respond to a range of different stress conditions. Genes specifically derepressed in tup12(-) mutants require the Ssn6 protein for their repression. As for Tupl.2, Ssn6 is also required for efficient adaptation to KCI- and CaCl2-mediated stress. We conclude that Tup11 and Tup12 are at least partly functionally diverged and suggest that the Tup12 and Ssn6 proteins have adopted a specific role in regulation of the stress response.

  • 9.
    Ferreira, Monica E.
    et al.
    Södertörn University, School of Life Sciences, Molecular biology.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences, Chemistry.
    Nilsson, Johan
    Södertörn University, School of Life Sciences, Molecular biology.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology.
    WD40 Domain Divergence Is Important for Functional Differences between he Fission Yeast Tup11 and Tup12 Co-Repressor Proteins2010In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 6, article id e11009Article in journal (Refereed)
    Abstract [en]

    We have previously demonstrated that subsets of Ssn6/Tup target genes ave distinct requirements for the Schizosaccharomyces pombe homologs of he Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very igh level of divergence in the histone interacting repression domains f the two proteins suggested that determinants distinguishing Tup11 and up12 might be located in this domain. Here we have combined hylogenetic and structural analysis as well as phenotypic haracterization, under stress conditions that specifically require up12, to identify and characterize the domains involved in up12-specific action. The results indicate that divergence in the epression domain is not generally relevant for Tup12-specific function. nstead, we show that the more highly conserved C-terminal WD40 repeat omain of Tup12 is important for Tup12-specific function. Surface amino cid residues specific for the WD40 repeat domain of Tup12 proteins in ifferent fission yeasts are clustered in blade 3 of the propeller-like tructure that is characteristic of WD40 repeat domains. The Tup11 and up12 proteins in fission yeasts thus provide an excellent model system or studying the functional divergence of WD40 repeat domains.

  • 10.
    Ferreira, Monica E
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Hermann, Stefan
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Prochasson, P
    Stowers Institute for Medical Research, Kansas City, USA.
    Workman, J L
    Stowers Institute for Medical Research, Kansas City, USA.
    Berndt, Kurt D
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Athony P H
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Mechanism of transcription factor recruitment by acidic activators2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 23, p. 21779-21784Article in journal (Refereed)
    Abstract [en]

    Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex.

  • 11.
    Ferreira, Monica E.
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Nilsson, Johan
    Södertörn University, School of Life Sciences, Molecular biology.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences, Chemistry. Karolinska Institutet.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Protein domains underlying functional divergence between the Tup11 and Tup12 co-repressor proteins in fission yeastManuscript (preprint) (Other academic)
  • 12.
    Ferreira, Monica E.
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska instiutet.
    Prochasson, Philippe
     Stowers Institute for Medical Research, Kansas City, MO, USA.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences, Chemistry. Karolinska institutet.
    Workman, Jerry L.
    Stowers Institute for Medical Research, Kansas City, MO, USA.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska institutet.
    Activator-binding domains of the SWI/SNF chromatin remodeling complex characterized in vitro are required for its recruitment to promoters in vivo2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 9, p. 2557-2565Article in journal (Refereed)
    Abstract [en]

    Interaction between acidic activation domains and the activator-binding domains of Swi1 and Snf5 of the yeast SWI/SNF chromatin remodeling complex has previously been characterized in vitro. Although deletion of both activator-binding domains leads to phenotypes that differ from the wild-type, their relative importance for SWI/SNF recruitment to target genes has not been investigated. In the present study, we used chromatin immunoprecipitation assays to investigate the individual and collective importance of the activator-binding domains for SWI/SNF recruitment to genes within the GAL regulon in vivo. We also investigated the consequences of defective SWI/SNF recruitment for target gene activation. We demonstrate that deletion of both activator-binding domains essentially abolishes galactose-induced SWI/SNF recruitment and causes a reduction in transcriptional activation similar in magnitude to that associated with a complete loss of SWI/SNF activity. The activator-binding domains in Swi1 and Snf5 make approximately equal contributions to the recruitment of SWI/SNF to each of the genes studied. The requirement for SWI/SNF recruitment correlates with GAL genes that are highly and rapidly induced by galactose.

  • 13.
    Flinn, Elizabeth M
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Intstitutet.
    Wallberg, A E
    Hermann, Stefan
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Grant, P A
    Workman, J L
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Recruitment of Gen5-containing complexes during c-Myc-dependent gene activation - Structure and function aspects2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 26, p. 23399-23406Article in journal (Refereed)
    Abstract [en]

    The N-terminal domain of c-Myc plays a key role in cellular transformation and is involved in both activation and repression of target genes as well as in modulated proteolysis of c-Myc via the proteasome. Given this functional complexity, it has been difficult to clarify the structures within the N terminus that contribute to these different processes as well as the mechanisms by which they function. We have used a simplified yeast model system to identify the primary determinants within the N terminus for W chromatin remodeling of a promoter, (ii) gene activation from a chromatin template in vivo, and (iii) interaction with highly purified Gcn5 complexes as well as other chromatin-remodeling complexes in vitro. The results identify two regions that contain autonomous chromatin opening and gene activation activity, but both regions are required for efficient interaction with chromatin-remodeling complexes in vitro. The conserved Myc boxes do not play a direct role in gene activation, and Myc box II is not generally required for in vitro interactions with remodeling complexes. The yeast SAGA complex, which is orthologous to the human GCN5-TRRAP complex that interacts with Myc in human cells, plays a role in Myc-mediated chromatin opening at the promoter but may also be involved in later steps of gene activation.

  • 14. Haas, S A
    et al.
    Hild, M
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Hain, T
    Talibi, D
    Vingron, M
    Genome-scale design of PCR primers and long oligomers for DNA microarrays2003In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 19, p. 5576-5581Article in journal (Refereed)
    Abstract [en]

    During the last years, the demand for custom-made cDNA chips/arrays as well as whole genome chips is increasing rapidly. The efficient selection of gene-specific primers/oligomers is of the utmost importance for the successful production of such chips. We developed GenomePRIDE, a highly flexible and scalable software for designing primers/oligomers for large-scale projects. The program is able to generate either long oligomers (40-70 bases), or PCR primers for the amplification of gene-specific DNA fragments of user-defined length. Additionally, primers can be designed in-frame in order to facilitate large-scale cloning into expression vectors. Furthermore, GenomePRIDE can be adapted to specific applications such as the generation of genomic amplicon arrays or the design of fragments specific for alternative splice isoforms. We tested the performance of GenomePRIDE on the entire genomes of Listeria monocytogenes (1584 gene-specific PCRs, 48 long oligomers) as well as of eukaryotes such as Schizosaccharomyces pombe (5006 gene-specific PCRs), and Drosophila melanogaster (21306 gene-specific PCRs). With its computing speed of 1000 primer pairs per hour and a PCR amplification success of 99%, GenomePRIDE represents an extremely cost- and time-effective program.

  • 15.
    Hermann, Stefan
    et al.
    Södertörn University, Avdelning Naturvetenskap.
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap.
    How transcriptional activators bind target proteins2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 43, p. 40127-40132Article in journal (Refereed)
    Abstract [en]

    The product of the proto-oncogene c-myc influences many cellular processes through the regulation of specific target genes. Through its transactivation domain (TAD), c-Myc protein interacts with several transcription factors, including TATA-binding protein (TBP). We present data that suggest that in contrast to some other transcriptional activators, an extended length of the c-Myc TAD is required for its binding to TBP. Our data also show that this interaction is a multistep process, in which a rapidly forming low affinity complex slowly converts to a more stable form. The initial complex formation results from ionic or polar interactions, whereas the slow conversion to a more stable form is hydrophobic in nature. Based on our results, we suggest two alternative models for activation domain/target protein interactions, which together provide a single universal paradigm for understanding activator-target factor interactions.

  • 16.
    Johnsson, Anna
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Durand-Dubief, Mickael
    Karolinska Intitutet.
    Xue-Franzen, Yongtao
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Rönnerblad, Michelle
    Karolinska Institutet.
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    HAT-HDAC interplay modulates global histone H3K14 acetylation in gene-coding regions during stress2009In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 10, no 9, p. 1009-1014Article in journal (Refereed)
    Abstract [en]

    Histone acetylation and deacetylation are important for gene regulation. The histone acetyltransferase, Gcn5, is an activator of transcriptional initiation that is recruited to gene promoters. Here, we map genome-wide Gcn5 occupancy and histone H3K14ac at high resolution. Gcn5 is predominantly localized to coding regions of highly transcribed genes, where it collaborates antagonistically with the class-II histone deacetylase, Clr3, to modulate H3K14ac levels and transcriptional elongation. An interplay between Gcn5 and Clr3 is crucial for the regulation of many stress-response genes. Our findings suggest a new role for Gcn5 during transcriptional elongation, in addition to its known role in transcriptional initiation.

  • 17.
    Johnsson, Anna E.
    et al.
    Södertörn University, School of Life Sciences, Molecular biology.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology.
    The role of specific HAT-HDAC interactions in transcriptional elongation2010In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 9, no 3, p. 467-471Article in journal (Refereed)
    Abstract [en]

    We previously reported genome-wide evidence that the Gcn5 histone cetyltransferase (HAT) is located in the transcribed region of highly xpressed genes and that it plays an important role in transcriptional longation in the fission yeast, Schizosaccharomyces pombe (EMBO Reports 009; 10: 1009-14). Furthermore, the specific interplay between Gcn5 and he Clr3 histone deacetylase (HDAC) controls the acetylation levels of ysine-14 in histone H3 in the same class of highly expressed genes. utants of histone H3 that cannot be acetylated at residue 14 show imilar stress phenotypes to those observed for mutants lacking Gcn5. In his Extra View article we review these findings in relation to related iterature and extend important aspects of the original study. Notably, cn5 and Gcn5-dependent acetylation of histone H3K14 tend to be more nriched in the upstream regions of genes that require Gcn5 for correct xpression compared to genes that are independent of Gcn5. This suggests critical role of Gcn5 in the transcriptional initiation of these enes. Gcn5 is however most highly enriched in the transcribed regions f these gene sets but there is no difference between Gcn5-dependent and cn5-independent gene sets. Thus we suggest that Gcn5 plays an important ut redundant role in the transcriptional elongation of these genes. The ir2 HDAC has a similar genomic localization and enzymatic activity to lr3. We studied gcn5 Delta sir2 Delta double mutants that do not show a uppressed phenotype in relation to gcn5 Delta single mutants, compared o gcn5 Delta clr3 Delta mutants that do, in order to better understand he specificity of the interplay between Gcn5 and Clr3. In some classes f non-highly expressed genes the clr3 Delta mutant tends to restore evels of histone H3K14 acetylation in the double mutant strain more ffectively than sir2 Delta.

  • 18.
    Johnsson, Anna
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Xue-Franzen, Yongtao
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Intitutet.
    Lundin, Maria
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Stress-specific role of fission yeast Gcn5 histone acetyltransferase in programming a subset of stress response genes2006In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 5, no 8, p. 1337-1346Article in journal (Refereed)
    Abstract [en]

    Gcn5 is a coactivator protein that contributes to gene activation by acetylating specific lysine residues within the N termini of histone proteins. Gcn5 has been intensively studied in the budding yeast, Saccharomyces cerevisiae, but the features of genes that determine whether they require Gcn5 during activation have not been conclusively clarified. To allow comparison with S. cerevisiae, we have studied the genome-wide role of Gcn5 in the distantly related fission yeast, Schizosaccharomyces pombe. We show that Gcn5 is specifically required for adaptation to KCl- and CaCl2-mediated stress in S. pombe. We have characterized the genome-wide gene expression responses to KCl stress and show that Gcn5 is involved in the regulation of a subset of stress response genes. Gcn5 is most clearly associated with KCl-induced genes, but there is no correlation between Gcn5 dependence and the extent of their induction. Instead, Gen5-dependent KCl-induced genes are specifically enriched in four different DNA motifs. The Gcn5-dependent KCl-induced genes are also associated with biological process gene ontology terms such as carbohydrate metabolism, glycolysis, and nicotinamide metabolism that together constitute a subset of the ontology parameters associated with KCl-induced genes.

  • 19. Lind, U
    et al.
    Greenidge, P
    Gillner, M
    Koehler, K F
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Carlstedt-Duke, J
    Functional probing of the human glucocorticoid receptor steroid-interacting surface by site-directed mutagenesis - Gln-642 plays an important role in steroid recognition and binding2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 25, p. 19041-19049Article in journal (Refereed)
    Abstract [en]

    To elucidate which amino acids in the glucocorticoid receptor ligand-binding domain might be involved in determining steroid binding specificity by interaction with the D-ring of glucocorticoids, we have performed site-directed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-739 based on their proximity to the steroid in a model structure. Mutations of these residues affected steroid binding affinity, specificity, and/or steroid-dependent transactivation. The results indicate that these residues are located in close proximity to the ligand and appear to play a role in steroid recognition and/or transactivating sensitivity, possibly by changes in the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. Mutation of Gln-642 resulted in a marked decrease in affinity for steroids containing a 17 alpha-OH group. This effect was alleviated by the presence of a 16 alpha-CH3 group to a varying degree. Thr-739 appears to form a hydrogen bond with the 21-OH group of the steroid, as well as possibly forming hydrophobic interactions with the steroid, Met-EGO and Met-639 appear to form hydrophobic interactions with the D-ring of the steroid, although the nature of these interactions cannot be characterized in more detail at this point.

  • 20. Lind, U.
    et al.
    Greenidge, P.
    Gustafsson, J. -A
    Wright, Anthony P. H.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Instiute.
    Carlstedt-Duke, J.
    Valine 571 functions as a regional organizer in programming the glucocorticoid receptor for differential binding of glucocorticoids and mineralocorticoids1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 26, p. 18515-18523Article in journal (Refereed)
    Abstract [en]

    The glucocorticoid receptor (GR) interacts specifically with glucocorticoids, whereas its closest relative, the mineralocorticoid receptor (MR), interacts with both glucocorticoids and mineralocorticoids, such as aldosterone. To investigate the mechanism underlying the glucocorticoid/mineralocorticoid specificity of the GR, we used a yeast model system to screen for GR ligand-binding domain mutants, substituted with MR residues in the segment 565-574, that can be efficiently activated by aldosterone. In all such increased activity mutants, valine 571 was replaced by methionine, even though most mutants also contained substitutions of other residues with their MR counterparts. Further analysis in yeast and COS-7 cells has revealed that the identity of residue 571 determines the behavior of other MR substituted residues in the 565-574 segment. Generally, MR substitutions in this region are only consistent with aldosterone binding if residue 571 is also replaced with methionine (MR conformation). If residue 571 is valine (GR conformation), most other MR substitution mutants drastically reduce interaction with both mineralocorticoid and glucocorticoid hormones. Based on these functional data, we hypothesize that residue 571 functions as a regional organizer involved in discriminating between glucocorticoid and mineralocorticoid hormones. We have used a molecular model of the GR ligand-binding domain in an attempt to interpret our functional data in structural terms.

  • 21.
    Lundberg, Max
    et al.
    Lund University.
    Boss, John
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology. Karolinska Institute, Karolinska University Hospital.
    Canbäck, Björn
    Lund University.
    Liedvogel, Miriam
    Lund University.
    Larson, Keith W
    Lund University.
    Grahn, Mats
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology.
    Åkesson, Susanne
    Lund University.
    Bensch, Staffan
    Lund University.
    Wright, Anthony Ph
    Karolinska Institute, Karolinska University Hospital.
    Characterisation of a transcriptome to find sequence differences between two differentially migrating subspecies of the willow warbler Phylloscopus trochilus.2013In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 14, article id 330Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Animal migration requires adaptations in morphological, physiological and behavioural traits. Several of these traits have been shown to possess a strong heritable component in birds, but little is known about their genetic architecture. Here we used 454 sequencing of brain-derived transcriptomes from two differentially migrating subspecies of the willow warbler Phylloscopus trochilus to detect genes potentially underlying traits associated with migration.

    RESULTS: The transcriptome sequencing resulted in 1.8 million reads following filtering steps. Most of the reads (84%) were successfully mapped to the genome of the zebra finch Taeniopygia gutatta. The mapped reads were situated within at least 12,101 predicted zebra finch genes, with the greatest sequencing depth in exons. Reads that were mapped to intergenic regions were generally located close to predicted genes and possibly located in uncharacterized untranslated regions (UTRs). Out of 85,000 single nucleotide polymorphisms (SNPs) with a minimum sequencing depth of eight reads from each of two subspecies-specific pools, only 55 showed high differentiation, confirming previous studies showing that most of the genetic variation is shared between the subspecies. Validation of a subset of the most highly differentiated SNPs using Sanger sequencing demonstrated that several of them also were differentiated between an independent set of individuals of each subspecies. These SNPs were clustered in two chromosome regions that are likely to be influenced by divergent selection between the subspecies and that could potentially be associated with adaptations to their different migratory strategies.

    CONCLUSIONS: Our study represents the first large-scale sequencing analysis aiming at detecting genes underlying migratory phenotypes in birds and provides new candidates for genes potentially involved in migration.

  • 22.
    Lundberg, Max
    et al.
    Lund University.
    Liedvogel, Miriam
    Lund University / Max Planck Institute for Evolutionary Biology, Plön, Germany.
    Larson, Keith
    Umeå University.
    Sigeman, Hanna
    Lund University.
    Grahn, Mats
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Environmental Science.
    Wright, Anthony Ph
    Karolinska Institute, Karolinska University Hospital.
    Åkesson, Susanne
    Lund University.
    Bensch, Staffan
    Lund University.
    Genetic differences between willow warbler migratory phenotypes are few and cluster in large haplotype blocks2017In: Evolution Letters, ISSN 2056-3744, Vol. 1, no 3, p. 155-168Article in journal (Refereed)
    Abstract [en]

    It is well established that differences in migratory behavior between populations of songbirds have a genetic basis but the actual genes underlying these traits remains largely unknown. In an attempt to identify such candidate genes we de novo assembled the genome of the willow warbler Phylloscopus trochilus, and used whole-genome resequencing and a SNP array to associate genomic variation with migratory phenotypes across two migratory divides around the Baltic Sea that separate SW migrating P. t. trochilus wintering in western Africa and SSE migrating P. t. acredula wintering in eastern and southern Africa. We found that the genomes of the two migratory phenotypes lack clear differences except for three highly differentiated regions located on chromosomes 1, 3, and 5 (containing 146, 135, and 53 genes, respectively). Within each migratory phenotype we found virtually no differences in allele frequencies for thousands of SNPs, even when comparing geographically distant populations breeding in Scandinavia and Far East Russia (>6000 km). In each of the three differentiated regions, multidimensional scaling-based clustering of SNP genotypes from more than 1100 individuals demonstrates the presence of distinct haplotype clusters that are associated with each migratory phenotype. In turn, this suggests that recombination is absent or rare between haplotypes, which could be explained by inversion polymorphisms. Whereas SNP alleles on chromosome 3 correlate with breeding altitude and latitude, the allele distribution within the regions on chromosomes 1 and 5 perfectly matches the geographical distribution of the migratory phenotypes. The most differentiated 10 kb windows and missense mutations within these differentiated regions are associated with genes involved in fatty acid synthesis, possibly representing physiological adaptations to the different migratory strategies. The ∼200 genes in these regions, of which several lack described function, will direct future experimental and comparative studies in the search for genes that underlie important migratory traits.

  • 23.
    Neely, K E
    et al.
    Pennsylvania State University, Pennsylvania, USA.
    Hassan, A H
    Pennsylvania State University, Pennsylvania, USA.
    Wallberg, Annika E
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Steger, D J
    Pennsylvania State University, Pennsylvania, USA.
    Cairns, B R
    University of Utah School of Medicine, Salt Lake City, USA.
    Wright, Athony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Workman, J L
    Pennsylvania State University, Pennsylvania, USA.
    Activation domain-mediated targeting of the SWI/SNF complex to promoters stimulates transcription from nucleosome arrays1999In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 4, no 4, p. 649-655Article in journal (Refereed)
    Abstract [en]

    The yeast SWI/SNF complex is required for the transcription of several yeast genes and has been shown to alter nucleosome structure in an ATP-dependent reaction. In this study, we show that the complex stimulated in vitro transcription from nucleosome templates in an activation domain-dependent manner. Transcription stimulation by SWI/SNF required an activation domain with which it directly interacts. The acidic activation domains of VP16, Gcn4, Swi5, and Hap4 interacted directly with the purified SWI/SNF complex and with the SWI/SNF complex in whole-cell extracts. The similarity of activation domain interactions and transcriptional stimulation between SWI/SNF and the SAGA histone acetyltransferase complex may account for their apparent overlapping functions in vivo.

  • 24.
    Nilsson, Johan
    et al.
    Södertörn University, School of Life Sciences, Molecular biology.
    Grahn, Mats
    Södertörn University, School of Life Sciences, Environmental science. Södertörn University, School of Life Sciences, Biology.
    Wright, Anthony Ph
    Södertörn University, School of Life Sciences, Molecular biology.
    Proteome-wide evidence for enhanced positive Darwinian selection within intrinsically disordered regions in proteins2011In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 12, no 7, p. R65-Article in journal (Refereed)
    Abstract [en]

    ABSTRACT: BACKGROUND: Understanding the adaptive changes that alter the function of proteins during evolution is an important question for biology and medicine. The increasing number of completely sequenced genomes from closely related organisms, as well as individuals within species, facilitates systematic detection of recent selection events by means of comparative genomics. RESULTS: We have used genome-wide strain-specific single nucleotide polymorphism data from 64 strains of budding yeast (Saccharomyces cerevisiae or Saccharomyces paradoxus) to determine whether adaptive positive selection is correlated with protein regions showing propensity for different classes of structure conformation. Data from phylogenetic and population genetic analysis of 3746 gene alignments consistently shows a significantly higher degree of positive Darwinian selection in intrinsically disordered regions of proteins compared to regions of alpha helix, beta sheet or tertiary structure. Evidence of positive selection is significantly enriched in classes of proteins whose functions and molecular mechanisms can be coupled to adaptive processes and these classes tend to have a higher average content of intrinsically unstructured protein regions. CONCLUSIONS: We suggest that intrinsically disordered protein regions may be important for the production and maintenance of genetic variation with adaptive potential and that they may thus be of central significance for the evolvability of the organism or cell in which they occur.

  • 25.
    Nugent, Rebecca L.
    et al.
    University of Southern California.
    Johnsson, Anna
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Intitutet.
    Fleharty, Brian
    Stowers Institute for Medical Research.
    Gogol, Madelaine
    Stowers Institute for Medical Research.
    Xue-Franzen, Yongtao
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Seidel, Chris
    Stowers Institute for Medical Research.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Forsburg, Susan L.
    University of Southern California.
    Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation2010In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, article id 59Article in journal (Refereed)
    Abstract [en]

    Background: Histone acetyltransferase enzymes (HATs) are implicated in egulation of transcription. HATs from different families may overlap in arget and substrate specificity. esults: We isolated the elp3(+) gene encoding the histone cetyltransferase subunit of the Elongator complex in fission yeast and haracterized the phenotype of an Delta elp3 mutant. We examined genetic nteractions between Delta elp3 and two other HAT mutants, Delta mst2 nd Delta gcn5 and used whole genome microarray analysis to analyze heir effects on gene expression. onclusions: Comparison of phenotypes and expression profiles in single, ouble and triple mutants indicate that these HAT enzymes have verlapping functions. Consistent with this, overlapping specificity in istone H3 acetylation is observed. However, there is no evidence for verlap with another HAT enzyme, encoded by the essential mst1(+) gene.

  • 26.
    Provost, P
    et al.
    Karolinska Institute.
    Silverstein, Rebecca A
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Dishart, D
    Karolinska Institute.
    Walfridsson, Julian
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Djupedal, Ingela
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Kniola, Barbara
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Samuelsson, B
    Karolinska Institute.
    Radmark, O
    Karolinska Institute.
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Dicer is required for chromosome segregation and gene silencing in fission yeast cells2002In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 99, no 26, p. 16648-16653Article in journal (Refereed)
    Abstract [en]

    RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1(+)) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into approximate to23 nucleotide fragments in vitro, and dcr1Delta cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1(+) was required for silencing of two genes containing a conserved motif.

  • 27.
    Shiue, Chiou-Nan
    et al.
    Karolinska Institutet.
    Arabi, Azadeh
    Karolinska Institutet.
    Wright, Anthony P H
    Karolinska Institutet.
    Nucleolar organization, growth control and cancer2010In: Epigenetics, ISSN 1559-2294, E-ISSN 1559-2308, Vol. 5, no 3, p. 200-205Article in journal (Refereed)
    Abstract [en]

    The nucleolus is a dynamic region of the nucleus that is disassembled and reformed each cell cycle and whose size is correlated with cell growth rate. Nucleolar size is a prognostic measure of cancer disease severity and increasing evidence suggests a causative role of nucleolar lesions in many cancers. In recent work (Shiue et al. Oncogene 28, 1833-42, 2009) we showed that the c-Myc oncoprotein induces changes in the higher order structure of rDNA chromatin in the nucleolus of growth stimulated quiescent rat cells. Here we show that c-Myc induces similar changes in human cells, that c-Myc plays a role in the overall structural integrity of the nucleolus and that c-Myc and its antagonistic partner Mad1 interact to program the epigenetic status of rDNA chromatin. These changes are discussed in relation to current knowledge about nucleolar structure as well as the organization of chromosomes and transcription factories in nuclear regions outside the nucleolus.

  • 28.
    Shiue, Chiounan N
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Berkson, Rachel G.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Anthony P H
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    c-Myc induces changes in higher order rDNA structure on stimulation of quiescent cells2009In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 28, no 16, p. 1833-1842Article in journal (Refereed)
    Abstract [en]

    c-Myc is an oncogenic transcription factor capable of activating transcription by all three nuclear RNA polymerases, thus acting as a high-level coordinator of protein synthesis capacity and cell growth rate. c-Myc recruits RNA polymerase I-related transcription factors to the rDNA when quiescent cells are stimulated to re-enter the cell cycle. Using a model system of cell lines with variable c-Myc status, we show that on stimulation c-Myc rapidly induces gene loop structures in rDNA chromatin that juxtapose upstream and downstream rDNA sequences. c-Myc activation is both necessary and sufficient for this change in rDNA chromatin conformation. c-Myc activation induces association of TTF-1 with the rDNA, and c-Myc is physically associated with induced rDNA gene loops. The origins of two or more rDNA gene loops are closely juxtaposed, suggesting the possibility that c-Myc induces nucleolar chromatin hubs. Induction of rDNA gene loops may be an early step in the reprogramming of quiescent cells as they re-enter the growth cycle.

  • 29.
    Shiue, Chiou-Nan
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Anthony P.
    c-Myc induces recruitment of rDNA genes to the nuclear matrix in response to cell growth stimulationManuscript (preprint) (Other academic)
  • 30. Wallberg, A E
    et al.
    Neely, K E
    Hassan, A H
    Gustafsson, J A
    Workman, J L
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap.
    Recruitment of the SWI-SNF chromatin remodeling complex as a mechanism of gene activation by the glucocorticoid receptor tau 1 activation domain2000In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 20, no 6, p. 2004-2013Article in journal (Refereed)
    Abstract [en]

    The SWI-SNF complex has been shown to alter nucleosome conformation in an ATP-dependent manner, leading to increased accessibility of nucleosomal DNA to transcription factors. In this study, we show that the SWI-SNF complex can potentiate the activity of the glucocorticoid receptor (GR) through the N-terminal transactivation domain, tau 1, in both yeast and mammalian cells. GR-sl can directly interact with purified SWI-SNF complex, and mutations in tau 1 that affect the transactivation activity in vivo also directly affect tau 1 interaction with SWI-SNF. Furthermore, the SWI-SNF complex can stimulate tau 1-driven transcription from chromatin templates in vitro, Taken together, these results support a model in which the GR can directly recruit the SWI-SNF complex to target promoters during glucocorticoid-dependent gene activation. We also provide evidence that the SWI-SNF and SAGA complexes represent independent pathways of tau 1-mediated activation but play overlapping roles that are able to compensate for one another under some conditions.

  • 31. Wallberg, A E
    et al.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap.
    Gustafsson, J A
    Chromatin-remodeling complexes involved in gene activation by the glucocorticoid receptor2001In: Vitamines and Hormones, ISSN 0083-6729, Vol. 60, p. 75-122Article in journal (Refereed)
  • 32. Wärnmark, A
    et al.
    Treuter, E
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Gustafsson, J A
    Activation functions 1 and 2 of nuclear receptors: Molecular strategies for transcriptional activation2003In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 17, no 10, p. 1901-1909Article in journal (Refereed)
    Abstract [en]

    Nuclear receptors (NRs) comprise a family of ligand inducible transcription factors. To achieve transcriptional activation of target genes, DNA-bound NRs directly recruit general transcription factors (GTFs) to the preinitiation complex or bind intermediary factors, so-called coactivators. These coactivators often constitute subunits of larger multiprotein complexes that act at several functional levels, such as chromatin remodeling, enzymatic modification of histone tails, or modulation of the preinitiation complex via interactions with RNA polymerase II and GTFs. The binding of NR to coactivators is often mediated through one of its activation domains. Many NRs have at least two activation domains, the ligand-independent activation function (AF)-1, which resides in the N-terminal domain, and the ligand-dependent AF-2, which is localized in the C-terminal domain. In this review, we summarize and discuss current knowledge regarding the molecular mechanisms of AF-1- and AF-2-mediated gene activation, focusing on AF-1 and AF-2 conformation and coactivator binding.

  • 33. Wärnmark, A
    et al.
    Wikström, Anja
    KTH.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Gustafsson, J A
    Härd, T
    The N-terminal regions of estrogen receptor alpha and beta are unstructured in vitro and show different TBP binding properties2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 49, p. 45939-45944Article in journal (Refereed)
    Abstract [en]

    The N-terminal regions of the estrogen receptor ve (ER alpha -N) and beta (ER beta -N) were expressed and purified to homogeneity. Using NAM and circular dichroism spectroscopy, we conclude that both ER alpha -N and ER beta -N are unstructured in solution. The TATA box-binding protein (TBP) has been shown previously to interact with ERa-N in vitro and to potentiate ER-activated transcription. We used surface plasmon resonance and circular dichroism spectroscopy to confirm and further characterize the ER-N-TBP interaction. Our results show that the intrinsically unstructured ERa-N interacts with TBP, and suggest that structural changes are induced in ERa-N upon TBP interaction. Conformational changes upon target factor interaction have not previously been demonstrated for any N-terminal region of nuclear receptors. In addition, no binding of ER beta -N to TBP was detected. This difference in TBP binding could imply differential recruitment of target proteins by ERa-N and ER beta -N. The affinity of the ER alpha -N-TBP interaction was determined to be in the micromolar range (K-D = 10(-6) to 10(-5) m). Our results support models of TBP as a target protein for the N-terminal activation domain of ER alpha. Further, our results suggest that target proteins can induce and/or stabilize ordered structure in N-terminal regions of nuclear receptors upon interaction.

  • 34.
    Wärnmark, Anette
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Intsitute.
    Gustafsson, J A
    Karolinska Institute.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Architectural principles for the structure and function of the glucocorticoid receptor tau 1 core activation domain2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 20, p. 15014-15018Article in journal (Refereed)
    Abstract [en]

    A 58-amino acid region mediates the core transactivation activity of the glucocorticoid receptor tau 1 activation domain. This tau 1 core domain is unstructured in aqueous buffers, but in the presence of trifluoroethanol three Alpha-helical segments are induced. Two of these putative structural modules have been tested in different combinations with regard to transactivation potential in vivo and binding capacity to the coactivators in vitro, The results show that whereas single modules are not transcriptionally active, any combination of two or three modules is sufficient, with trimodular constructs having the highest activity. However, proteins containing one, two, or three segments bind Ada2 and cAMP-response element-binding protein with similar affinity. A single segment is thus able to bind a target factor but cannot transactivate target genes significantly. The results are consistent with models in which activation domains are comprised of short activation modules that allow multiple interactions with coactivators. Our results also suggest that an increased number of modules may not result in correspondingly higher affinity but instead that the concentration of binding sites is increased, which gives rise to a higher association rate. This is consistent with a model where the association rate for activator-target factor interactions rather than the equilibrium constant is the most relevant measure of activator potency.

  • 35.
    Xue, Yongtao
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    Haas, S A
    Max-Plank Institute for Molecular Genetics, Berlin, Germany.
    Brino, L
    Eurogentec SA, Seraing, Belgium.
    Gusnanto, A
    Karolinska Institute.
    Reimers, M
    Karolinska Institute.
    Talibi, D
    Eurogentec SA, Seraing, Belgium.
    Vingron, M
    Max-Plank Institute for Molecular Genetics, Berlin, Germany.
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    Wright, Anthony P H
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift2004In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 21, no 1, p. 25-39Article in journal (Refereed)
    Abstract [en]

    Completion of the fission yeast genome sequence has opened up possibilities for post-genomic approaches. We have constructed a DNA microarray for genome-wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR-amplified from a genomic DNA template, that represent >99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3'-end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25degreesC to 36degreesC, conditions widely used when studying temperature-sensitive mutants. Obligingly, the vast majority of genes do not change more than two-fold, supporting the widely held view that temperature-shift experiments specifically reveal phenotypes associated with temperature-sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat-shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here.. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On-line Supplementary Information and are also available commercially.

  • 36.
    Xue-Franzen, Yongtao
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Johnsson, Anna
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Intitutet.
    Brodin, David
    Karolinska Institutet.
    Henriksson, Johan
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Bürglin, Thomas R.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles2010In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, article id 200Article in journal (Refereed)
    Abstract [en]

    Background: Gcn5 is a transcriptional coactivator with histone cetyltransferase activity that is conserved with regard to structure as ell as its histone substrates throughout the eukaryotes. Gene egulatory networks within cells are thought to be evolutionarily iverged. The use of evolutionarily divergent yeast species, such as S. erevisiae and S. pombe, which can be studied under similar nvironmental conditions, provides an opportunity to examine the nterface between conserved regulatory components and their cellular pplications in different organisms. esults: We show that Gcn5 is important for a common set of stress esponses in evolutionarily diverged yeast species and that the activity f the conserved histone acetyltransferase domain is required. We define group of KCl stress response genes in S. cerevisiae that are pecifically dependent on Gcn5. Gcn5 is localised to many Gcn5-dependent enes including Gcn5 repressed targets such as FLO8. Gcn5 regulates ivergent sets of KCl responsive genes in S. cerevisiae and S. pombe. enome-wide localization studies showed a tendency for redistribution of cn5 during KCl stress adaptation in S. cerevisiae from short genes to he transcribed regions of long genes. An analogous redistribution was ot observed in S. pombe. onclusions: Gcn5 is required for the regulation of divergent sets of Cl stress-response genes in S. cerevisiae and S. pombe even though it s required a common group of stress responses, including the response o KCl. Genes that are physically associated with Gcn5 require its ctivity for their repression or activation during stress adaptation, roviding support for a role of Gcn5 as a corepressor as well as a oactivator. The tendency of Gcn5 to re-localise to the transcribed egions of long genes during KCl stress adaptation suggests that Gcn5 lays a specific role in the expression of long genes under adaptive onditions, perhaps by regulating transcriptional elongation as has been een for Gcn5 in S. pombe. Interestingly an analogous redistribution of cn5 is not seen in S. pombe. The study thus provides important new nsights in relation to why coregulators like Gcn5 are required for the orrect expression of some genes but not others.

  • 37.
    Xue-Franzen, Yongtao
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Kjaerulff, Soren
    University of Copenhagen, Copenhagen, Denmark.
    Holmberg, Christian
    University of Copenhagen, Copenhagen, Denmark.
    Wright, Anthony
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Nielsen, Olaf
    University of Copenhagen, Copenhagen, Denmark.
    Genomewide identification of pheromone-targeted transcription in fission yeast2006In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, p. 303-, article id 303Article in journal (Refereed)
    Abstract [en]

    Background: Fission yeast cells undergo sexual differentiation in response to nitrogen starvation. In this process haploid M and P cells first mate to form diploid zygotes, which then enter meiosis and sporulate. Prior to mating, M and P cells communicate with diffusible mating pheromones that activate a signal transduction pathway in the opposite cell type. The pheromone signalling orchestrates mating and is also required for entry into meiosis. Results: Here we use DNA microarrays to identify genes that are induced by M-factor in P cells and by P-factor in M-cells. The use of a cyr1 genetic background allowed us to study pheromone signalling independently of nitrogen starvation. We identified a total of 163 genes that were consistently induced more than two-fold by pheromone stimulation. Gene disruption experiments demonstrated the involvement of newly discovered pheromone-induced genes in the differentiation process. We have mapped Gene Ontology ( GO) categories specifically associated with pheromone induction. A direct comparison of the M- and P-factor induced expression pattern allowed us to identify cell-type specific transcripts, including three new M- specific genes and one new P-specific gene. Conclusion: We found that the pheromone response was very similar in M and P cells. Surprisingly, pheromone control extended to genes fulfilling their function well beyond the point of entry into meiosis, including numerous genes required for meiotic recombination. Our results suggest that the SteII transcription factor is responsible for the majority of pheromone-induced transcription. Finally, most cell-type specific genes now appear to be identified in fission yeast.

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