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Studies of transcription factor domains and their interactions with other transcription factors
Södertörn University, School of Life Sciences, Molecular biology. Karolinska institutet.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The studies in this thesis deal with different questions concerning interactions of functional domains of factors involved in transcriptional regulation. The first study of this thesis is focused on the target factor binding mechanism of transcriptional activators. Many activators in evolutionary distant species are classified as acidic based on a high content of acidic residues in the activation domain and intrinsically unstructured in solution. Our results indicate that such activation domains interact with target factors through coupled binding and folding of the activation domain after an initial ionic interaction, and demonstrate the generality of this binding mechanism. We propose that target interaction through coupled binding and folding of the recruiting domain is important for the role of activators as regulators of transcription. In the following study we show that deletion of two regions that mediate interaction with activators in vitro prevents promoter recruitment of the SWI/SNF chromatinremodeling complex in vivo, and causes strongly reduced transcriptional activity of the corresponding genes. This study validates direct interaction between the Swi1- and Snf5 activator binding domains of the S. cerevisiae SWI/SNF complex and activators previously demonstrated in vitro, and importantly indicates that the activator binding domains are essential for the ability of SWI/SNF to function as co-activator. In the last study we investigate which domains are involved in distinct in vivo function of the paralogous co-repressors Tup11 and Tup12 of the Ssn6/Tup complex in S. pombe. Tup11 and Tup12 have been shown to differ in importance in context of a common complex for subsets of Ssn6/Tup target genes, and it was proposed that this might depend on divergence in the histone-interaction domain. Here we show that distinct in vivo roles of Tup12 do not depend on differences in the highly diverged histoneinteraction domain, but mainly on differences in the overall highly conserved WD40 repeat domain, which putatively mediates interaction with repressors and target factors such as histone modifying complexes and components of the transcriptional machinery. We propose that clusters of amino acids, putatively located in blade 3 of the WD40 repeat domain, could be important for interaction with distinct target factors of Tup11 and Tup12. Furthermore, we show that the stoichiometry of the Ssn6/Tup complex is likely to change under CaCl2 stress, by a mechanism involving changes in the relative cellular levels of the complex components.

Place, publisher, year, edition, pages
Stockholm: Karolinska institutet , 2009. , 46 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:sh:diva-31106ISBN: 9789174095333OAI: oai:DiVA.org:sh-31106DiVA: diva2:1045406
Public defence
2009-06-11, MB505, Alfred Nobels allé 7, Huddinge, 15:00 (English)
Opponent
Supervisors
Available from: 2016-11-09 Created: 2016-11-09 Last updated: 2016-11-09Bibliographically approved
List of papers
1. Mechanism of transcription factor recruitment by acidic activators
Open this publication in new window or tab >>Mechanism of transcription factor recruitment by acidic activators
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2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 23, 21779-21784 p.Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-14456 (URN)10.1074/jbc.M502627200 (DOI)000229557900015 ()15826952 (PubMedID)2-s2.0-20444475372 (ScopusID)
Available from: 2011-12-23 Created: 2011-12-23 Last updated: 2016-11-09Bibliographically approved
2. Activator-binding domains of the SWI/SNF chromatin remodeling complex characterized in vitro are required for its recruitment to promoters in vivo
Open this publication in new window or tab >>Activator-binding domains of the SWI/SNF chromatin remodeling complex characterized in vitro are required for its recruitment to promoters in vivo
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2009 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 9, 2557-2565 p.Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-13900 (URN)10.1111/j.1742-4658.2009.06979.x (DOI)000264882700012 ()19476494 (PubMedID)2-s2.0-64149100814 (ScopusID)
Available from: 2011-12-14 Created: 2011-12-14 Last updated: 2016-11-09Bibliographically approved
3. Protein domains underlying functional divergence between the Tup11 and Tup12 co-repressor proteins in fission yeast
Open this publication in new window or tab >>Protein domains underlying functional divergence between the Tup11 and Tup12 co-repressor proteins in fission yeast
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-31105 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2016-11-09 Created: 2016-11-09 Last updated: 2016-11-09Bibliographically approved

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