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PreprintCited by 4Genomics and Chromatin DynamicsFungal and yeast genetics researchProtein Structure and Dynamics

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Functional characterization and comparative analysis of gene repression-mediating domains interacting with yeast pleiotropic corepressors Sin3, Cyc8 and Tup1

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Lettow, Julia, Kliewe, Felix, Aref, Rasha, Schüller, Hans-Joachim·2023·Current Genetics
DOI 10.21203/rs.3.rs-2475911/v1PMC PMC10163088

Preprint — published as 10.1007/s00294-023-01262-6

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Abstract

Transcriptional corepressors Sin3, Cyc8 and Tup1 are important for downregulation of gene expression by recruiting various histone deacetylases once they gain access to defined genomic locations by interaction with pathway-specific repressor proteins. In this work we systematically investigated whether 17 yeast repressor proteins (Cti6, Dal80, Fkh1, Gal80, Mig1, Mot3, Nrg1, Opi1, Rdr1, Rox1, Sko1, Ume6, Ure2, Xbp1, Yhp1, Yox1 and Whi5) representing several unrelated regulatory pathways are able to bind to Sin3, Cyc8 and Tup1. Our results show that paired amphipathic helices 1 and 2 (PAH1 and PAH2) of Sin3 are functionally redundant for some regulatory pathways. WD40 domains of Tup1 proved to be sufficient for interaction with repressor proteins. Using length variants of selected repressors, we mapped corepressor interaction domains (CIDs) in vitro and assayed gene repression in vivo. Systematic comparison of CID minimal sequences allowed us to define several related positional patterns of hydrophobic amino acids some of which could be confirmed as functional important by site-directed mutagenesis. Although structural predictions indicated that certain CIDs may be α-helical, most repression domains appear to be randomly structured and must be considered as intrinsically disordered regions (IDR) adopting a defined conformation only by interaction with a corepressor.

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Fig. 1 In vitro interaction of selected transcriptional repressor proteins with pleiotropic corepressors and gene repression by lexA fusions in vivo. For pull-down assays, GST fusions with expression cassettes encoding full-length proteins were used (exception: Ume6 aa 508–594, shown to able to bind PAH2 of Sin3; Kadosh and Struhl 1997). GST fusion proteins were immobilized on GSH sepharose and incubated with bacterial protein extracts, containing HA-Sin3 (pSW11; aa 1–1536, full-length), HA-Cyc8 (pFK77, aa 1–398, encoding TPR motifs) or HA-Tup1 (pFK76; full-length). Gene repression in vivo was measured in transformants of strain RTS-lexA (reporter gene lexAOp-CYC1-lacZ) containing plasmids which encode lexA fusions. Specific β-galactosidase activities are given in nmol oNPG hydrolyzed per min per mg of protein. Protein extracts prepared from at least 12 independent transformants were assayed. GST and lexA fusion plasmids are compiled in Supporting Online Material (Table S2). In vivo gene repression (RF, repression factor) was calculated as the ratio of specific β-galactosidase activities measured in transformants with empty vector pRT-lexA and individual lexA-repressor fusions, respectively. n. t. not tested, SD standard deviation, + in vitro interaction, - no interaction
Fig. 1 In vitro interaction of selected transcriptional repressor proteins with pleiotropic corepressors and gene repression by lexA fusions in vivo. For pull-down assays, GST fusions with expression cassettes encoding full-length proteins were used (exception: Ume6 aa 508–594, shown to able to bind PAH2 of Sin3; Kadosh and Struhl 1997). GST fusion proteins were immobilized on GSH sepharose and incubated with bacterial protein extracts, containing HA-Sin3 (pSW11; aa 1–1536, full-length), HA-Cyc8 (pFK77, aa 1–398, encoding TPR motifs) or HA-Tup1 (pFK76; full-length). Gene repression in vivo was measured in transformants of strain RTS-lexA (reporter gene lexAOp-CYC1-lacZ) containing plasmids which encode lexA fusions. Specific β-galactosidase activities are given in nmol oNPG hydrolyzed per min per mg of protein. Protein extracts prepared from at least 12 independent transformants were assayed. GST and lexA fusion plasmids are compiled in Supporting Online Material (Table S2). In vivo gene repression (RF, repression factor) was calculated as the ratio of specific β-galactosidase activities measured in transformants with empty vector pRT-lexA and individual lexA-repressor fusions, respectively. n. t. not tested, SD standard deviation, + in vitro interaction, - no interactionCC BY · PubMed Central
Fig. 2 Interaction of gene-specific repressor proteins with functional domains of Sin3. GST fusions with full-length repressor proteins (for expression plasmids cf. Supporting Online Table S2) were immobilized on GSH sepharose and incubated with protein extracts from yeast transformants, containing epitope-tagged Sin3 length variants 1–300 (PAH1; pCW83), 301–600 (PAH2; pYJ91), 601–950 (PAH3 + HID1; pYJ90), 801–1100 (HID1; pYJ89) and 1100–1536 (PAH4; pMP20), respectively. GST devoid of repressor fusion served as a negative control. HID HDAC interaction domain; PAH, paired amphipathic helix, + in vitro interaction, - no interaction
Fig. 2 Interaction of gene-specific repressor proteins with functional domains of Sin3. GST fusions with full-length repressor proteins (for expression plasmids cf. Supporting Online Table S2) were immobilized on GSH sepharose and incubated with protein extracts from yeast transformants, containing epitope-tagged Sin3 length variants 1–300 (PAH1; pCW83), 301–600 (PAH2; pYJ91), 601–950 (PAH3 + HID1; pYJ90), 801–1100 (HID1; pYJ89) and 1100–1536 (PAH4; pMP20), respectively. GST devoid of repressor fusion served as a negative control. HID HDAC interaction domain; PAH, paired amphipathic helix, + in vitro interaction, - no interactionCC BY · PubMed Central
Fig. 3 Interaction of selected gene-specific repressor proteins with N- and C-terminal length variants of Tup1. a Structural features of Tup1 and position of HA-tagged length variants used for interaction studies. b GST fusions with repressor proteins alpha2, Yox1, Gal80, Ure2, Cti6 and Dal80 (for expression plasmids cf. Supporting Online Table S2) were immobilized on GSH sepharose and incubated with bacterial protein extracts, containing epitope-tagged Tup1 length variants 1–339 (no WD40 repeats; pVS1), 340-713 (WD40 repeats 1–7; pVS2) and 599–713 (WD40 repeats 6 and 7; pVS5), respectively
Fig. 3 Interaction of selected gene-specific repressor proteins with N- and C-terminal length variants of Tup1. a Structural features of Tup1 and position of HA-tagged length variants used for interaction studies. b GST fusions with repressor proteins alpha2, Yox1, Gal80, Ure2, Cti6 and Dal80 (for expression plasmids cf. Supporting Online Table S2) were immobilized on GSH sepharose and incubated with bacterial protein extracts, containing epitope-tagged Tup1 length variants 1–339 (no WD40 repeats; pVS1), 340-713 (WD40 repeats 1–7; pVS2) and 599–713 (WD40 repeats 6 and 7; pVS5), respectivelyCC BY · PubMed Central
Fig. 4 Molecular analysis of Yox1 interaction with corepressors and gene repression by lexA fusions. Yox1 length variants were fused with GST, immobilized on GSH sepharose and incubated with bacterial protein extracts, containing HA-Sin3 (pJL34; aa 1-480, comprising PAH1 and PAH2), HA-Cyc8 (pFK77, aa 1-398, encoding TPR motifs) or HA-Tup1 (pFK76; full-length). GST expression plasmids and lexA fusion plasmids encoding length variants or missense mutations of Yox1 are compiled in Supporting Online Material (Table S2). Gene repression in vivo was measured in transformants of strain RTS-lexA (reporter gene lexAOp-CYC1-lacZ). Empty vector pRT-lexA devoid of effector domains was used as a negative control for maximal reporter gene expression. Specific β-galactosidase activities are given in nmol oNPG hydrolyzed per min per mg of protein. Protein extracts prepared from at least 12 independent transformants were assayed. HD homeodomain, n. t. not tested, RF repression factor, SD standard deviation, + in vitro interaction, - no interaction
Fig. 4 Molecular analysis of Yox1 interaction with corepressors and gene repression by lexA fusions. Yox1 length variants were fused with GST, immobilized on GSH sepharose and incubated with bacterial protein extracts, containing HA-Sin3 (pJL34; aa 1-480, comprising PAH1 and PAH2), HA-Cyc8 (pFK77, aa 1-398, encoding TPR motifs) or HA-Tup1 (pFK76; full-length). GST expression plasmids and lexA fusion plasmids encoding length variants or missense mutations of Yox1 are compiled in Supporting Online Material (Table S2). Gene repression in vivo was measured in transformants of strain RTS-lexA (reporter gene lexAOp-CYC1-lacZ). Empty vector pRT-lexA devoid of effector domains was used as a negative control for maximal reporter gene expression. Specific β-galactosidase activities are given in nmol oNPG hydrolyzed per min per mg of protein. Protein extracts prepared from at least 12 independent transformants were assayed. HD homeodomain, n. t. not tested, RF repression factor, SD standard deviation, + in vitro interaction, - no interactionCC BY · PubMed Central
Fig. 5 Molecular analysis of corepressor interaction in vitro and gene repression in vivo by lexA fusions of repressors Dal80 (a), Xbp1 (b) and Ure2 (c). Length variants of repressor proteins were fused with GST, immobilized on GSH sepharose and incubated with bacterial protein extracts, containing HA-Sin3 (pJL34; aa 1-480, comprising PAH1 and PAH2), HA-Cyc8 (pFK77, aa 1-398, encoding TPR motifs) or HA-Tup1 (pFK76; full-length). GST expression plasmids and lexA fusion plasmids encoding length variants of Dal80, Xbp1 and Ure2 are compiled in Supporting Online Material (Table S2). Gene repression in vivo was measured in transformants of strain RTS-lexA (reporter gene lexAOp-CYC1-lacZ) which contain plasmids encoding lexA fusions. Empty vector pRT-lexA was used as a negative control for maximal reporter gene expression. Specific β-galactosidase activities are given in nmol oNPG hydrolyzed per min per mg of protein. Protein extracts prepared from at least 12 independent transformants were assayed. ZF zinc finger, MR Mbp1-related DNA-binding domain, n. t. not tested, RF repression factor, SD standard deviation, + in vitro interaction, - no interaction
Fig. 5 Molecular analysis of corepressor interaction in vitro and gene repression in vivo by lexA fusions of repressors Dal80 (a), Xbp1 (b) and Ure2 (c). Length variants of repressor proteins were fused with GST, immobilized on GSH sepharose and incubated with bacterial protein extracts, containing HA-Sin3 (pJL34; aa 1-480, comprising PAH1 and PAH2), HA-Cyc8 (pFK77, aa 1-398, encoding TPR motifs) or HA-Tup1 (pFK76; full-length). GST expression plasmids and lexA fusion plasmids encoding length variants of Dal80, Xbp1 and Ure2 are compiled in Supporting Online Material (Table S2). Gene repression in vivo was measured in transformants of strain RTS-lexA (reporter gene lexAOp-CYC1-lacZ) which contain plasmids encoding lexA fusions. Empty vector pRT-lexA was used as a negative control for maximal reporter gene expression. Specific β-galactosidase activities are given in nmol oNPG hydrolyzed per min per mg of protein. Protein extracts prepared from at least 12 independent transformants were assayed. ZF zinc finger, MR Mbp1-related DNA-binding domain, n. t. not tested, RF repression factor, SD standard deviation, + in vitro interaction, - no interactionCC BY · PubMed Central

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Metadata

Authors
Lettow, J., Kliewe, F., Aref, R., & Schüller, H.-J.
Affiliations
  • Universität Greifswald (DE)
  • Universitätsmedizin Greifswald (DE)
  • Ain Shams University (EG)
Corresponding author
Schüller, Hans-Joachim
ORCID
Venue
Current Genetics
Publisher
Research Square Platform LLC
Published
2023-01-18
Volume
69
Issue
2-3
Pages
127-139
ISSN
0172-8083 (ISSN-L)
DOI
10.21203/rs.3.rs-2475911/v1
PMC
PMC10163088
OpenAlex
W4322720308
Cited by
4
References
1
Citations by year
2026: 1 · 2024: 2 · 2023: 1
FWCI
0.35
Type
journal article
Language
en
Open access
Licence
Locations
  • Current Genetics (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Greater South Information System
Funders
Universität Greifswald
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
Keywords
corepressors, Sin3, Tup1, gene repression, repressor protein, WD40 domain, intrinsically disordered regions, Saccharomyces cerevisiae, histone deacetylase, protein-protein interaction domains
MeSH terms
Carrier Proteins, DNA-Binding Proteins, Glutathione Peroxidase, Nuclear Proteins, Phosphatidate Phosphatase, Prions, Repressor Proteins, Saccharomyces cerevisiae, Transcription Factors, Transcription, Genetic, Gene Expression Regulation, Fungal, Cell Cycle Proteins, Saccharomyces cerevisiae Proteins, Basic-Leucine Zipper Transcription Factors, GATA Transcription Factors, Forkhead Transcription Factors, Co-Repressor Proteins

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CoChat. (2026). Citation check: Functional characterization and comparative analysis of gene repression-mediating domains interacting with yeast pleiotropic corepressors Sin3, Cyc8 and Tup1. https://app.cochat.ai/papers/10.21203/rs.3.rs-2475911/v1
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Lettow, J., Kliewe, F., Aref, R., & Schüller, H.-J. (2023). Functional characterization and comparative analysis of gene repression-mediating domains interacting with yeast pleiotropic corepressors Sin3, Cyc8 and Tup1. Current Genetics. https://doi.org/10.21203/rs.3.rs-2475911/v1