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A D53 repression motif induces oligomerization of TOPLESS corepressors and promotes assembly of a corepressor-nucleosome complex

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Science Advances  02 Jun 2017:
Vol. 3, no. 6, e1601217
DOI: 10.1126/sciadv.1601217
  • Fig. 1 D53(794–808) is a functional EAR motif.

    (A) Domain map of D53, with the position of three putative EAR motifs indicated. The segments with homology to the N-cap, the two AAA domains, and the middle domain (MD) of Clp-disaggregation machines are indicated. (B) AlphaScreen interaction assays between biotinylated peptides representing the three putative D53 EAR motifs and His6Sumo-tagged TPR2 TPD. (C) M2H assay between VP16 activation domain-fused TPL/TPR and Gal4 DBD-fused full-length wild-type (WT) D53 and triple EAR L→A mutant d53 [mEAR1, mEAR2, mEAR3; see mutated leucine residues in (A)]. RLU, relative luciferase units. (D) M2H assay of the interaction between the TPDs of the three TPL proteins and the 15-residue EAR-3 motif shown in (A) and (B) fused to the Gal4 DBD. ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001, one-way analysis of variance (ANOVA) with Tukey’s post hoc test; n = 3; error bars = SEM.

  • Fig. 2 Mutations in both EAR-2 and EAR-3 partially compromise D53 repressor activity in protoplasts and D53-mediated SL signaling in transgenic rice.

    (A) D53 EAR mutant repressor activities. Gal4 DBD-D53-VP16 expression plasmids were cotransfected with a 35S promoter–Gal4 binding site (UAS)–firefly luciferase (LUC) reporter plasmid and a 35S promoter–Renilla luciferase reference plasmid into rice protoplasts; different letters above the columns indicate statistically significant differences between groups [Tukey’s honest significant difference (HSD) test, P < 0.05]. All data are presented as means ± SD. (B) Wild-type and mutant d53-overexpressing transgenic plants, tagged with 3×FLAG. (C and D) Tiller number (C) and height (D) of T2 generation transgenic plants. n = 15 plants; error bars = SEM; different letters at the top of each column indicate a significant difference at P < 0.05 determined by Tukey’s HSD test.

  • Fig. 3 Structure of TPR2 TPD in complex with D53 EAR-2 peptide.

    (A) Structure overview showing two TPD tetramers with an EAR-2 peptide at the TPD tetramer-tetramer interface (dashed rectangle). EAR motif peptides are shown as green stick models, the TPDs are shown as cartoon models, and the TPD Zn2+ ions are shown as gray spheres. (B) Close-up of the interface; key amino acids and bonds are shown and labeled. D794 and Q804 of the EAR-2 motif (letters in parentheses) were not resolved in the structure. (C) Main interacting residues between TPR2 TPD and the D53 EAR-2 motif peptide. Brown, TPD monomer 1; cyan, TPD monomer 2; pink, TPD monomer from interacting tetramer [same color code in (A)]; bold, key interaction residues. aa, amino acids; VdW, van der Waals bond (4.5 Å cutoff); H-bond, hydrogen bond (3.5 Å cutoff).

  • Fig. 4 Only mutations that affect binding of both TPD–EAR-2 interfaces disrupt the TPD–EAR-2 interaction.

    (A) TPD L111A/L130A/L179A/I195A is unable to bind the D53 EAR-2 motif. AlphaScreen interaction assay of His6Sumo-TPR2 TPD wild-type and mutant proteins with biotinylated D53 EAR-2 peptide. n = 3; error bars = SD. See fig. S4B for SDS gel with protein loading controls. (B and C) Summary of AlphaScreen homologous competition assays of D53 EAR-2 mutant peptides (see fig. S10). Concentration of untagged wild-type and D53 EAR-2 mutant peptides required to reduce 50% of the AlphaScreen biotin-D53/His6Sumo-TPD binding signal (IC50). n = 3; error bars = SD.

  • Fig. 5 D53 EAR-2 peptide induces TPR2 TPD tetramer-tetramer interaction.

    (A) Cartoon presentation of AlphaScreen oligomerization assay (see Materials and Methods for details). Biotin-tagged TPR2 TPD and His6Sumo-tagged TPR2 TPD were incubated in the absence and presence of increasing concentrations of untagged D53 EAR-2 peptide. (B) Assay with wild-type and Y798A D53 EAR-2 peptide. (C) Assay with wild-type and L111A/L130A His6Sumo-TPD. n = 3; error bars = SD. (D) DLS of TPR2 TPD in solution in the absence and presence of NINJA and D53 wild-type and Y798A EAR-2 peptides.

  • Fig. 6 The tpl-1 (N176H) mutation can be partially rescued by mutations in interface-2.

    (A and B) Interface between two wild-type (N176) TPD tetramers (A) or with modeled N176H (tpl-1) mutation (B). The EAR-2 peptide is shown as orange line model, with EAR-2 L304 and its interacting residues shown in stick presentation. (C) Mutations in interface 1 residues Y68 and K71 rescue the aggregation phenotype of TPD N176H. Coomassie-stained protein gel [see (F) for size marker] of lysates of His6Sumo-TPD–expressing cells and lysate supernatant after centrifugation. (D) AlphaScreen interaction between biotin-D53 EAR-2 peptide and wild-type and mutant His6Sumo-TPD (n = 3; error bars = SD). (E) Overlay of size exclusion chromatography (SEC) profiles of TPR2 TPD, His6Sumo-TPD, and TPD/His6Sumo-TPD. mAU, milliabsorbance units. (F) TPD N176H destabilizes wild-type TPD. Different amounts of wild-type His6Sumo-TPD (WT) and untagged TPD N176H (mt) were mixed, incubated on ice for 4 hours, and centrifuged. Supernatants were analyzed by SDS–polyacrylamide gel electrophoresis (PAGE). Note that the TPD ratios are based on the amounts of pure WT and mt protein in supernatants following centrifugation (compare 1:0 and 0:1 ratios).

  • Fig. 7 TPR2 TPD binds reconstituted nucleosomes and histone tails.

    (A) AlphaScreen interaction between 100 nM biotin-tagged nucleosomes (nucl.) and 100 nM of the His6Sumo-tagged TPDs of TPL, TPR1, and TPR2. The interaction between the TPR2 TPD and the NINJA EAR motif serves as a positive control, and the interaction between the nucleosome and the unrelated nuclear receptor SHP serves as a negative control. Note that the AlphaScreen signal is dependent on proximity and therefore generates a weaker signal for biotinylated nucleosomes than for small biotinylated peptides. (B and C) AlphaScreen interaction between 100 nM His6Sumo-tagged TPR2 TPD and 100 nM biotin-tagged unmodified (B) or modified (C) histone tail peptides. n = 3; error bars = SD.

  • Fig. 8 D53 EAR-2 peptide stabilizes the binding between TPR2 TPD and recombinant nucleosomes.

    (A) AlphaScreen interaction between 100 nM biotin-tagged nucleosomes and 100 nM His6Sumo-tagged TPR2 TPD in the absence and presence of increasing concentrations of untagged D53 EAR-2 peptide. n = 3; error bars = SD. (B) Speculative model for chromatin compaction through D53 EAR-2–mediated TPD oligomerization.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/6/e1601217/DC1

    table S1. X-ray data collection and refinement statistics for TPR2-TPD + D53 EAR-2 peptide complex structures.

    table S2. D53 EAR-2 peptide increases the average hydrodynamic radius of the TPR2 TPD.

    fig. S1. Sequence alignment of the EAR motifs of D53 homologs from monocots and dicots.

    fig. S2. Sequence alignment of the TPD from monocots and dicots.

    fig. S3. Interaction of D53 EAR-2 peptide with the TPDs of TPL, TPR1, and TPR2.

    fig. S4. Expression and protein loading controls.

    fig. S5. M2H interaction between EAR-3–containing D53 fragments and TPL proteins.

    fig. S6. Fo-Fc omit map of the bound EAR peptide contoured at 2σ in the binding pocket of the TPR2 TPD.

    fig. S7. Mutations in TPR2 TPD site 1 do not abolish binding of the D53 EAR motif.

    fig. S8. D53 EAR-2 peptide binds site 1 in TPR2 TPD site 2 mutation L179A/L195A.

    fig. S9. TPR2 TPD L111A/L130A/L179A/I195A fails to bind the D53 EAR-2 motif.

    fig. S10. Relative binding strength of wild-type and mutant D53 EAR-2 motif peptides.

    fig. S11. The D53 EAR-2 peptide does not change the stability of the TPR2 TPD.

    fig. S12. The EAR peptides of SMXL6, SMXL7, and SMXL8 do not induce TPR2 TPD tetramer-tetramer interaction.

    fig. S13. Analysis of GFP-D53 and GFP-D53/TPD complexes by fluorescence-detection SEC.

    fig. S14. D53 homology model.

  • Supplementary Materials

    This PDF file includes:

    • table S1. X-ray data collection and refinement statistics for TPR2-TPD + D53 EAR-2 peptide complex structures.
    • table S2. D53 EAR-2 peptide increases the average hydrodynamic radius of the TPR2 TPD.
    • fig. S1. Sequence alignment of the EAR motifs of D53 homologs from monocots and dicots.
    • fig. S2. Sequence alignment of the TPD from monocots and dicots.
    • fig. S3. Interaction of D53 EAR-2 peptide with the TPDs of TPL, TPR1, and TPR2.
    • fig. S4. Expression and protein loading controls.
    • fig. S5. M2H interaction between EAR-3–containing D53 fragments and TPL proteins.
    • fig. S6. Fo-Fc omit map of the bound EAR peptide contoured at 2σ in the binding pocket of the TPR2 TPD.
    • fig. S7. Mutations in TPR2 TPD site 1 do not abolish binding of the D53 EAR motif.
    • fig. S8. D53 EAR-2 peptide binds site 1 in TPR2 TPD site 2 mutation L179A/L195A.
    • fig. S9. TPR2 TPD L111A/L130A/L179A/I195A fails to bind the D53 EAR-2 motif.
    • fig. S10. Relative binding strength of wild-type and mutant D53 EAR-2 motif peptides.
    • fig. S11. The D53 EAR-2 peptide does not change the stability of the TPR2 TPD.
    • fig. S12. The EAR peptides of SMXL6, SMXL7, and SMXL8 do not induce TPR2 TPD tetramer-tetramer interaction.
    • fig. S13. Analysis of GFP-D53 and GFP-D53/TPD complexes by fluorescencedetection SEC.
    • fig. S14. D53 homology model.

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