A systematic interactome of Saccharomyces cerevisiae SET1C expands its functional landscape and identifies candidate regulatory connections
- Marseille Cancer Research Center (CRCM), U1068 Inserm, UMR7258 CNRS, Aix Marseille University, Institut Paoli-Calmettes, France
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Republic of Korea
- Ochang Institute of Biological and Environmental Science, Korea Basic Science Institute, Republic of Korea
- Wellcome Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, United Kingdom
- Institut de recherches cliniques de Montréal, Center for Genetic and Neurological Diseases, Canada
- Département de biochimie et médecine moléculaire, Faculté de Médicine, Université de Montréal, Canada
- Division of Experimental Medicine, Faculty of Medicine, McGill University, Canada
- School of Life and Environmental Sciences, Deakin University, Australia
Figures
Figure 1 with 1 supplement
Schematic representation of the Set1 and subunit major interactors identified in the systematic yeast two-hybrid (Y2H) screens.
(A) Schematic representation of Set1 FL and Set1 fragments 1–754 and 754–1081. (B) Set1 FL, 1–754 and 754–1081 major Y2H interactors. (C) Interacting regions of Set1 with Swd2, Shg1, and Spp1. (D) Swd2, Spp1, Shg1 major Y2H interactors. (E) Swd1, Swd3, Bre2, Sdc1 major Y2H interactors. The term ‘interactor’ is used to mean a high-confidence two-hybrid interaction, with the limitations that this entails. The color reflects the predicted biological score (see Materials and methods). Red, highest confidence; blue, high confidence; green, good confidence.
Figure 1—figure supplement 1
Schematic representation of SET1C/COMPASS.
These yeast two-hybrid (Y2H) screens revealed the interaction of Mer2 with Spp1 (Acquaviva et al., 2013a) and of Rbp1-CTD with the N-terminal region of Set1 (Bae et al., 2020).
Figure 2
Set1 1–754 interacts with RGG proteins and the importin Kap104.
(A) RGG proteins and import/export proteins interacting with Set1 1–754, Set1 754–1081, and Spp1, Shg1, and Sdc1. (B) Set1 interacting domain (SID) (blue) within Kap104. Heat-like repeats 9 and 10 are represented in purple. (C) PY-NLS in the N-terminal region of Set1. (D) AlphaFold modeling of a seven subunit Set1C (Set1-Bre2-Sdc1(x2)-Swd1-Swd3-Spp1) and Kap104. A representative model is shown using the following color code: Kap104 SID 359–621 (white, the remaining Kap104 residues are hidden). Set1 N-term 1–754 (yellow), Set1 C-term 755–1079 (red); Set1 PY-NLS 41–90 (blue); Swd1 (green); Swd3 (cyan); Bre2 (raspberry); Sdc1 (subunit 1, gray); Sdc1 (subunit 2, chocolate); Spp1 (violet).
Figure 3
The SET1C yeast two-hybrid (Y2H) interactome identifies proteins involved in RNA biogenesis.
All Y2H interactors are described in Supplementary file 2. The processes linked to RNA metabolism in which the different interactors are involved are shown in the figure. The green lines linking Prp22 to Set1FL/Set1 754–1080 and Prp8 to Set1 FL and Spp1 indicate interactions with a high degree of confidence.
Figure 4
Set1 is co-precipitated with Prp22 in vivo.
(A) Co-immunoprecipitation experiments were performed in W303 expressing chromosomally encoded Myc-Set1 (Dehé et al., 2006a) and Prp22AID-FLAG (Mendoza-Ochoa et al., 2019). Prp22-AID-6FLAG was pulled down using a FLAG antibody. IgG was used as a negative control. Input, non-bound, and immunoprecipitation (IP) samples were loaded. The western blot was probed either with an antibody against the MYC epitope tag (left) or against the FLAG epitope tag (right). Myc-Set1 and Prp22AID-FLAG are indicated by * and °, respectively. (B, C) The same experiment was carried out in the absence or presence of RNase. A strain with 9MYC-Set1 and no flag-tagged Prp22 was used as a negative control. The western blots were probed with anti-MYC and anti-FLAG, respectively.
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Figure 4—source data 1
Source data for the co-immunoprecipitation experiments shown in Figure 4.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig4-data1-v1.zip
Figure 5 with 1 supplement
Set1 is SUMOylated.
6His-SUMO-conjugated proteins were purified from cells transformed (+) or not transformed (−) with a plasmid encoding 6His-SUMO under control of the CUP1 promoter. Cell lysates (input) and Ni-purified material (elutes) were analyzed by western blotting with an anti-MYC antibody (A) or an anti-GAL4 antibody (B–D). Analysis of 6His-SUMO-conjugated forms of (A) genomically MYC-tagged Set1 or (B) GB-Set1 transformed cells was performed (left panels); in both cases, SUMO expression and efficiency of purification were controlled using an anti-SUMO antibody (right panels). (C) SUMOylation analysis of Set1 fragment F3+F4 (aa. 351–956) wild-type (WT) and the K769R mutant. (D) SUMOylation analysis of Set1 fragment F5 (aa. 956–1080) WT, single mutants K1055R and K1060R, and the double mutant K1055R/K1060R mutant. Unmodified MYC-Set1 and GAL4-Set1 in both the (–) and (+) His-SUMO eluates are most likely due to the stickiness of unmodified Set1 to the beads.
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Figure 5—source data 1
Source data for the western blots shown in Figure 5.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig5-data1-v1.zip
Figure 5—figure supplement 1
Set1 SUMOylation analysis.
(A) Schematic representation of the F1-F5 Set1 fragments. (B) List of predicted SUMOylated sites by GPS-SUMO 1.0 (Zhao et al., 2014). The predicted SUMOylated lysine is bold underlined.
Figure 6 with 1 supplement
SET1C interacts in vitro with Snf2C-AT-hook.
(A) A schematic diagram depicting the domains of Snf2 and the Snf2 fragments used in this study, along with the SDS-PAGE/Coomassie staining of the purified GST-tagged Snf2 fragments. (B, C) GST pull-down assay using purified GST-tagged Snf2 fragments. The purified SET1C (B) or SET1C-C762 complex (C) was mixed with GST-tagged Snf2 fragments, followed by GST pull-down, and the bound proteins were analyzed by immunoblotting. (D) A schematic diagram illustrating Snf2 fragments with a more detailed breakdown of the AT-hook domain, along with the SDS-PAGE/Coomassie Blue staining of the purified Snf2 fragments. The lysines present in the AT-hook are represented by the letter K. (E) GST pull-down assay using purified GST-tagged Snf2 fragments and SET1C.
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Figure 6—source data 1
Source data for the Coomassie blue–stained gels, western blot, and autoradiographs presented in Figure 6.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig6-data1-v1.zip
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Figure 6—source data 2
Uncropped and labelled gel image corresponding to Figure 6.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig6-data2-v1.pptx
Figure 6—figure supplement 1
Purification and activity of SET1C and SET1C-C762.
(A) A schematic diagram of the subunits used for the reconstitution of SET1C and Set1-C762 complexes. (B) SDS-PAGE/Coomassie Blue staining of purified SET1C. (C) In vitro methyltransferase assay using SET1C and free histone H3. (D) SDS-PAGE/Coomassie Blue staining of purified Set1-C762 complex. (E) In vitro methyltransferase assay using the Set1-C762 complex and free histone H3.
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Figure 6—figure supplement 1—source data 1
Source data for the Coomassie blue–stained gels and autoradiographs presented in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Uncropped and labelled gel image corresponding to Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig6-figsupp1-data2-v1.pptx
Figure 7 with 2 supplements
Snf2 is methylated in tandem AT-hook domain by reconstituted Set1C.
(A) Schematic representation of Snf2 fragments. (B) In vitro methyltransferase assay using SET1C incubated with the indicated Snf2 fragments. 3H-SAM was used as a methyl donor and methylated proteins were detected by autoradiography. The band marked with a red star is a degradation product of Snf2C. (C) Schematic diagram of N-terminal truncated SET1 complexes (left) and in vitro methyltransferase assay with GST-Snf2-AT-hook and truncated SET1 complexes. (D) Schematic diagram showing the positions of all lysines in Snf2-AT-hook and the further cleaved fragments of Snf2-AT-hook. The red box indicates the two lysines that are acetylated by Gcn5. (E) Coomassie staining of purified Snf2 fragments (lower) and an in vitro methyltransferase assay using these fragments with SET1C (upper). (F) In vitro methyltransferase assay by SET1C when each of the four lysines in the C-terminal region of the Snf2-AT-hook is substituted with arginine or when both lysines known to be acetylated by Gcn5 are substituted. (G) A schematic diagram showing the position of the RG-repeat region and the design of Snf2-AT-hook with RG-repeat truncation. (H, I) GST pull-down assay (H) and in vitro methyltransferase assay (I) using purified SET1C and GST-Snf2-AT-hook with or without RG-repeats.
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Figure 7—source data 1
Source data for the Coomassie blue–stained gels, autoradiographs and western blot presented in Figure 7.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig7-data1-v1.zip
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Figure 7—source data 2
Uncropped and labelled gel image corresponding to Figure 7.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig7-data2-v1.pptx
Figure 7—figure supplement 1
15 lysines of Snf2-AT-hook were replaced with arginine one by one.
Purified Snf2-AT-hook wild-type and 15 mutant proteins were subjected to Coomassie staining (upper) and in vitro methyltransferase assay with SET1C (lower).
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Figure 7—figure supplement 1—source data 1
Source data for the Coomassie blue–stained gels and autoradiographs presented in Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig7-figsupp1-data1-v1.zip
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Figure 7—figure supplement 1—source data 2
Uncropped and labelled gel image corresponding to Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig7-figsupp1-data2-v1.pptx
Figure 7—figure supplement 2
Lysine mutations in the AT-Hook Motifs (K1488, K1494, K1498, and K1526) do not suppress Methylation of the Snf2-B3 Fragment.
(A) Designed AT-hook mutants with quadra mutation of all four lysines (K1488, K1494, K1498, K1526) or triple mutations among these four lysines. K1494 and K1498 are known to be acetylated by Gcn5. (B) In vitro methyltransferase assay using reconstituted SET1C and Snf2-AT-hook mutants.
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Figure 7—figure supplement 2—source data 1
Source data for the Coomassie blue–stained gels and autoradiographs presented in Figure 7—figure supplement 2.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig7-figsupp2-data1-v1.zip
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Figure 7—figure supplement 2—source data 2
Uncropped and labelled gel image corresponding to Figure 7—figure supplement 2.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig7-figsupp2-data2-v1.pptx
Figure 8
The arginines in the RG-repeat of Snf2 are methylated by reconstituted SET1C.
(A) A diagram showing the wild-type (WT) Snf2-B3 fragment and the Mut Snf2-B3 with all four lysines substituted with alanine. (B) Coomassie staining of purified WT and Mut Snf2-B3. (C) Mass spectrometry experiment design to identify the methylation sites of Snf2-B3. (D) Coomassie staining of Mut Snf2-B3 after methylation reaction and an additional purification step using Ni-NTA. The band corresponding to Mut Snf2-B3 (~18 KDa) was excised and used for mass spectrometry analysis. The ~37 KDa band observed in lanes 5–8 appears to be a SET1C subunit that binds nonspecifically to Ni-NTA, likely SWD2 based on its size. (E) Mass spectrometry analysis result of Snf2-B3 methylation sites revealed that multiple arginines in the RG-repeat were methylated. Amino acid sequence of Snf2 highlighting the arginines methylated by SET1. The sequence of the B3 fragment is shown in bold, and the arginines methylated by SET1 are marked in red. Methylation and demethylation are denoted as M and DM, respectively.
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Figure 8—source data 1
Source data for the Coomassie blue–stained gels presented in Figure 8.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig8-data1-v1.zip
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Figure 8—source data 2
Uncropped and labelled gel image corresponding to Figure 8.
- https://cdn.elifesciences.org/articles/109886/elife-109886-fig8-data2-v1.pptx
Figure 9 with 2 supplements
The arginines of the motif ARTSTRGR within the AT-hook are methylated in vivo in a Set1-dependent way.
(A) Set1 interacts in vivo with Snf2 and Snf2-∆RG. Myc-tagged Snf2 and Snf2∆RG were immunoprecipitated with 9E10 Myc antibodies (see Materials and methods) and revealed with either 9E10 (upper panel) or Set1 antibodies (lower panel). (B) Snf2C complex was purified from WT and set1∆ strains, separated on a 4–12% Bis-Tris gel and silver stained (left); the presence of Snf2-GFP is detected by western blotting with anti-GFP antibody (right). The area corresponding to Snf2-GFP was excised from the gel and used for mass spectrometry analysis. Peptides flanking the RG repeats (1485–1549) with their post-translational modifications (PTMs) are shown in Figure 9—figure supplement 2. (C) Panel C shows a focus on the amino sequence flanking the RG repeats. The positions of residues from D1485 to V1549 are indicated on the figure. Peptides identified after digestion of Snf2-GFP are indicated in color with their identified PTM indicated by the color code shown in the top of the panel. The small numbers above the amino acids indicate the probability of localization according to the MS2 peaks. It should be noted that for wild-type K1488 and R1490, a peptide with dimethylation is detected, but no discriminating MS2 peak allows us to conclude whether K or R are dimethylated. This is also the case for dimethylation on R1528 and R1535. The results presented represent the observed PTMs from two independent experiments, each containing three replicates (Supplementary file 6).
Figure 9—figure supplement 1
Volcano plot describing the enrichment of proteins in the purifications of Snf2-GFP compared to the control (no tag) (see Supplementary file 5) created by Perseus.
(A) In a wild-type strain, (B) in the set1∆ strain. The curve represents the significant limit of quantification (S0=5 and false discovery rate [FDR] = 0.01).
Figure 9—figure supplement 2
Peptides identified after Snf2-GFP digestion for wild-type (WT) and set1Δ are indicated with their identified post-translational modification (PTM).
The red numbers indicate the probability of localization according to the MS2 peaks for the preceding amino acid.
Appendix 1—figure 1
Chromatin regulators identified in all the two-hybrid screens.
The lines indicate the individual proteins involved in the yeast two-hybrid (Y2H) interaction. Red line refers to a very high-confidence Y2H interaction. All Y2H interactors are described in Supplementary file 2. Interactors are grouped according to the complex to which they belong.
Appendix 1—figure 2
SID (blue) and RGG motif (green) within the RGG proteins.
The interaction domains indicated represent the minimal overlapping DNA sequence present in multiple independent yeast two-hybrid (Y2H) interacting clones of the same gene. Each genomic fragment of a Y2H clone was analyzed, and the shared overlapping region for a given gene was determined to be the only common element among all interacting clones. As such, this region represents the minimal sequence required for interaction.
Appendix 1—figure 3
SET1C yeast two-hybrid (Y2H) interactors involved in DNA transactions.
The processes in which Y2H interactors are involved are shown in the figure. Red and blue lines refer to the confidence of the Y2H interaction.
Appendix 1—figure 4
Spp1 interacts weakly with Mcm2 in vitro.
(A) Schematic representation of Mcm2. The minimal region of interaction with Spp1 is located at the extreme C-terminus of Mcm2. (B) Pull-down assays were performed with GST, GST-Spp1, and GST-Mcm2 fusion proteins. 35S-labeled in vitro-translated proteins served as preys; input shows 10% of radioactive material included in binding reactions.
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Appendix 1—figure 4—source data 1
Source data for the autoradiography shown in Appendix 1—figure 4.
- https://cdn.elifesciences.org/articles/109886/elife-109886-app1-fig4-data1-v1.zip
Appendix 1—figure 5
Spp1 and Sdc1 interact with Nis1 in vitro.
Pull-down assays were performed with GST, GST-Spp1, GST-Sdc1, and GST-Shg1 fusion proteins as indicated at the bottom of the panels. 35S-labeled in vitro-translated proteins served as preys; input shows 10% of radioactive material included in binding reactions. The asterisk marks a band that is an undefined Set1 in vitro translation product, and it is marked so that it is not confused with full-length Bre2. The arrows point to the expected full-length in vitro translation products. Nis1 (46 KDa), Bre2 (58 KDa), Set1 (124 KDa).
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Appendix 1—figure 5—source data 1
Source data for the autoradiograph corresponding to Appendix 1—figure 5.
- https://cdn.elifesciences.org/articles/109886/elife-109886-app1-fig5-data1-v1.zip
Appendix 1—figure 6
Identification of SET1C yeast two-hybrid (Y2H) interactors showing changes in localization following hypoxia.
SET1C interactors that change localization are shown. In hypoxia, 243 proteins change their localization (Henke et al., 2011). Several components of the SWI/SNF complex were found to rapidly change location (from nucleus to cytosol) in hypoxia (Dastidar et al., 2012).
Appendix 1—figure 7
SET1C yeast two-hybrid (Y2H) interactors are involved in stress responses and metabolism pathways.
The processes in which Y2H interactors are involved are shown in the figure. Red arrows indicate high-confidence Y2H interactions.
Tables
Appendix 1—table 1
H3K4- like proteins.
Shown are selected proteins containing sequences similar to the modification site found in histone H3. An exhaustive list can be found in Supplementary file 3.
| protein | size (kDa) | motif | function |
|---|---|---|---|
| Histone H3 | 35 | ARTKQT | core histone protein required for chromatin assembly |
| Nrm1 | 57 | KTKQT | transcriptional co-repressor of MBF-regulated gene expression |
| Nrd1 | 84 | RSKQ | transcription termination and 3' end maturation of nonpolyadenylated RNAs |
| Rix1 | 107 | KTKQS | processing of ITS2 sequences from 35 S pre-rRNA |
| Dbf2 | 86 | RTKQ | transcription and stress response, part of a network of genes in exit from mitosis |
| Dbf20 | 86 | RTKQ | late nuclear division, one of the mitotic exit network (MEN) proteins |
| Not5 | 86 | KTKQ | transcription initiation, elongation and in mRNA degradation |
| Mcm2 | 120 | ARTK | DNA replication |
| Prp8 | 300 | RTKQ | second catalytic step of splicing; mutations of human Prp8 cause retinitis pigmentosa |
Additional files
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Supplementary file 1
Plasmids and yeast strains used in this study.
- https://cdn.elifesciences.org/articles/109886/elife-109886-supp1-v1.xlsx
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Supplementary file 2
All SET1C interactors identified in the 10 yeast two-hybrid (Y2H) screens.
The interactors common to several subunits and to common Set1 fragments are shown on sheet 13 of the table.
- https://cdn.elifesciences.org/articles/109886/elife-109886-supp2-v1.xlsx
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Supplementary file 3
Mass spectrometry analysis of TAP-Nis1 affinity purification.
- https://cdn.elifesciences.org/articles/109886/elife-109886-supp3-v1.xlsx
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Supplementary file 4
H3K4-like domain proteins.
We used the scansite search algorithm (http://scansite.mit.edu) and systematically identified sequence motifs that are related to the Set1C modification site in histone H3.
- https://cdn.elifesciences.org/articles/109886/elife-109886-supp4-v1.xlsx
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Supplementary file 5
Protein composition of the Snf2-GFP complex in WTand set1∆ strain.
- https://cdn.elifesciences.org/articles/109886/elife-109886-supp5-v1.xlsx
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Supplementary file 6
Snf2 post-translational modifications in either wild-type (WT) or set1∆ strains.
- https://cdn.elifesciences.org/articles/109886/elife-109886-supp6-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/109886/elife-109886-mdarchecklist1-v1.docx
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Appendix 1—figure 4—source data 1
Source data for the autoradiography shown in Appendix 1—figure 4.
- https://cdn.elifesciences.org/articles/109886/elife-109886-app1-fig4-data1-v1.zip
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Appendix 1—figure 5—source data 1
Source data for the autoradiograph corresponding to Appendix 1—figure 5.
- https://cdn.elifesciences.org/articles/109886/elife-109886-app1-fig5-data1-v1.zip
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A systematic interactome of Saccharomyces cerevisiae SET1C expands its functional landscape and identifies candidate regulatory connections
eLife 15:RP109886.
https://doi.org/10.7554/eLife.109886.3
