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. Author manuscript; available in PMC: 2025 May 5.
Published in final edited form as: J Proteome Res. 2024 Oct 22;23(11):5016–5029. doi: 10.1021/acs.jproteome.4c00498

Distinct regions within SAP25 recruit O-linked glycosylation, DNA demethylation, and ubiquitin ligase and hydrolase activities to the Sin3/HDAC complex

Pratik Goswami a,§, Charles AS Banks b,§, Janet Thornton b,#, Bethany D Bengs c, Mihaela E Sardiu c, Laurence Florens b, Michael P Washburn b,#,*
PMCID: PMC12051219  NIHMSID: NIHMS2074420  PMID: 39435885

Abstract

Sin3 is an evolutionarily conserved repressor protein complex mainly associated with histone deacetylase (HDAC) activity. Many proteins are part of Sin3/HDAC complexes, and the function of most of these members remains poorly understood. SAP25, a previously identified Sin3A associated protein of 25 kDa, has been proposed to participate in regulating gene expression programs involved in the immune response but the exact mechanism of this regulation is unclear. SAP25 is not expressed in HEK293 cells, which hence serve as a natural knockout system to decipher the molecular functions uniquely carried out by this Sin3/HDAC subunit. Using molecular, proteomic, protein engineering, and interaction network approaches, we show that SAP25 interacts with distinct enzymatic and regulatory protein complexes in addition to Sin3/HDAC. Additional proteins uniquely recovered from the Halo-SAP25 pull-downs included the SCF E3 ubiquitin ligase complex SKP1/FBXO3/CUL1 and the ubiquitin carboxyl-terminal hydrolase 11 (USP11). Furthermore, mutational analysis demonstrates that distinct regions of SAP25 participate in its interaction with USP11, OGT/TETs, and SCF(FBXO3). These results suggest that SAP25 may function as an adaptor protein to coordinate the assembly of different enzymatic complexes to control Sin3/HDAC-mediated gene expression. The data were deposited with the MASSIVE repository with the identifiers MSV000093576 and MSV000093553.

Keywords: SAP25, Sin3/HDAC, O-GlcNAc transferase, E3 ubiquitin ligase, SCF(FBXO3), quantitative proteomics, topological scoring, protein interaction network

Graphical Abstract

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Introduction

Mapping networks of protein interactions helps us to understand the structure of protein complexes, how they might regulate biological processes, and the impact of the aberrant function of protein complexes in different diseases 1. Affinity Purification Mass Spectrometry (AP-MS) is one of the main approaches to map protein interaction and involves using an exogenously expressed affinity-tagged bait to capture endogenous prey proteins. Proteins copurifying with the bait are identified and quantified using mass spectrometry 2, 3. In addition, AP-MS data provides significant information regarding protein-protein interactions under physiological conditions 4. Hub proteins are important in human protein interaction networks where they interact with many additional proteins. Human proteins like RUVBL1 and RUVBL2 (RuvB Like AAA ATPase 1/2) 5, Histone Deacetylases 1 and 2 (HDAC1/2) 6 and SIN3A and SIN3B (switch-independent 3 (SIN3) transcriptional regulator family member A/B) 7 are among many with this characteristic. Each of these proteins is reported to interact with multiple individual proteins in addition to being in multiple protein complexes 5-7. Next, determining how individual protein complexes are assembled within a cell is an important area of research.

Human SIN3A acts as a scaffolding protein, forming a framework upon which Sin3 complexes are assembled. Sin3 co-repressors bind with HDACs, enabling Sin3-mediated transcriptional repression 8. The Sin3/HDAC family of complexes is associated with differentiation, apoptosis, proliferation, tumorigenesis and has a role in the development of cancers 9. The integrity of the Sin/HDAC complex is crucial for central nervous system development 8, methionine catabolism, and glycolysis/gluconeogenesis 10. It also plays a significant role in breast cancer metastasis 11, activating autophagy 12, contributing to pulmonary arterial hypertension 13, and influencing triple-negative breast cancer 14. In addition to the SIN3A/B paralogues, several proteins bind with the Sin/HDAC complexes including RBBP4/RBBP7 and HDAC1/2, which are shared with other transcriptional repressor complexes, and the Sin3-associated proteins SAP30/SAP30L (Sin3A associated protein 30/L), SUDS3 (Sin3 histone deacetylase corepressor complex component SDS3), BRMS1/BRMS1L (Breast Cancer Metastasis-Suppressor 1/1L) , ING1/2 (Inhibitor Of Growth Protein 1/2), ARID4A/B (AT-rich interacting domain 4A/B), SAP18 (Sin3A Associated Protein 18), FAM60A (Family with sequence similarity 60A), and SAP25 (Sin3A Associated Protein 25) 15, 16, 17, 18. While Sin3/HDAC complexes mainly regulate gene transcription 19 by deacetylating histone tails and altering the chromatin environment, several components of the Sin3/HDAC complex interact with proteins carrying various enzymatic activities 18, 20, 21. Specifically, SIN3A has been linked to OGT, an O-linked N-acetylglucosamine transferase 19 and the TET (ten eleven translocation enzymes) DNA demethylases 20, 21.

First reported in 2006, the SIN3A-associated protein of 25 kDa SAP25 was described associated with the mouse mSIN3/HDAC complex and shown to participate in transcriptional repression 18. More recently, we showed that human SAP25 captured complexes contain the Sin3 subunits SUDS3 and SAP30 3. SAP25 has also been associated with various additional components of epigenetic-modifying complexes, such as OGT and TET218, 21, 22, with an L254F-OGT variant upregulating SAP25 in X-linked intellectual disability 23. Furthermore, Tet2 purified complexes were found to contain both OGT and Sin3 complex components SAP25, SIN3A, and SAP30 21. In addition, John and coworkers recently proposed that IFIT1 regulates gene expression programs of the immune response by coordinating the removal of Sin3 repressor complexes containing SAP25from the IFNB1, IRF7, and IRF8 promoters 24. Finally, a genome-wide DNA methylation study listed the SAP25 gene in the top 10 positions with differentially methylated CpG sites in patients with active inflammatory bowel diseases 25,while altered expression of SAP25 was observed in microglia 26, contributing to Alzheimer's disease 27.

Here we focus on the SAP25 protein to elucidate its protein interaction network and role within the Sin3/HDAC complex. First, we conducted affinity purifications of proteins from cells stably expressing Halo-Tag SAP25 and analyzed purified proteins using mass spectrometry. Our analysis of proteins associated with SAP25 revealed its involvement not only with the Sin3/HDAC complex, but also with additional proteins with enzymatic function. Next, we generated a series of mutant proteins to determine whether these additional complexes could associate with SAP25 independently of Sin3/HDAC. Overall, our results highlight the involvement of SAP25 as an adaptor protein capable of independently recruiting enzymatic activities to the Sin3/HDAC, potentially deploying a range of activities involved in chromatin modification and remodeling, beyond histone deacetylation.

Material & Methods

Materials

Magne® HaloTag® beads (G7281), HaloTag® polyclonal antibody (G9281) and TMRDirect™ fluorescent ligand (G2991) were purchased from Promega. AcTEV protease (#12575015) was from Thermo Fisher Scientific. Rabbit anti-SAP30 (ab125187) and rabbit anti-SIN3A (ab3479) polyclonal antibodies were from Abcam (Cambridge, United Kingdom). SNAP-Capture magnetic beads (S9145S) were from NEB (Ipswich, MA). Salt Active Nuclease (SAN) was from ArcticZymes (Tromso, Norway). Rabbit anti-FBXO3 (HPA002467) was from Atlas Antibodies. Rabbit anti-HDAC1 (10197-1-AP) was from Proteintech. Rabbit anti-SAP25 (HPA062610-100UL), and mouse anti-FLAG (F3165-.2MG) were from Sigma. IRDye® 800CW labeled goat anti-Rabbit (926-3211) and IRDye® 680LT labeled goat anti-Mouse (926-68020) secondary antibodies were from LI-COR Biosciences. FLAG-OGT in pcDNA5FRT 28 was a gift from the laboratory of Joan and Ron Conaway.

Cloning sequences to express Halo affinity tagged versions of SAP25 and Sin3 complex subunits

A codon optimized synthetic sequence (SAP25 in pIDTSmart (Amp)) coding for a 199 aa sequence matching SAP25 (originally annotated as NP_001162153.1) was used to amplify various sequences coding for different versions of SAP25 using the primers listed in Supporting Information (cloning of full-length SAP25 in pFN21A was described previously 3). This 199 AA sequence also matches the more recently annotated version NP_001335606.1 (SAP25 isoform 3). For transient expression, the resulting PCR products were subcloned into either pFN21A (Promega) or Halo pcDNA5/FRT PacI PmeI (described previously 29). The construction of all stable cell lines except for Halo-SIN3A and Halo-SAP25 has been described previously 3, 7. Halo-SIN3A was cloned from ORF Clone # FHC11647 (Promega) by first mutating the codon for amino acid 109 from GCT (A) to GTT (V) to match the translated sequence to NP_056292. The resulting sequence was then cloned into CMVd2 pcDNA5 PacI PmeI (described previously 30) using the primers listed in Supporting Information. Stable cell line construction is described in Banks et al. (2018) 3. Halo-SAP25 was also cloned into CMVd2 pcDNA5 PacI PmeI for construction of a stable cell line. For expression of FLAG-OGT, the mutations in the OGT1 insert (N764D and K1010E) in the original plasmid were corrected using site directed mutagenesis. For expression of FBXO3, an FBXO3(NM_012175) ORF Clone was purchased from GenScript (Cat. #OHU25332D), and the open reading frame amplified using the primers listed in Supporting Information. The resulting PCR product was subcloned into SNAP-FLAG-pcDNA5 3.

Preparation of whole cell extracts

For transient expression of mutant versions of SAP25, whole cell lysates were prepared from approximately 1 x 107 293T cells transiently transfected with the constructs indicated in the figure legends as described previously 29. Cells were harvested 48 hours after transfection and washed twice with ice-cold PBS. Cell pellets were frozen, thawed, and resuspended in approximately 300 μl lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton® X-100, 0.1% sodium deoxycholate, 0.1 mM benzamidine HCl, 55 μM phenanthroline, 10 μM bestatin, 20 μM leupeptin, 5 μM pepstatin A and 1 mM PMSF. The resulting lysate was passed through a 26-gauge needle 10 times and centrifuged at 21, 000 x g for 30 minutes at 4°C. The supernatant was diluted with 700 μl of TBS (25 mM Tris-HCl pH 7.4, 137 mM NaCl, 2.7 mM KCl). Extracts were prepared from Flp-In™-293 stable cell lines as described previously 29.

Purification of recombinant protein complexes

Lysates were incubated overnight at 4°C with Magne® HaloTag® beads prepared from 0.1 ml bead slurry (transiently transfected cells) or 0.2 ml of bead slurry (stable cell lines). The beads were isolated using a DynaMag-2 Magnet and the supernatant discarded. Beads were washed 4 times in buffer containing TBS (25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 2.7 mM KCl) and 0.05% Nonidet® P40. To elute bound proteins, beads were incubated in 100 μl elution buffer (25 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 1 mM DTT, and 2 Units AcTEV) for 2h at 25°C. To purify SNAP-tagged proteins, cell lysates were allowed to incubate overnight at 4 °C with SNAP-Capture magnetic beads followed by washing with wash buffer as described above. Elution of bound proteins was accomplished by incubating the beads with a buffer containing 50 mm Tris·HCl pH 8.0, 0.5 mm EDTA, 1 mm DTT, and 1 Unit of PreScission Protease at 4 °C overnight.

Digestion of proteins for mass spectrometry

HaloTag® purified protein complexes were TCA precipitated and resuspended in buffer containing 100 mM Tris-HCl, pH 8.5 and 8 M urea. Disulfide bonds were reduced with 5 mM TCEP (tris (2-carboxyethyl) phosphine) for 30 minutes at room temperature and bond reformation prevented by alkylation with 10 mM chloroacetamide for 30 minutes at room temperature in the dark. Proteins were initially digested with the addition of 0.1 μg of Lys-C followed by incubation in a ThermoMixer® (Eppendorf) for 6 hours at 37°C with shaking. Samples were diluted to 2 M urea by the addition of an appropriate volume of 100 mM Tris-HCl, pH 8.5. Proteins were further digested with the addition of 2 mM CaCl2 and 0.5 μg trypsin, and subsequent incubation in a ThermoMixer® at 37°C overnight. Reactions were quenched with the addition of formic acid (5% final concentration) prior to mass spectrometry analysis.

Liquid chromatography mass spectrometry analysis

Digested peptides were analyzed using an LTQ Linear Ion Trap mass spectrometer (Thermo Scientific) run in positive ion mode connected to an Agilent 1100 HPLC system. Samples were first loaded offline onto 3-phase microcapillary MudPIT columns as described previously 31 and eluted into the mass spectrometer using a series of 10 ~2-hour MudPIT steps. Chromatography gradients used a combination of Buffer A (5% acetonitrile, 0.1% formic acid), Buffer B (80% acetonitrile, 0.1% formic acid), and Buffer C (500 mM ammonium acetate, 5% acetonitrile, 0.1% formic acid).

Processing of tandem mass spectrometry data

Resulting .raw files were converted to .ms2 files using RAWDistiller v. 1.0. The ProLuCID algorithm version 1.3.5 was used for searching. Spectra from stable cell line experiments were matched to a database containing 48080 human protein sequences downloaded from the National Center for Biotechnology Information (NCBI) (2022-04-12 release), together with 427 common contaminant sequences and shuffled versions of all sequences to estimate false discovery rates (FDRs). The total number of sequences searched was 97014. Spectra from transient transfection experiments were searched with the same database, but with the addition of 8 sequences for recombinant mutant versions of SAP25 (together with 8 further shuffled sequences). Data were searched for peptides with a static modification of 57.0215 on cysteine residues (carbamidomethylation) and a variable modification of 15.9949 on methionine residues (oxidation). A mass tolerance of 500 ppm was used for both precursor and fragment ions. Only fully tryptic peptides were considered.

The entire in-house software suite (kite), used for the processing and analysis of the mass spectrometry datasets, is available in Zenodo 32. We used the in-house software program swallow in combination with DTASelect v. 1.9 33 to filter peptide spectrum matches (PSMs) resulting in mean spectrum, peptide, and protein FDRs below 5% (Tables S1B-M and S2B-I). The mean spectral FDR for the 36 MudPIT runs (transient transfection experiments) was 0.714% ± 0.263% (standard deviation), and the mean protein FDR was 3.958% ± 1.838% (standard deviation). For stable cell line experiments, the mean spectral FDR for the 54 MudPIT runs was 0.468% ± 0.173% (standard deviation), and the mean protein FDR was 2.133% ± 1.139% (standard deviation). The minimum peptide length was 7 amino acids. Proteins identified in different runs were compared using sandmartin 32, Contrast 33, and NSAF7 software. The parsimony option in Contrast was used to remove proteins that were subsets of others.

Experimental design and statistical rationale

AP-MS analyses required at least three biological replicates (Tables S1-S2). The number of biological replicates for analyses involving a stable cell line was: 5 (Halo-control); 5 (Halo-SAP25). For analyses using transient cells the number of biological replicates employed was: 9 (Halo-control); 4 (Halo-SAP25); 4 (Halo-SAP25 Δ21-40); 4 (Halo-SAP25 Δ110-119); 3 (Halo-SAP25 P114-117A); 3 (Halo-Sap25 LL149-150AA); 3 (Halo-SAP25 16-30); 3 (Halo-SAP25 50-130); 3 (Halo-SAP25 131-168). We employed QPROT, for analyzing differential protein expression which is based on protein intensity data 34. A high-confidence interaction was confirmed when the interaction parameters satisfied the conditions of log2FC > 2 and Z-statistic > 3.

Fluorescence microscopy

HEK293T cells were seeded to 40% confluency in MatTek culture dishes and incubated at 37 °C in 5% CO2 for 24 hours. Cells were then subsequently transfected with plasmid expressing Halo-SAP25. HaloTag® TMRDirect™ was introduced to the culture medium at a concentration of 20 nm to label Halo-tagged proteins. The cells were then incubated overnight at 37 °C in 5% CO2. Next day, cells were stained with Hoechst dye for 1 hour to label nuclei followed by washing with Opti-MEM® reduced serum medium. The cells were then visualized using an LSM-700 Falcon confocal microscope.

Topological scoring

Topological scoring was performed for each protein as described previously 2, 35. Proteins significantly enriched with log2FC > 2 and Z-statistics > 3 were selected as input to the TopS Shiny application, available at GitHub - WashburnLab/Topological-score-TopS- . This application uses the mean spectral counts for each bait across all baits to derive TopS score, signifying a probability of binding. Proteins were filtered out based on TopS value greater than 10 (Table S3).

Results

SAP25 captures both the Sin3 complex and additional enzymatic and regulatory protein complexes.

To determine SAP25 interacting partners, we first constructed a cell line stably expressing a recombinant version of SAP25 (NP_001335606.1) fused with an N-terminal Halo-tag (Halo-SAP25) in Flp-In™-293 cells. We used the weaker CMVd2 promoter in our Sin3 complex cell lines to express the bait proteins at a level that more closely reflects endogenous protein expression levels, even though SAP25 is not endogenously expressed in HEK293 cells 3. Five biological replicates of the resulting cell extracts from Halo-SIN3A and Halo-SAP25 cell lines were subjected to Halo affinity purification and the isolated proteins identified using Multidimensional Protein Identification Technology (MudPIT) 3, 36. We next used QPROT statistical analysis 34 to compare enrichment of proteins detected in Halo-SAP25 samples with proteins detected in controls (Fig. 1A). This analysis defined a set of 143 Halo-SAP25 enriched proteins with log2FC > 2 and Z statistic > 3 (Fig. 1A and Table S1A, rows 5-148).

Figure 1: SAP25 captures a distinct subset of proteins that are not part of the Sin3 complex.

Figure 1:

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A. Significance plot showing proteins enriched by stably expressed Halo-SAP25 (Flp-In™-293 cells) compared with controls (log2FC > 2, Z statistic > 3). Significantly enriched proteins include subunits of the Sin3 complex, as well as an O-linked glycosylase (OGT), DNA demethylases (TET2/3), an E3 ubiquitin ligase complex (FBXO3, SKP1, CUL1), and ubiquitin hydrolase USP11 (Table S1A). B. Expression of SAP25 associated proteins in HEK293 cells in the absence of Halo-SAP25. FPKM for the indicated transcripts are taken from our previously published RNAseq dataset available from the NCBI GEO repository under accession number GSE79656. Values are the mean of three biological replicates and error bars represent standard deviation. C. Sin3 and other enzymatic and regulatory proteins observed in purifications from Halo-SAP25 or from 11 other stable cell lines expressing Halo-tagged Sin3 complex subunits. Values are log2FC (enrichment compared with Flp-In™-293 cell control purifications) (Table S1A). The comparative heatmap was generated using the Morpheus online tool https://software.broadinstitute.org/morpheus). Clustering was performed with Euclidean distance and average linkage.

As expected, a GO term enrichment analysis of these SAP25 associated proteins using ClueGO (pV < 0.01) identified a highly enriched (pV < 0.0005) cluster of GO terms related to “Sin3-type complex” (Figure S1, pink nodes). Among the proteins identified in this cluster were the 9 Sin3 subunits we had previously identified as core Sin3 complex subunits (SIN3A, SIN3B, SUDS3, SAP30, SAP30L, HDAC1, HDAC2, RBBP4 and RBBP7) 3. In total, in addition to SAP25, we identified all 17 Sin3 complex subunits that we had previously identified copurifying with Halo-SAP30 3 in our SAP25 purifications with relatively high abundance (dNSAF values > 0.0003) and reproducibly across biological replicates (Table S1B). This association of human SAP25 with the Sin3 complex was consistent both with a previous study in which exogenously expressed mouse SAP25 was recognized as an integral component of the mSin3 complex 3, 18, and with our previous observation by Western blotting that exogenously expressed human SAP25 captured the Sin3 subunits SUDS3 and SAP30 3.

In addition to the Sin3 complex, we also identified the components of several protein complexes that had not previously been found to copurify with SAP25. Importantly, we identified three types of proteins with enzymatic functions: 1) components of the E3 ubiquitin ligase complex SCF(FBXO3), 2) the O-Linked N-Acetylglucosamine (GlcNAc) transferase OGT together with its established binding partners TET2 and TET3, and 3) the deubiquitinase USP11 (Fig. 1A). These proteins have previously been shown to regulate gene transcription 19, 22, 37, 38. The observation of OGT with SAP25 is also consistent with several investigations that suggest that Sin3 complex, SAP25, OGT, and TET family proteins (TET1, TET2, and TET3) collaborate in regulating transcription 19, 21, 22, 39.

A subset of the SAP25 interactome does not copurify with the Sin3 complex in the absence of SAP25

Next, we wanted to understand whether these complexes were copurifying with the Sin3 complex in the absence of SAP25. We had previously determined that SAP25 is not expressed in HEK293 cells 3, which hence serve as a natural “knockout” system to decipher interactions in the presence or absence of SAP25 (Fig. 1B). Therefore, we expanded our proteomic profiling and compared the Halo-SAP25 purification with purifications using other Sin3 subunits as baits. For this, we used eleven Sin3 complex-associated baits previously studied in our lab 7. These include: SIN3A, SUDS3, SAP30, SAP30L, ARID4A, ARID4B, BRMS1, BRMS1L, ING1, ING2, and SAP130 (Tables S1C-M). These proteins were stably expressed in Flp-In™-293 cells with an N-terminal Halo-tag. Proteins copurifying with the Sin3 subunit baits were again identified by MudPIT, and their enrichment compared with controls (log2FC) calculated using the QPROT algorithm (Table S1A).

Having determined enrichment values, we compared these values for Sin3 complex subunits, SCF(FBXO3), OGT/TET, and USP11 across the different Sin3 complex baits (Fig. 1C). Specifically, we used hierarchical clustering performed with Euclidean distance as a metric and average linkage as a method. First, we observed that all baits purified Sin3 complex as we expected. Second, we found that enzymatic complexes SCF(FBXO3) and OGT/TET2/3 were not observed with any other Sin3 complex baits, suggesting that their association with SAP25 is independent of the Sin3 complex. Similarly, USP11 was also present in SAP25 purifications and absent from most other Sin3 complex purifications, although we detected USP11 with Halo-SAP30L and to a lesser extent with Halo-SAP130. Interestingly, although we had previously failed to detect SAP18 in purified Sin3 complexes 3, here we also observed that Halo-ARID4A and Halo-SUDS3 each captured modest amounts of SAP18. We next checked that by expressing Halo-SAP25 we were not simply switching on expression of the new interactors making them available for copurification with SAP25/Sin3. For this we looked at RNAseq data for HEK293 cells (in the absence of exogenous protein expression) that we had generated in a previous study 40. This confirmed that although SAP25 is not expressed in HEK293 cells, SCF(FBXO3) subunits, USP11, OGT and TET proteins are all robustly expressed (Fig 1B). Thus, they are potentially available in cells in the absence of SAP25. In conclusion, the hierarchical clustering analysis supports a model in which SAP25 associates with several complexes separate from the Sin3 complex.

Distinct SAP25 regions are responsible for its interactions with Sin3A or with other enzymatic complexes.

Having determined that a subset of proteins appeared to associate uniquely with Halo-SAP25, and not with other Sin3 subunit baits in the absence of SAP25, we hypothesized that this set of proteins might bind to one or more regions within SAP25 distinct from the region that interacts with the Sin3 complex. To investigate this, we transiently expressed different mutant versions of Halo-SAP25 in 293T cells for AP-MS analysis. Analysis of transiently expressed proteins is advantageous as it allows faster screenings of different mutants 40, 41.

First, we expressed wild type SAP25 fused to an N-terminal Halo tag in293T cells (Halo-SAP25) and tested expression by fluorescence confocal microscopy and by SDS-PAGE analysis of purified complexes (Fig. 2A). The presence of SAP25 in the nucleus was consistent with its association with Sin3 complex proteins, which are known to participate in transcription repression. In addition, we also observed a pool of Halo-SAP25 in the cytoplasm. This cytoplasmic fraction is consistent with the observation by Shiio et al. 18 of a cytoplasmic pool of recombinant mouse SAP25.

Figure 2: Different regions of SAP25 are important for capture of either the Sin3 complex or the other enzymatic and regulatory proteins.

Figure 2:

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A. Localization and purification of Halo-SAP25 in 293T cells. Left: Halo-SAP25 is labeled with HaloTag® TMRDirect™ fluorescent ligand (red) with DNA (nuclei) stained with Hoechst dye (blue). Right: SDS-PAGE/silver stain analysis of proteins captured by Halo-SAP25 transiently expressed in 293T cells. B. Halo-tagged mutant versions of SAP25 were constructed as indicated for expression in 293T cells prior to AP-MS analysis. C. Hierarchical clustering of Sin3 complex proteins and other enzymatic and regulatory proteins observed in purifications using wild-type and mutant versions of SAP25 baits expressed in 293T cells. Values are log2FC with negative and missing values set to zero (Table S2A). The comparative heatmap was generated using the Morpheus online tool (https://software.broadinstitute.org/morpheus).53). Clustering was performed with Euclidean distance and average linkage. D. Topological analysis of SAP25 mutants. TopS scores are calculated from spectral counts assigned to proteins detected in the purifications indicated (Table S3).

Next, we asked whether different regions of SAP25 might be important for recruiting different complexes, or alternatively, whether one region in SAP25 was responsible for capturing multiple complexes. We examined the ability of mutant versions of Halo-SAP25 to enrich subunits of the different complexes compared with controls. Initially, we tested: 1) two deletion mutants (Halo-SAP25 Δ21-40 and Halo-SAP25 Δ110-119), 2) two substitution mutants (SAP25 LL149/150AA and SAP25 P114-117A), and 3) three truncation mutants covering N-terminal, middle and C-terminal regions within SAP25 (Fig. 2B). We used the QPROT algorithm 34 to calculate log2FC values for enriched proteins based on spectral count values (Table S2A, with the detailed lists of SAP25-associated proteins for each SAP25 construct AP-MS analysis provided in Tables S2B-I). We performed hierarchical clustering on the subset of proteins we were interested in (Fig. 2C).

First, we noticed that the components of the unique Sin3 subunits (excluding the core subunits RBBP4/7 and HDAC1/2 – which are shared with other complexes such as NuRD) clustered separately as did OGT/TET2/3 and USP11. Second, we noticed that each of our truncated versions of SAP25 enriched for a different complex: Halo-SAP25 16-30 enriches for SCF(FBXO3), but not OGT or Sin3 (Fig. 2C, lane 7); Halo-SAP25 50-130 enriches for OGT/TET and USP11 but not FBXO3 or Sin3 (Fig. 2C, lane 8); and SAP25 131-168 enriches for Sin3 subunits but not SCF(FBXO3) or OGT/TET (Fig. 2C, lane 5). Third, we noticed that our deletion mutants lost binding with components of one specific complex but not the others. For example, Halo-SAP25 Δ21-40 loses binding to FBXO3 and CUL1 but retains binding to Sin3, OGT/TET, and USP11 (Fig. 2C, lane 1). Halo-SAP25 Δ110-119 loses binding to OGT/TET but retains binding to Sin3, SCF(FBXO3), and USP11 (Fig. 2C, lane 3). Fourth, we noticed that the substitution mutant Halo-SAP25 LL149/150AA, which we generated based on a mutant of mouse SAP25, which was previously shown to lose binding to Sin3 complex 18, similarly lost binding to Sin3 complex in our experiments (Fig. 2C, lane 6). Remarkably, this mutant was still able to capture SCF(FBXO3), OGT, and USP11.

For further confirmation that the different mutant versions of SAP25 were able to capture different subsets of proteins, we also calculated TopS values 2 for Sin3 complex subunits, SCF(FBXO3), OGT/TET, and USP11 (Fig. 2D). TopS generates positive or negative scores (Table S3) for proteins based on spectral counts and these scores indicate whether the proteins are over or underrepresented in one purification compared with others 2. For the truncation mutants, we found that Sin3 complex subunits were overrepresented in Halo-SAP25 131-168 purifications (dark red segments), that USP11 was overrepresented in Halo-SAP25 50-130 purifications, and that SCF(FBXO3) was overrepresented in Halo-SAP25 16-30 purifications. In addition, we found that both SCF(FBXO3) and OGT were overrepresented in the Halo-SAP25 LL149/150AA purification, that is, their binding to SAP25 was not compromised by the substitution of these two leucine residues.

In summary, our initial analyses of these different mutant versions of Halo-SAP25 supported our hypothesis that different regions of SAP25 were likely involved in recruitment of Sin3 complex, SCF(FBXO3), USP11, and OGT/TET. Having made these observations, we next performed follow-up analyses to examine dNSAF (quantitative) values for each complex in turn and perform additional experiments to further examine the association of each complex with SAP25.

A SAP25 C-terminal region associates with the Sin3 complex

Having determined that the region important for Sin3 recruitment was likely in the C-terminal half of SAP25, we evaluated the presence of the Sin3 complex by examining the dNSAF (abundance) values for Sin3 subunits detected in Halo-SAP25 WT, Halo-SAP25 131-168, and Halo-SAP25 LL149/150AA purifications (Fig. 3A). We found that Sin3 subunits were present in both Halo-SAP25-WT and Halo-SAP25 131-168 purifications (dark blue and light blue bars). In contrast, the majority of Sin3 subunits were not detected in Halo-SAP25 LL149-50AA purifications (Fig. 3A – absence of pink bars). Interestingly, small amounts of the subunits HDAC1/2 and RBBP4/7, which are shared among several HDAC complexes, were still detected in these SAP25 LL149/150AA mutant purifications. The increased abundance of Sin3 in the Halo-SAP25 131-168 compared with Halo-SAP25 WT purifications is likely due to the increase in bait abundance. The complete lists of proteins copurifying with these mutants are provided in Tables S2B, S2F, and S2H.

Figure 3: A SAP25 C-terminal region captures the Sin3 complex.

Figure 3:

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A. SAP25 region 131-168 is sufficient and amino acids 149-150 are required for interaction with the Sin3 complex. Quantitative (dNSAF) values are shown for Sin3 complex proteins identified in Halo-SAP25 WT, Halo-SAP25 131-168, and Halo-SAP25 LL149-150AA purifications. Bait proteins were transiently expressed in 293T cells. Values are averages of at least three biological replicates and error bars represent standard deviation. B. Sin3 complex proteins interact with Halo-SAP25 but not Halo-SAP25 LL149-150AA. 293T cell lysates transfected with either Halo control, Halo-SAP25 or Halo-SAP25 LL149-150AA were subjected to Halo affinity capture. Copurified proteins were resolved by SDS-PAGE and identified by Western blotting using the indicated antibodies. C. Inferred position of SAP25-Sin3 complex binding.

Next, to support our initial findings, we again expressed Halo-control, Halo-SAP25, and Halo-SAP25 LL149/150AA in293T cells and this time analyzed the eluates from the resulting purifications by SDS-PAGE followed by Western blotting. We detected Sin3 complex proteins SIN3A, HDAC1, and SAP30 as copurifying with Halo SAP25 but not with Halo-SAP25 LL149/150AA (Fig 3B - compare the last 2 lanes). This supports that the SAP25 leucine residues 149 and 150 are required for its interaction with Sin3 complex proteins (Fig. 3C).

The SCF(FBXO3) complex interacts with an N-terminal region of SAP25

In addition to the Sin3 complex, our AP-MS studies had indicated several potential unique interacting partners for SAP25 (Fig. 1A). Among these, we found that SAP25 captures components of the E3 ubiquitin ligase SCF(FBXO3), which promotes ubiquitination 38. SCF(FBXO3) is built from the substrate recognition protein FBXO3, the adaptor protein SKP1, the cullin CUL1, and a small (12 kDa) ring-box protein RBX1 38. Previously, SCF(FBXO3) has been shown to target the autoimmune regulator AIRE for ubiquitination, increasing an association between AIRE and the transcription elongation factor pTEFb, and promoting proper expression of AIRE regulated genes 42. The association between SCF(FBXO3) and transcriptional regulator Sin3 was intriguing as SCF(FBXO3) had not previously copurified with any of the other Sin3 complex proteins studied by our group (Fig. 1C) 7.

To confirm that SCF(FBXO3) was recruited to SAP25 independently of the Sin3 complex, we asked which region of SAP25 might be important for the association between SCF(FBXO3) and SAP25. For this, we compared mean dNSAF values of SCF(FBXO3) complex proteins purified using the Halo tagged baits SAP25 WT, SAP25 16-30 and SAP25 Δ21-40 (Fig. 4A). We observed that both SAP25 WT and SAP25 16-30 capture three of the SCF(FBXO3) subunits, FBXO3, SKP1, and CUL1 (Fig. 4A, dark and light blue bars). In contrast, Halo-SAP25 Δ21-40 failed to capture the FBXO3 and CUL1 subunits of SCF(FBXO3) and captured substantially less of the SKP1 subunit, but this mutant could still capture SIN3A (Fig. 4A, pink bars). This is consistent with a model in which the SCF(FBXO3) complex binds SAP25 independently of the Sin3 complex.

Figure 4: Association between SCF(FBXO3) and an N-terminal region of SAP25.

Figure 4:

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A. SAP25 region 16-30 is sufficient and region 21-40 is required for interaction with SCF(FBXO3). Quantitative (dNSAF) values for SAP25, SCF(FBXO3) complex subunits, and SIN3A are shown for Halo-SAP25 WT Halo-SAP25 16-30 and Halo-SAP25 Δ21-40purifications. Bait proteins were transiently expressed in 293T cells. Values are the average of at least three biological replicates and error bars represent standard deviation. B. FBXO3 captures SAP25 but no other Sin3 complex subunits. SNAP-FLAG-FBXO3 was co-expressed with the indicated Halo-tagged proteins and the resulting lysates subjected to SNAP affinity purification. Copurified proteins were identified by SDS-PAGE followed by Western blotting using the indicated antibodies. C. Amino acid positions within SAP25 region 16-30 are important for FBXO3 capture. Proteins captured by either Halo-SAP25 or Halo-SAP25 S26/27/40D T35E were resolved by SDS-PAGE and detected by Western blotting using the indicated antibodies. Mutated amino acids within an N-terminal region are highlighted in red. D. Inferred position within SAP25 required for FBXO3 binding. Possible abrogation of SAP25/FBXO3 interaction by modified residues within the binding region are indicated by red Ps.

To provide additional evidence that SCF(FBXO3) does not interact directly with the Sin3 complex, we built a recombinant version of FBXO3 with an N-terminal SNAP tag and tested its ability to capture either SAP25 or Sin3 complex components SIN3A or HDAC2 (Fig 4B). We transfected 293T cells with SNAP-FLAG-FBXO3 and either Halo-SAP25, HDAC2-Halo or Halo-SIN3A and subjected the resulting cell lysates to SNAP affinity purification. After purification, as expected, we detected the presence of Halo-SAP25 in cells transfected with SNAP-FLAG-FBXO3. In contrast, SNAP-FLAG-FBXO3 could not capture HDAC2-Halo or Halo-SIN3A in the absence of SAP25 (Fig. 4B). To further support that the region including amino acids 21-40 of SAP25 is important for recruitment of FBXO3, we used a Halo-SAP25 S26/27/40D T35E mutant, which contains four point-mutations within the SAP25 region 21-40. These amino acid substitutions are designed to mimic possible phosphorylation of these serine/threonine residues. The resulting Western blot analysis shows that endogenous FBXO3 copurifies with Halo-SAP25 WT but not with the Halo-SAP25 S26/27/40D T35E mutant (Fig. 4C). This supports that SAP25 amino acids within region 21-40 are important for controlling the association between SAP25 and FBXO3 (Fig. 4D).

OGT interacts with a central region of SAP25 not required for recruitment of either the SCF(FBXO3) or Sin3 complexes

Transcription factors can be modified on serine or threonine residues by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides 43, 44, 45, 46. Previous evidence suggests that the enzyme responsible for catalyzing this reaction, O-GlcNAc transferase (OGT), is associated with murine Sin3 complexes. Specifically, in vitro translated mSin3A was captured by GST-OGT, and HA-OGT coimmunoprecipitated mSin3A in Cos-7 cells 19.In addition, direct interaction of TET2 and TET3 with OGT has also been observed and linked with transcription regulation 22, 47. However, we had not previously detected OGT in our Sin3 purifications (Tables S1C-M) and the mechanism of recruitment of OGT to the Sin3 complex is still not fully understood.

Following our initial observation that Halo-SAP25 Δ110-119 could enrich Sin3 complex but not OGT (Fig. 2C), we considered whether OGT might be recruited to SAP25 independently of the Sin3 complex via a central region within SAP25. To examine whether SAP25 could capture OGT independently of the other complexes, we plotted dNSAF values for several representative proteins of these complexes for the SAP25 WT, SAP25 50-130, and Halo-SAP25 Δ110-119 purifications (Fig. 5A). We found that while SAP25 WT and SAP25 50-130 could both capture OGT and OGT binding partner TET2, the mutant with a disrupted central region (Halo-SAP25 Δ110-119) failed to capture OGT and TET2. The interaction between SAP25 50-130 and OGT was unlikely to be via either Sin3 or SCF(FBXO3) complexes as neither representative subunits SIN3A nor FBXO3 were detected in SAP25-50-130 purifications. Interestingly, USP11 was detected in all purifications, suggesting a different binding mechanism between SAP25 and USP11.

Figure 5: OGT/TET interacts with a central SAP25 region.

Figure 5:

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A. SAP25 region 50-130 is sufficient and region 110-119 is required for OGT/TET capture. Quantitative (dNSAF) values are shown for selected proteins identified in purifications prepared using the indicated Halo-tagged versions of SAP25. Values are averages of at least three biological replicates and error bars represent standard deviation. B. Halo-SAP25 captures FLAG-OGT. FLAG-OGT was expressed with or without Halo-SAP25 in 293T cells. Proteins identified in the resulting Halo-affinity purifications (eluted by TEV cleavage of Halo) were identified by SDS-PAGE followed by Western blot analysis using the indicated antibodies. C. Effect of disrupting sub-regions within Halo-SAP25 110-119 on OGT capture. FLAG-OGT was co-expressed with the indicated versions of SAP25, and the resulting Halo purifications analyzed by SDS-PAGE followed by Western blotting using the indicated antibodies. D. SAP25 region required for OGT binding.

Finally, we made further mutations in the vicinity of the SAP25 110-119 region and tested the ability of two new versions of SAP25, Halo-SAP25 LL112/113AA and SLK109-111AAA to capture recombinant FLAG-OGT. Having confirmed that Halo-SAP25 WT could capture FLAG-OGT as expected (Fig. 5B), we observed that, although we captured negligible FLAG-OGT with Halo-SAP25 P114-117A (Fig. 5C), mutations just before this region (SLK109-111AAA and LL112-113AA) did not abrogate SAP25 capture of OGT. Thus, although some changes to the SAP25 110-119 region disrupt the SAP25/OGT interaction, other modest changes do not.

In conclusion, both our AP-MS data and Western blot analysis support that OGT can be recruited to SAP25 independently of the Sin3 complex. We consistently identified OGT in SAP25 purifications (Fig. 1, Fig. 2C) together with the OGT binding partners TET2 and TET3 21, 22, but did not identify OGT copurifying with any other Sin3 complex baits, including Halo-SIN3A, in the absence of SAP25 (Fig. 1B). We mapped SAP25 region 110-119 as necessary for recruitment of OGT but not the Sin3 complex, and we determined that SAP25 50-130 is sufficient for OGT capture but not for Sin3 complex capture (Fig. 5). These results suggest that SAP25 may play a vital role in recruiting OGT to the Sin3 complex to participate in modulating post-translational modifications through O-linkage of the monosaccharide N-acetylglucosamine (O-GlcNAc).

Discussion

HEK293 cells as a natural knock-out line for SAP25.

We have previously published several studies to characterize the interaction network between Sin3 complex components 7 using a robust AP-MS approach combining Halo affinity purification with multidimensional protein identification technology (MudPIT). Although SAP25 had been reported as a bona fide component of Sin3 by Shiio et al. in 2006 18, we were never able to detect endogenous SAP25 in Sin3 complexes isolated via other Sin3 subunits. However, exogenously expressed Halo-SAP25 did capture Sin3 components 3. RNASeq analysis revealed that SAP25 was not expressed in the HEK293 cells we used for these affinity purifications 3. Intriguingly, the O-GlcNAc transferase OGT and TET1/2/3 DNA methylases, which are known to be associated with Sin3/HDAC 19-21, were also never detected from these various purifications from Flp-In™-293 cells, except in the Halo-SAP25 dataset. Therefore, SAP25 appears to be necessary for the recruitment of these enzymatic complexes to Sin3/HDAC. To test this hypothesis further, we used 293T cells as a natural SAP25 knockout cell line to identify interaction partners uniquely pulled down by SAP25 and dissect how truncations, deletions, and point-mutations within the SAP25 sequence affect these interactions without the competition for interaction from endogenous full-length SAP25.

To identify SAP25 unique interactors, we compared the Halo-SAP25 copurified proteins with the ones identified from AP-MS analysis of eleven Halo-tagged Sin3Sin3 complex baits (Fig.1). In addition to the recovery of OGT and TET1/2/3 only in the presence of exogenous SAP25, two additional proteins/protein complexes with established enzymatic activities captured our attention: an FBXO3 based SCF ubiquitin ligase and a deubiquitinase (USP11), both of which have yet to be linked to the Sin3/HDAC complex. Having established that Halo-SAP25 captures unique interactors, we considered the mechanisms that might explain this. One possibility was that SAP25 facilitates binding of these other complexes to other Sin3 complex subunits. A second possibility was that SAP25 functions as an adaptor protein that provides a platform for the recruitment of different complexes via different regions of SAP25.

To test the possibility that SAP25 functions as an adaptor protein, we set out to map the regions of SAP25 responsible for capturing the different protein complexes. From these studies we suggest a model in which distinct discrete regions of SAP25 are responsible for docking with Sin3, SCF(FBXO3), OGT/TET, and USP11 (Fig. 6). Taken together, these findings suggest a distinct role for SAP25 as an adaptor protein that can separately recruit SCF(FBXO3), USP11, and OGT/TET to the Sin3 complex. Having established that SAP25 likely provides a platform for various complexes to assemble independently but in proximity, we now consider the possible roles these complexes might play while docked to SAP25. Four of these complexes are involved in protein post-translational modification – ubiquitination (SCF(FBXO3), deubiquitination (USP11), O-GlcNAcylation (OGT), or deacetylation (Sin3/HDAC) – while the TET proteins are DNA demethylases (Fig. 6). Although it is well established that the Sin3 complex represses gene transcription by deacetylating histones, the targets of the other complexes when recruited to Sin3/HDACs by SAP25 are not known.

Figure 6. Overview of the regions of SAP25 required or sufficient for capture of SCF(FBXO3), OGT, or the Sin3 complex.

Figure 6.

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A-B. Quantitative comparison of Sin3 complex, SCF(FBXO3) complex, USP11, and OGT captured by the indicated versions of SAP25 expressed in 293T cells. Values on the Circos diagrams are mean dNSAF x 100,000 (calculated from a minimum of three biological replicates). Subunits shared between the Sin3 and NuRD complexes are indicated by green ribbons. C. Predicted structure of SAP25 (AlphaFold AF-Q8TEE9-F1) with regions 16-30 (yellow), 50-130 (red), 131-168 (blue) and lysines 149/150 (blue atoms) indicated.

Association of SAP25/Sin3 with USP11.

USP11 is a ubiquitin hydrolase that post-translationally modifies target proteins by removing ubiquitin. Proteins can be ubiquitinated through conjugation to either the N-terminus or to one of the seven lysines within ubiquitin. Although K48 linked ubiquitination is well known for targeting proteins for destruction by the proteosome, cleavage assays have indicated that USP11 favors targeting K6, K11, K33, and K63 linked ubiquitin chains 48. Of these, K63 ubiquitination has been well characterized and is thought to coordinate a variety of processes including DNA repair (reviewed in 49). Timely transcriptional repression is important for initiating DNA repair and several lines of evidence have also linked USP11 to transcriptional repressor complexes.

First, USP11 was shown to physically interact with the components MTA2, MBD3, and HDAC2 of the transcriptional repressor NuRD 37. The HDAC1/2 histone deacetylases and RBBP4/7 subunits form a core complex shared between NuRD and Sin3/HDAC complexes (Fig. 6A-B – green ribbons). In the same study, NuRD and USP11 were also recruited together to UV laser induced sites of DNA damage where USP11 was shown to deubiquitinate histones H2A (K119Ub) and H2B (K120Ub). Ting and co-workers proposed that USP11 might be influencing the DNA repair process by helping to remove repair factors during the later periods of DNA damage response 37. In addition to NuRD, USP11 was found to interact with the PCGF2 (MEL18) subunit of the polycomb repressive complex PRC1 50. PRC1 complex acts as an E3 ubiquitin ligase to ubiquitinate histone H2AK119. In this context, USP11 was not seen to affect H2A ubiquitination, but rather to target PRC1 complex subunits (MEL18, BMI1, and RING1) for deubiquitination. Maertens and coworkers proposed that here, USP11 was modulating the degradation of these PRC1 subunits, and hence the availability of PRC1 for repression of target genes such as INK4a. Finally, USP11 targets and stabilizes the promyelocytic protein PML 51. PML interacts directly with the Sin3 complex components mSin3A and HDAC1 52 and a pool of SAP25 has also been found colocalized with PML in PML nuclear bodies 18 consistent with a possible role for SAP25 in recruiting USP11 to the Sin3 complex for targeting Sin3-bound PML. To conclude, USP11 has been found accompanying several complexes involved in transcriptional repression including NuRD, PRC1 and PML, and may target either complex subunits or histones (H2A/B) for deubiquitination.

Association of SAP25/Sin3 with SCF(FBXO3).

In contrast to USP11, the SCF(FBXO3) complex functions as an E3 ubiquitin ligase, but like USP11, SCF(FBXO3) has also been observed to interact with PML via the FBXO3 subunit 53. Shima and coworkers proposed that SCF(FBXO3) does not target PML itself, but rather targets transcriptional coactivators (p300 and HIPK2) for ubiquitination and degradation. PML interaction with SCF(FBXO3) inhibits this degradation of coactivators, stabilizing transcription. This raises the possibility that SAP25 also controls SCF(FBXO3) targeting of coactivators. SCF(FBXO3) also targets the transcription factor AIRE via the FBXO3 subunit 42. Shao and coworkers found that although SCF(FBXO3) does appear to target the transcription factor for degradation, ubiquitination of AIRE also increases its binding to transcription elongation factor P-TEFb, ensuring proper transcription elongation of AIRE regulated genes. Finally, SCF(FBXO3) may play a role in controlling inflammation. Specifically, FBXO3 targets another F-box protein FBX12 for degradation, stimulating inflammation 54. Similarly, SAP25 has also been proposed to help control expression of inflammatory genes 24.

Association of SAP25/Sin3 with OGT and TET proteins.

OGT, an enzyme that catalyzes the addition of O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides 19, is essential for cell survival 55, and is enriched in the nucleus where it plays a functional role in regulating transcription (reviewed in 56). Nuclear OGT may target histones. Specifically, O-GlcNAcylation of histone H2B by OGT promotes its monoubiquitination at K120 (the same residue deubiquitinated by USP11) 57, possibly to create an anchor to recruit the E3 ubiquitin ligase RNF20/40.

Several lines of evidence have previously suggested that OGT can act cooperatively with the Sin3 complex. Initially, Yang et al. tested the ability of OGT to capture in vitro translated murine Sin3A and determined that OGT contacts the mSin3A PAH4 domain. By recruiting Gal4-Sin3A to promoters with or without exogenous OGT, they then demonstrated that OGT and mSin3A act synergistically to repress transcription 19. Although they found that OGT and Sin3A interact in vitro, the exact mechanism of OGT recruitment to the Sin3 complex in cells might be more complex as we needed SAP25 for capture of OGT together with SIN3A (Fig. 1B). Later studies also confirmed a role for murine Sin3A in functioning cooperatively with OGT. Having characterized TET1 and TET2 as OGT interactors in mouse ES cell nuclei, Vella et al. purified complexes via OGT and found them to contain mSin3A, HDAC1 and TET1/2 58. After showing that OGT and TET colocalize at transcription start sites, with TET being required for OGT recruitment to promoters, they confirmed O-GlcNAcylation of both TET and mSin3A and proposed them as OGT targets.

Other lines of evidence have focused on the interplay between the Sin3 complex and the TET family of demethylases. Using mouse ES cells (which do express SAP25), mouse TET2 was recently found copurifying with Sin3, SAP25, and OGT, with evidence suggesting a role for TET2 (unrelated to its catalytic activity) in recruiting Sin3 complex to active enhancers 21. In contrast to mouse ES cell experiments, and in line with our observations, Chen et al 59 detected OGT but not Sin3 complex subunits copurifying with either tagged TET2 or TET3 purified from 293T cells (which we have shown lack SAP25) 3. Our finding that SAP25 acts as a bridge between OGT and Sin3 explains the absence of Sin3 proteins in these TET2/3 purifications. It is possible that a different recruitment mechanism governs the association of TET1 with Sin3. TET1, OGT and Sin3 complex subunits do copurify 60, but recently, TET1 was shown to interact with the SIN3A PAH1 domain (the same domain that interacts with SAP25) 20. A separate recruitment mechanism for TET1 might explain why we find TET2/3 but not TET1 copurifying with SAP25 (Fig. 1B).

Conclusions.

Our work reveals the interplay between SAP25, several complexes with enzymatic and regulatory functions, and the Sin3 complex. We hypothesize that SAP25 is acting as an adaptor protein and recruits these complexes to the Sin3 complex, possibly to regulate transcription by modulating post-translational and DNA modifications. Whether or not these are distinct SAP25 complexes remains unknown. Evidence from previous studies suggests that SAP25 may act as a docking protein as it does not display any recognizable DNA-binding motifs 18. Our topological and comparative proteomics analysis of Sin3 complex baits supports our hypothesis. Recently, SAP25 and HDAC were linked with the expression of inflammation and antiviral response genes 24. Furthermore, FBXO3, a distinctive interacting partner of SAP25, is associated with breast cancer metastasis induced by PI3K and is associated with USP4 stabilization 61. The presence of USP11 in SAP25 purifications could indicate a role for SAP25 in coordinating transcriptional repression by Sin3/HDAC with DNA damage repair. These results suggest promising directions for further research into the function of SAP25, which could help to determine whether SAP25 functions to recruit these enzymatic activities to the Sin3 complex to regulate transcriptional repression.

Supplementary Material

Supplementary Information
Supplementary Table 1
Supplementary Table 2
Supplementary Table 4
Supplementary Table 3

Acknowledgements

The research presented here was supported by the Stowers Institute for Medical Research and the National Institute of General Medical Sciences of the National Institutes of Health R35GM145240 (M.P.W.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Supporting Information

Description of Supporting Tables, Primers used to construct vectors, Figure S1. (PDF)

Detailed information on stable cell line affinity purification results. (Table S1)

Detailed information on wild type and mutant transient cell line affinity purification results (Table S2)

Detailed information on Topological Scoring (Table S3)

Detailed information regarding MassIVE locations of all runs used in this study (Table S4)

Original Images of Figures. (PDF)

The authors declare that they have no conflicts of interest related to the work presented here.

Data availability

We have reported some of the raw mass spectrometry data used in this study previously (all searches are new to this study). A summary of the runs used here, together with details of where they were first reported is provided in Table S4. Mass spectrometry data has been deposited in the MassIVE repository (http://massive.ucsd.edu) with the data identifiers MSV000093553 (transient transfection data) and MSV000093576 (stable cell line data). Original data underlying this manuscript generated at the Stowers Institute can be accessed after publication from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-2438.

References

  • (1).Morris JH; Knudsen GM; Verschueren E; Johnson JR; Cimermancic P; Greninger AL; Pico AR Affinity purification-mass spectrometry and network analysis to understand protein-protein interactions. Nat Protoc 2014, 9 (11), 2539–2554. DOI: 10.1038/nprot.2014.164 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (2).Sardiu ME; Gilmore JM; Groppe BD; Dutta A; Florens L; Washburn MP Topological scoring of protein interaction networks. Nat Commun 2019, 10 (1), 1118. DOI: 10.1038/s41467-019-09123-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (3).Banks CAS; Thornton JL; Eubanks CG; Adams MK; Miah S; Boanca G; Liu X; Katt ML; Parmely TJ; Florens L; et al. A Structured Workflow for Mapping Human Sin3 Histone Deacetylase Complex Interactions Using Halo-MudPIT Affinity-Purification Mass Spectrometry. Mol Cell Proteomics 2018, 17 (7), 1432–1447. DOI: 10.1074/mcp.TIR118.000661 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (4).Meyer K; Selbach M. Quantitative affinity purification mass spectrometry: a versatile technology to study protein-protein interactions. Front Genet 2015, 6, 237. DOI: 10.3389/fgene.2015.00237 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (5).Sardiu ME; Cai Y; Jin J; Swanson SK; Conaway RC; Conaway JW; Florens L; Washburn MP Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics. Proc Natl Acad Sci U S A 2008, 105 (5), 1454–1459. DOI: 10.1073/pnas.0706983105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (6).Banks CAS; Miah S; Adams MK; Eubanks CG; Thornton JL; Florens L; Washburn MP Differential HDAC1/2 network analysis reveals a role for prefoldin/CCT in HDAC1/2 complex assembly. Sci Rep 2018, 8 (1), 13712. DOI: 10.1038/s41598-018-32009-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (7).Adams MK; Banks CAS; Thornton JL; Kempf CG; Zhang Y; Miah S; Hao Y; Sardiu ME; Killer M; Hattem GL; et al. Differential Complex Formation via Paralogs in the Human Sin3 Protein Interaction Network. Mol Cell Proteomics 2020, 19 (9), 1468–1484. DOI: 10.1074/mcp.RA120.002078 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (8).Latypova X; Vincent M; Mollé A; Adebambo OA; Fourgeux C; Khan TN; Caro A; Rosello M; Orellana C; Niyazov D; et al. Haploinsufficiency of the Sin3/HDAC corepressor complex member SIN3B causes a syndromic intellectual disability/autism spectrum disorder. Am J Hum Genet 2021, 108 (5), 929–941. DOI: 10.1016/j.ajhg.2021.03.017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (9).Verza FA; Das U; Fachin AL; Dimmock JR; Marins M. Roles of Histone Deacetylases and Inhibitors in Anticancer Therapy. Cancers (Basel) 2020, 12 (6). DOI: 10.3390/cancers12061664 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (10).Liu M; Saha N; Gajan A; Saadat N; Gupta SV; Pile LA A complex interplay between SAM synthetase and the epigenetic regulator SIN3 controls metabolism and transcription. J Biol Chem 2020, 295 (2), 375–389. DOI: 10.1074/jbc.RA119.010032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (11).Li W; Zhang Z; Liu X; Cheng X; Zhang Y; Han X; Zhang Y; Liu S; Yang J; Xu B; et al. The FOXN3-NEAT1-SIN3A repressor complex promotes progression of hormonally responsive breast cancer. J Clin Invest 2017, 127 (9), 3421–3440. DOI: 10.1172/JCI94233 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (12).Wu J; Huang X; Li X; Zhou H; Chen X; Chen Y; Guo Y; Huang J; Huang H; Huang Z; et al. Suppression of the long non-coding RNA LINC01279 triggers autophagy and apoptosis in lung cancer by regulating FAK and SIN3A. Discov Oncol 2024, 15 (1), 3. DOI: 10.1007/s12672-023-00855-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (13).Jankowski K; Jagana V; Bisserier M; Hadri L. Switch-Independent 3A: An Epigenetic Regulator in Cancer with New Implications for Pulmonary Arterial Hypertension. Biomedicines 2023, 12 (1). DOI: 10.3390/biomedicines12010010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (14).Kwon YJ; Petrie K; Leibovitch BA; Zeng L; Mezei M; Howell L; Gil V; Christova R; Bansal N; Yang S; et al. Selective Inhibition of SIN3 Corepressor with Avermectins as a Novel Therapeutic Strategy in Triple-Negative Breast Cancer. Mol Cancer Ther 2015, 14 (8), 1824–1836. DOI: 10.1158/1535-7163.MCT-14-0980-T [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (15).Buljan M; Banaei-Esfahani A; Blattmann P; Meier-Abt F; Shao W; Vitek O; Tang H; Aebersold R. A computational framework for the inference of protein complex remodeling from whole-proteome measurements. Nat Methods 2023, 20 (10), 1523–1529. DOI: 10.1038/s41592-023-02011-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (16).Marcum RD; Hsieh J; Giljen M; Justice E; Daffern N; Zhang Y; Radhakrishnan I. A capped Tudor domain within a core subunit of the Sin3L/Rpd3L histone deacetylase complex binds to nucleic acid G-quadruplexes. J Biol Chem 2022, 298 (2), 101558. DOI: 10.1016/j.jbc.2021.101558 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (17).Kumari S; Rehman A; Chandra P; Singh KK Functional role of SAP18 protein: From transcriptional repression to splicing regulation. Cell Biochem Funct 2023. DOI: 10.1002/cbf.3830 [DOI] [PubMed] [Google Scholar]
  • (18).Shiio Y; Rose DW; Aur R; Donohoe S; Aebersold R; Eisenman RN Identification and characterization of SAP25, a novel component of the mSin3 corepressor complex. Mol Cell Biol 2006, 26 (4), 1386–1397. DOI: 10.1128/mcb.26.4.1386-1397.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (19).Yang X; Zhang F; Kudlow JE Recruitment of O-GlcNAc Transferase to Promoters by Corepressor mSin3A: Coupling Protein O-GlcNAcylation to Transcriptional Repression. Cell 2002, 110 (1), 69–80. DOI: 10.1016/S0092-8674(02)00810-3. [DOI] [PubMed] [Google Scholar]
  • (20).Chandru A; Bate N; Vuister GW; Cowley SM Sin3A recruits Tet1 to the PAH1 domain via a highly conserved Sin3-Interaction Domain. Sci Rep 2018, 8 (1), 14689. DOI: 10.1038/s41598-018-32942-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (21).Flores JC; Sidoli S; Dawlaty MM Tet2 regulates Sin3a recruitment at active enhancers in embryonic stem cells. iScience 2023, 26 (7), 107170. DOI: 10.1016/j.isci.2023.107170 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (22).Deplus R; Delatte B; Schwinn MK; Defrance M; Méndez J; Murphy N; Dawson MA; Volkmar M; Putmans P; Calonne E; et al. TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS. Embo j 2013, 32 (5), 645–655. DOI: 10.1038/emboj.2012.357 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (23).Vaidyanathan K; Niranjan T; Selvan N; Teo CF; May M; Patel S; Weatherly B; Skinner C; Opitz J; Carey J; et al. Identification and characterization of a missense mutation in the O-linked β-N-acetylglucosamine (O-GlcNAc) transferase gene that segregates with X-linked intellectual disability. J Biol Chem 2017, 292 (21), 8948–8963. DOI: 10.1074/jbc.M116.771030 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (24).John SP; Sun J; Carlson RJ; Cao B; Bradfield CJ; Song J; Smelkinson M; Fraser IDC IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages. Cell Rep 2018, 25 (1), 95–106.e106. DOI: 10.1016/j.celrep.2018.09.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (25).McDermott E; Ryan EJ; Tosetto M; Gibson D; Burrage J; Keegan D; Byrne K; Crowe E; Sexton G; Malone K; et al. DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights into Disease Pathogenesis. J Crohns Colitis 2016, 10 (1), 77–86. DOI: 10.1093/ecco-jcc/jjv176 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (26).Hunter M; Spiller KJ; Dominique MA; Xu H; Hunter FW; Fang TC; Canter RG; Roberts CJ; Ransohoff RM; Trojanowski JQ; et al. Microglial transcriptome analysis in the rNLS8 mouse model of TDP-43 proteinopathy reveals discrete expression profiles associated with neurodegenerative progression and recovery. Acta Neuropathol Commun 2021, 9 (1), 140. DOI: 10.1186/s40478-021-01239-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (27).Torrealba E; Aguilar-Zerpa N; Garcia-Morales P; Díaz M. Compensatory Mechanisms in Early Alzheimer's Disease and Clinical Setting: The Need for Novel Neuropsychological Strategies. J Alzheimers Dis Rep 2023, 7 (1), 513–525. DOI: 10.3233/adr-220116 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (28).Cai Y; Jin J; Swanson SK; Cole MD; Choi SH; Florens L; Washburn MP; Conaway JW; Conaway RC Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex. J Biol Chem 2010, 285 (7), 4268–4272. DOI: 10.1074/jbc.C109.087981 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (29).Banks CA; Lee ZT; Boanca G; Lakshminarasimhan M; Groppe BD; Wen Z; Hattem GL; Seidel CW; Florens L; Washburn MP Controlling for gene expression changes in transcription factor protein networks. Mol Cell Proteomics 2014, 13 (6), 1510–1522. DOI: 10.1074/mcp.M113.033902 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (30).Banks CA; Boanca G; Lee ZT; Florens L; Washburn MP Proteins interacting with cloning scars: a source of false positive protein-protein interactions. Sci Rep 2015, 5, 8530. DOI: 10.1038/srep08530 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (31).Swanson SK; Florens L; Washburn MP Generation and analysis of multidimensional protein identification technology datasets. Methods Mol Biol 2009, 492, 1–20. DOI: 10.1007/978-1-59745-493-3_1 [DOI] [PubMed] [Google Scholar]
  • (32).Wen Z. kite: a software suite for processing and analysis of tandem mass spectrometry data. . 2022. 10.5281/zenodo. (accessed 5914885 (2022). v1.0.0. ). [DOI] [Google Scholar]
  • (33).Tabb DL; McDonald WH; Yates JR 3rd. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J Proteome Res 2002, 1 (1), 21–26. DOI: 10.1021/pr015504q [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (34).Choi H; Kim S; Fermin D; Tsou CC; Nesvizhskii AI QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics. J Proteomics 2015, 129, 121–126. DOI: 10.1016/j.jprot.2015.07.036 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (35).Sardiu ME; Florens L; Washburn MP Generating topological protein interaction scores and data visualization with TopS. Methods 2020, 184, 13–18. DOI: 10.1016/j.ymeth.2019.08.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (36).Wolters DA; Washburn MP; Yates JR 3rd. An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 2001, 73 (23), 5683–5690. DOI: 10.1021/ac010617e [DOI] [PubMed] [Google Scholar]
  • (37).Ting X; Xia L; Yang J; He L; Si W; Shang Y; Sun L. USP11 acts as a histone deubiquitinase functioning in chromatin reorganization during DNA repair. Nucleic Acids Res 2019, 47 (18), 9721–9740. DOI: 10.1093/nar/gkz726 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (38).Zhang Z; Bao Z; Gao P; Yao J; Wang P; Chai D. Diverse Roles of F-BoxProtein3 in Regulation of Various Cellular Functions. Front Cell Dev Biol 2021, 9, 802204. DOI: 10.3389/fcell.2021.802204 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (39).Ito R; Katsura S; Shimada H; Tsuchiya H; Hada M; Okumura T; Sugawara A; Yokoyama A. TET3-OGT interaction increases the stability and the presence of OGT in chromatin. Genes Cells 2014, 19 (1), 52–65. DOI: 10.1111/gtc.12107 [DOI] [PubMed] [Google Scholar]
  • (40).Banks CA; Boanca G; Lee ZT; Eubanks CG; Hattem GL; Peak A; Weems LE; Conkright JJ; Florens L; Washburn MP TNIP2 is a Hub Protein in the NF-kappaB Network with Both Protein and RNA Mediated Interactions. Mol Cell Proteomics 2016, 15 (11), 3435–3449. DOI: 10.1074/mcp.M116.060509 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (41).Wright JF Transient transfection methods for clinical adeno-associated viral vector production. Hum Gene Ther 2009, 20 (7), 698–706. DOI: 10.1089/hum.2009.064 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (42).Shao W; Zumer K; Fujinaga K; Peterlin BM FBXO3 Protein Promotes Ubiquitylation and Transcriptional Activity of AIRE (Autoimmune Regulator). J Biol Chem 2016, 291 (34), 17953–17963. DOI: 10.1074/jbc.M116.724401 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (43).Ozcan S; Andrali SS; Cantrell JE Modulation of transcription factor function by O-GlcNAc modification. Biochim Biophys Acta 2010, 1799 (5-6), 353–364. DOI: 10.1016/j.bbagrm.2010.02.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (44).Constable S; Lim JM; Vaidyanathan K; Wells L. O-GlcNAc transferase regulates transcriptional activity of human Oct4. Glycobiology 2017, 27 (10), 927–937. DOI: 10.1093/glycob/cwx055 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (45).Li H; Wang Y; Feng S; Chang K; Yu X; Yang F; Huang H; Wang Y; Li X; Guan F. Reciprocal regulation of TWIST1 and OGT determines the decitabine efficacy in MDS/AML. Cell Commun Signal 2023, 21 (1), 255. DOI: 10.1186/s12964-023-01278-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (46).Whisenhunt TR; Yang X; Bowe DB; Paterson AJ; Van Tine BA; Kudlow JE Disrupting the enzyme complex regulating O-GlcNAcylation blocks signaling and development. Glycobiology 2006, 16 (6), 551–563. DOI: 10.1093/glycob/cwj096 [DOI] [PubMed] [Google Scholar]
  • (47).Adams GE; Chandru A; Cowley SM Co-repressor, co-activator and general transcription factor: the many faces of the Sin3 histone deacetylase (HDAC) complex. Biochem J 2018, 475 (24), 3921–3932. DOI: 10.1042/bcj20170314 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (48).Harper S; Gratton HE; Cornaciu I; Oberer M; Scott DJ; Emsley J; Dreveny I. Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains. Biochemistry 2014, 53 (18), 2966–2978. DOI: 10.1021/bi500116x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (49).Lee BL; Singh A; Mark Glover JN; Hendzel MJ; Spyracopoulos L. Molecular Basis for K63-Linked Ubiquitination Processes in Double-Strand DNA Break Repair: A Focus on Kinetics and Dynamics. J Mol Biol 2017, 429 (22), 3409–3429. DOI: 10.1016/j.jmb.2017.05.029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (50).Maertens GN; El Messaoudi-Aubert S; Elderkin S; Hiom K; Peters G. Ubiquitin-specific proteases 7 and 11 modulate Polycomb regulation of the INK4a tumour suppressor. EMBO J 2010, 29 (15), 2553–2565. DOI: 10.1038/emboj.2010.129 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (51).Wu HC; Lin YC; Liu CH; Chung HC; Wang YT; Lin YW; Ma HI; Tu PH; Lawler SE; Chen RH USP11 regulates PML stability to control Notch-induced malignancy in brain tumours. Nat Commun 2014, 5, 3214. DOI: 10.1038/ncomms4214 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (52).Khan MM; Nomura T; Kim H; Kaul SC; Wadhwa R; Shinagawa T; Ichikawa-Iwata E; Zhong S; Pandolfi PP; Ishii S. Role of PML and PML-RARalpha in Mad-mediated transcriptional repression. Mol Cell 2001, 7 (6), 1233–1243. DOI: 10.1016/s1097-2765(01)00257-x [DOI] [PubMed] [Google Scholar]
  • (53).Shima Y; Shima T; Chiba T; Irimura T; Pandolfi PP; Kitabayashi I. PML activates transcription by protecting HIPK2 and p300 from SCFFbx3-mediated degradation. Mol Cell Biol 2008, 28 (23), 7126–7138. DOI: 10.1128/MCB.00897-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (54).Chen BB; Coon TA; Glasser JR; McVerry BJ; Zhao J; Zhao Y; Zou C; Ellis B; Sciurba FC; Zhang Y; et al. A combinatorial F box protein directed pathway controls TRAF adaptor stability to regulate inflammation. Nat Immunol 2013, 14 (5), 470–479. DOI: 10.1038/ni.2565 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (55).Shafi R; Iyer SP; Ellies LG; O'Donnell N; Marek KW; Chui D; Hart GW; Marth JD The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny. Proc Natl Acad Sci U S A 2000, 97 (11), 5735–5739. DOI: 10.1073/pnas.100471497 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (56).Yang X; Qian K. Protein O-GlcNAcylation: emerging mechanisms and functions. Nat Rev Mol Cell Biol 2017, 18 (7), 452–465. DOI: 10.1038/nrm.2017.22 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (57).Fujiki R; Hashiba W; Sekine H; Yokoyama A; Chikanishi T; Ito S; Imai Y; Kim J; He HH; Igarashi K; et al. GlcNAcylation of histone H2B facilitates its monoubiquitination. Nature 2011, 480 (7378), 557–560. DOI: 10.1038/nature10656 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (58).Vella P; Scelfo A; Jammula S; Chiacchiera F; Williams K; Cuomo A; Roberto A; Christensen J; Bonaldi T; Helin K; et al. Tet proteins connect the O-linked N-acetylglucosamine transferase Ogt to chromatin in embryonic stem cells. Mol Cell 2013, 49 (4), 645–656. DOI: 10.1016/j.molcel.2012.12.019 [DOI] [PubMed] [Google Scholar]
  • (59).Chen Q; Chen Y; Bian C; Fujiki R; Yu X. TET2 promotes histone O-GlcNAcylation during gene transcription. Nature 2013, 493 (7433), 561–564. DOI: 10.1038/nature11742 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (60).Shi FT; Kim H; Lu W; He Q; Liu D; Goodell MA; Wan M; Songyang Z. Ten-eleven translocation 1 (Tet1) is regulated by O-linked N-acetylglucosamine transferase (Ogt) for target gene repression in mouse embryonic stem cells. J Biol Chem 2013, 288 (29), 20776–20784. DOI: 10.1074/jbc.M113.460386 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (61).Xu J; Guo R; Wen N; Li L; Yi Y; Chen J; He Z; Yang J; Xiao ZJ; Niu M. FBXO3 stabilizes USP4 and Twist1 to promote PI3K-mediated breast cancer metastasis. PLoS Biol 2023, 21 (12), e3002446. DOI: 10.1371/journal.pbio.3002446 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information
NIHMS2074420-supplement-Supplementary_Information.pdf (640.7KB, pdf)
Supplementary Table 1
NIHMS2074420-supplement-Supplementary_Table_1.xlsx (4.4MB, xlsx)
Supplementary Table 2
NIHMS2074420-supplement-Supplementary_Table_2.xlsx (5.2MB, xlsx)
Supplementary Table 4
NIHMS2074420-supplement-Supplementary_Table_4.xlsx (17.6KB, xlsx)
Supplementary Table 3
NIHMS2074420-supplement-Supplementary_Table_3.xlsx (1,001KB, xlsx)

Data Availability Statement

We have reported some of the raw mass spectrometry data used in this study previously (all searches are new to this study). A summary of the runs used here, together with details of where they were first reported is provided in Table S4. Mass spectrometry data has been deposited in the MassIVE repository (http://massive.ucsd.edu) with the data identifiers MSV000093553 (transient transfection data) and MSV000093576 (stable cell line data). Original data underlying this manuscript generated at the Stowers Institute can be accessed after publication from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-2438.

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