Differential Complex Formation via Paralogs in the Human Sin3 Protein Interaction Network
2020; Elsevier BV; Volume: 19; Issue: 9 Linguagem: Inglês
10.1074/mcp.ra120.002078
ISSN1535-9484
AutoresMark K. Adams, Charles A.S. Banks, Janet L. Thornton, Cassandra G. Kempf, Ying Zhang, Sayem Miah, Yan Hao, Mihaela E. Sardiu, Maxime Killer, Gaye Hattem, Alexis Murray, Maria L. Katt, Laurence Florens, Michael P. Washburn,
Tópico(s)Histone Deacetylase Inhibitors Research
ResumoDespite the continued analysis of HDAC inhibitors in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The presence of two Sin3 paralogs in humans, SIN3A and SIN3B, may result in a heterogeneous population of Sin3 complexes and contributes to our poor understanding of the functional attributes of these complexes. Here, we profile the interaction networks of SIN3A and SIN3B to gain insight into complex composition and organization. In accordance with existing data, we show that Sin3 paralog identity influences complex composition. Additionally, chemical cross-linking MS identifies domains that mediate interactions between Sin3 proteins and binding partners. The characterization of rare SIN3B proteoforms provides additional evidence for the existence of conserved and divergent elements within human Sin3 proteins. Together, these findings shed light on both the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize. Despite the continued analysis of HDAC inhibitors in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The presence of two Sin3 paralogs in humans, SIN3A and SIN3B, may result in a heterogeneous population of Sin3 complexes and contributes to our poor understanding of the functional attributes of these complexes. Here, we profile the interaction networks of SIN3A and SIN3B to gain insight into complex composition and organization. In accordance with existing data, we show that Sin3 paralog identity influences complex composition. Additionally, chemical cross-linking MS identifies domains that mediate interactions between Sin3 proteins and binding partners. The characterization of rare SIN3B proteoforms provides additional evidence for the existence of conserved and divergent elements within human Sin3 proteins. Together, these findings shed light on both the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize. Over 13,000 or 70% of protein coding genes within the human genome have at least one paralog (1Ibn-Salem J. Muro E.M. Andrade-Navarro M.A. Co-regulation of paralog genes in the three-dimensional chromatin architecture.Nucleic Acids Res. 2017; 45: 81-91Crossref PubMed Scopus (32) Google Scholar). The acquisition of additional copies of a gene through duplication events provides opportunities for the development of unique gene products with distinct regulatory mechanisms (2Kondrashov F.A. Gene duplication as a mechanism of genomic adaptation to a changing environment.Proc. Biol. Sci. 2012; 279: 5048-5057Crossref PubMed Scopus (371) Google Scholar). Functional divergence can result from gene duplication and protein paralog identity can influence the composition of large protein complexes (3Link S. Spitzer R.M.M. Sana M. Torrado M. Völker-Albert M.C. Keilhauer E.C. Burgold T. Pünzeler S. Low J.K.K. Lindström I. Nist A. Regnard C. Stiewe T. Hendrich B. Imhof A. Mann M. Mackay J.P. Bartkuhn M. Hake S.B. PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex.Nat. Commun. 2018; 9: 4300Crossref PubMed Scopus (29) Google Scholar). However, the consequences of paralog switching are largely overlooked during the characterization of proteins, protein complexes, and protein interaction networks. Classically associated with transcriptional repression, the removal of histone lysine acetyl groups by the Sin3 histone deacetylase (HDAC) complexes represents a central mechanism whereby transcriptional status is regulated (4Adams G.E. Chandru A. Cowley S.M. Co-repressor, co-activator and general transcription factor: the many faces of the Sin3 histone deacetylase (HDAC) complex.Biochem. J. 2018; 475: 3921-3932Crossref PubMed Scopus (43) Google Scholar). Named for the scaffolding protein of the complexes, Sin3 complexes are well studied in Saccharomyces cerevisiae (5Carrozza M.J. Li B. Florens L. Suganuma T. Swanson S.K. Lee K.K. Shia W.J. Anderson S. Yates J. Washburn M.P. Workman J.L. Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription.Cell. 2005; 123: 581-592Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar, 6Carrozza M.J. Florens L. Swanson S.K. Shia W.J. Anderson S. Yates J. Washburn M.P. Workman J.L. Stable incorporation of sequence specific repressors Ash1 and Ume6 into the Rpd3L complex.Biochim. Biophys. Acta. 2005; 1731: 77-87Crossref PubMed Scopus (103) Google Scholar). However, in higher eukaryotes, the presence of additional components not found in lower eukaryotic forms of the Sin3 complexes likely increases the diversity of complex functions. Contributing to this expansion of components is the acquisition of paralogous genes encoding Sin3 proteins. The two Sin3 paralogs present within mammals, SIN3A and SIN3B, have undergone substantial divergence and maintain only 63% sequence similarity at the protein level in humans (supplemental Fig. S1). There is accumulating evidence that SIN3A and SIN3B are not functionally redundant within mammals. It has been shown that SIN3A can act as a suppressor of metastasis, whereas SIN3B can act as a pro-metastatic factor (7Lewis M.J. Liu J. Libby E.F. Lee M. Crawford N.P.S. Hurst D.R. SIN3A and SIN3B differentially regulate breast cancer metastasis.Oncotarget. 2016; 7: 78713-78725Crossref PubMed Scopus (21) Google Scholar). Additionally, genetic deletion of murine Sin3a results in early embryonic lethality whereas deletion of Sin3b induces late gestational lethality (8Cowley S.M. Iritani B.M. Mendrysa S.M. Xu T. Cheng P.F. Yada J. Liggitt H.D. Eisenman R.N. The mSin3A chromatin-modifying complex is essential for embryogenesis and T-cell development.Mol. Cell Biol. 2005; 25: 6990-7004Crossref PubMed Scopus (91) Google Scholar, 9David G. Grandinetti K.B. Finnerty P.M. Simpson N. Chu G.C. DePinho R.A. Specific requirement of the chromatin modifier mSin3B in cell cycle exit and cellular differentiation.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 4168-4172Crossref PubMed Scopus (84) Google Scholar). That SIN3A and SIN3B cannot compensate for the loss of one another provides evidence for paralog-specific functions within mammals and suggests that variations within the Sin3 complexes have functional consequences. Although the mechanisms responsible for divergent influences on development as well as cancer cell metastatic potential remain poorly understood, there is growing evidence that Sin3 paralog identity influences Sin3 complex composition (10Cantor D.J. David G. The potential of targeting Sin3B and its associated complexes for cancer therapy.Expert Opin. Ther. Targets. 2017; 21: 1051-1061Crossref PubMed Scopus (4) Google Scholar). Heterogeneity within a population of Sin3 complexes is not unprecedented as two distinct forms of the complex, known as Rpd3L (Sin3 large) and Rpd3S (Sin3 small) are found in S. cerevisiae. Whereas the ∼1.2 MDa Rpd3L complex localizes to gene promoter regions and influences transcription initiation, the ∼0.6 MDa Rpd3S complex is mostly found within actively transcribed genes and inhibits intragenic transcription (5Carrozza M.J. Li B. Florens L. Suganuma T. Swanson S.K. Lee K.K. Shia W.J. Anderson S. Yates J. Washburn M.P. Workman J.L. Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription.Cell. 2005; 123: 581-592Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar, 6Carrozza M.J. Florens L. Swanson S.K. Shia W.J. Anderson S. Yates J. Washburn M.P. Workman J.L. Stable incorporation of sequence specific repressors Ash1 and Ume6 into the Rpd3L complex.Biochim. Biophys. Acta. 2005; 1731: 77-87Crossref PubMed Scopus (103) Google Scholar). These two protein complexes share a common core of proteins, consisting of Rpd3, Sin3, and Ume1 (11Chen X.F. Kuryan B. Kitada T. Tran N. Li J.Y. Kurdistani S. Grunstein M. Li B. Carey M. The Rpd3 core complex Is a chromatin stabilization module.Curr. Biol. 2012; 22: 56-63Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), but are differentiated by their unique sets of subunits. Higher eukaryotes have genes encoding proteins that have homology with S. cerevisiae Sin3 complex components. Among proteins found in humans, HDAC1/HDAC2, SIN3A/SIN3B, and RBBP4/RBBP7 have homology to the S. cerevisiae core Sin3 complex components Rpd3, Sin3, and Ume1, respectively. In addition to possessing proteins that share homology with S. cerevisiae Sin3 core complex components, humans also have proteins that have homology to Rpd3L- and Rpd3S-specific components. SUDS3/BRMS1/BRMS1L, SAP30/SAP30L, and ING1/ING2 have homology to Rpd3L-specific components Sds3, Sap30, and Pho23, respectively (12Nourani A. Howe L. Pray-Grant M.G. Workman J.L. Grant P.A. Côté J. Opposite role of yeast ING family members in p53-dependent transcriptional activation.J. Biol. Chem. 2003; 278: 19171-19175Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 13Zhang Y. Sun Z.W. Iratni R. Erdjument-Bromage H. Tempst P. Hampsey M. Reinberg D. SAP30, a novel protein conserved between human and yeast, is a component of a histone deacetylase complex.Mol. Cell. 1998; 1: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 14Alland L. David G. Shen-Li H. Potes J. Muhle R. Lee H.C. Hou H. Chen K. DePinho R.A. Identification of mammalian Sds3 as an integral component of the Sin3/histone deacetylase corepressor complex.Mol. Cell Biol. 2002; 22: 2743-2750Crossref PubMed Scopus (90) Google Scholar). Components specifically found within Rpd3S, Rco1, and Eaf3, share homology with human PHF12 and MORF4L1, respectively (15Graveline R. Marcinkiewicz K. Choi S. Paquet M. Wurst W. Floss T. David G. The chromatin-associated Phf12 protein maintains nucleolar integrity and prevents premature cellular senescence.Mol. Cell Biol. 2017; 37: e00516-e00522Crossref Scopus (4) Google Scholar, 16Xu C. Cui G. Botuyan M.V. Mer G. Structural basis for the recognition of methylated histone H3K36 by the Eaf3 subunit of histone deacetylase complex Rpd3S.Structure. 2008; 16: 1740-1750Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Although SIN3A can clearly interact with Rpd3L component homologs, data supports the existence of SIN3B complexes that contain Rpd3S component homologs (17Varier R.A. Carrillo de Santa Pau E. van der Groep P. Lindeboom R.G.H. Matarese F. Mensinga A. Smits A.H. Edupuganti R.R. Baltissen M.P. Jansen P.W.T.C. ter Hoeve N. van Weely D.R. Poser I. van Diest P.J. Stunnenberg H.G. Vermeulen M. Recruitment of the mammalian histone-modifying EMSY complex to target genes is regulated by ZNF131.J. Biol. Chem. 2016; 291: 7313-7324Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 18Jelinic P. Pellegrino J. David G. A novel mammalian complex containing Sin3B mitigates histone acetylation and RNA polymerase II progression within transcribed loci.Mol. Cell Biol. 2011; 31: 54-62Crossref PubMed Scopus (57) Google Scholar). However, a combined analysis of Sin3 interaction partners to define modularity and identify mutual exclusivity within the network has not been performed. Using a combination of shotgun proteomics and chemical cross-linking MS (XL-MS), we profile the Sin3 interaction network. Our results outline the influence of paralog switching on complex construction. These findings define direct interactions within the Sin3 interaction network and identify divergent properties of the Sin3 paralogs. Expression vectors were prepared as described in Supplemental Methods. Stable cell lines were produced using Flp-In™-293 cells (Thermo Fisher Scientific, Waltham, MA), authenticated by STR profiling (FTA barcode: STR14169), and tested for mycoplasma using mycoplasma detection kits (American Type Culture Collection, Manassas, VA). The day before transfection, cells were plated at 50% confluency onto a 100 mm tissue culture plate containing DMEM and 10% FBS, then incubated at 37 °C in 5% CO2 overnight. The following day, cells were washed two times with Opti-MEM, then incubated with 8 ml Opti-MEM containing GlutaMAX supplement (Thermo Fisher Scientific). Plasmid DNA (4 µg total; 3.6 µg pOG44 + 0.4 µg DNA of interest) was added to 800 µL of Opti-MEM with GlutaMAX supplement along with 16 µL FuGENE® HD Transfection Reagent (Promega Corporation, Madison, WI), incubated for 15-30 min, then added dropwise to the prepared plate. One ml of FBS (Peak Serum, Inc, Wellington CO) was added the next morning. On day three of incubation, cells were split 1:10 and placed into selection media (DMEM/10% FBS/100 µg/ml Hygromycin B). Media was changed every 3 days for a total of three media changes. After 2 weeks, colonies were visible and picked for screening. Flp-In™-293 cell lines stably expressing HaloTag-SAP30, HaloTag-SAP30L, and HaloTag-SUDS3 were previously described (19Banks C.A.S. Thornton J.L. Eubanks C.G. Adams M.K. Miah S. Boanca G. Liu X. Katt M.L. Parmely T.J. Florens L. Washburn M.P. A structured workflow for mapping human Sin3 histone deacetylase complex interactions using Halo-MudPIT affinity-purification mass spectrometry.Mol. Cell. Proteomics. 2018; 17: 1432-1447Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 20Banks C.A.S. Zhang Y. Miah S. Hao Y. Adams M.K. Wen Z. Thornton J.L. Florens L. Washburn M.P. Integrative modeling of a Sin3/HDAC complex sub-structure.Cell Rep. 2020; 31: 107516Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Flp-In™-293 cell lines stably expressing HaloTag® fusion proteins were seeded at 40% confluency in 35 mm MatTek glass bottom dishes (MatTek Corporation, Ashland, MA) containing DMEM supplemented with penicillin-streptomycin solution, GlutaMAX supplement, and FBS to a final concentration of 10%. Cell media was supplemented with HaloTag® TMRDirect™ Ligand (Promega Corporation) to a final concentration of 20 nm 16–24 h after seeding. Cells were then cultured for an additional 16–24 h. Hoechst 33258 solution (Sigma Aldrich Corporation, St. Louis, MO) was added to culture dishes 80 min before imaging. Media conditions for transient transfection of 293T cells (American Type Culture Collection) with plasmid DNA were as stated for the imaging of the stable expression cell lines. Cells continued to grow 16–24 h after seeding at 40% confluency in 35 mm MatTek glass bottom dishes before transfection. Cells were transfected with Opti-MEM media containing 2.5 µg of plasmid, 5 µL LipofectAMINE™ LTX Reagent (Thermo Fisher Scientific), and 2.5 µL PLUS™ Reagent (Thermo Fisher Scientific). Cell media was supplemented with HaloTag® TMRDirect™ Ligand to a final concentration of 20 nm 16-24 h after transfection. After an additional 16–24 h of culture at 37 °C and 5% CO2, Hoechst 33258 solution was added to the culture dishes and incubation was continued for 1 h. Cells were washed and imaged in Opti-MEM media. Images were captured on a PerkinElmer Life Sciences UltraVIEW VoX spinning disk microscope (PerkinElmer, Inc., Waltham, MA), Axiovert 200M base (Carl Zeiss AG, Oberkochen, Germany), or an inverted LSM-700 point scanning confocal microscope controlled by Zeiss Zen software (Carl Zeiss AG). A 40× plan-apochromat (NA 1.4) oil objective was used to acquire images when operating the LSM-700 microscope. Detection wavelength ranges were 300–483 nm for Hoechst and 570–800 nm for HaloTag® TMRDirect™ Ligand. SP 490 and LP 490 filter sets were employed when imaging Hoechst and HaloTag® TMRDirect™ Ligand, respectively, on the LSM-700 microscope. Cells were lysed and recombinant proteins were isolated using Magne® HaloTag® Beads (Promega Corporation) as previously described (19Banks C.A.S. Thornton J.L. Eubanks C.G. Adams M.K. Miah S. Boanca G. Liu X. Katt M.L. Parmely T.J. Florens L. Washburn M.P. A structured workflow for mapping human Sin3 histone deacetylase complex interactions using Halo-MudPIT affinity-purification mass spectrometry.Mol. Cell. Proteomics. 2018; 17: 1432-1447Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Briefly, 2 confluent 850 cm2 culture vessels of Flp-In™-293 cells stably expressing a transgene were lysed and incubated with HL-SAN nuclease (ArcticZymes, Tromsø, Norway) at a final concentration of 2 U/ml for 2 h at 4 °C before protein enrichment. Recombinant protein was isolated via incubation with Magne® HaloTag® Beads and eluted with AcTEV™ Protease (Thermo Fisher Scientific). Affinity purified (AP) proteins were TCA precipitated, digested with Endoproteinase Lys-C or Recombinant Endoproteinase LysC (Promega Corporation), then digested further with Sequencing Grade Trypsin (Promega Corporation). Peptides were loaded onto triphasic MudPIT microcapillary columns as previously described (21Swanson S.K. Florens L. Washburn M.P. Generation and analysis of multidimensional protein identification technology datasets.Methods Mol. Biol. 2009; 492: 1-20Crossref PubMed Scopus (15) Google Scholar). Columns were placed in-line with an 1100 Series HPLC system (Agilent Technologies, Inc., Santa Clara, CA) coupled to a linear ion trap mass spectrometer (Thermo Fisher Scientific) and peptides were resolved using 10-step MudPIT chromatography as previously described (22Banks C.A.S. Kong S.E. Washburn M.P. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.Protein Expr. Purif. 2012; 86: 105-119Crossref PubMed Scopus (18) Google Scholar). For each replicate, 3 confluent 850 cm2 culture vessels of Flp-In™-293 cells stably expressing SIN3A-HaloTag or SIN3B_2-HaloTag were harvested. Protein was enriched using Magne® HaloTag® Beads and cross-linked with disuccinimidyl sulfoxide (DSSO) as previously described (20Banks C.A.S. Zhang Y. Miah S. Hao Y. Adams M.K. Wen Z. Thornton J.L. Florens L. Washburn M.P. Integrative modeling of a Sin3/HDAC complex sub-structure.Cell Rep. 2020; 31: 107516Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Briefly, DSSO (Cayman Chemical Company, Ann Arbor, MI) was added to samples to a final concentration of 5 mm while protein was immobilized on beads. Samples were incubated at room temperature for 40 min. Reactions were quenched with the addition of NH4HCO3 to a final concentration of 50 mm and samples were incubated an additional 15 min at room temperature. Recombinant proteins were eluted with AcTEV™ Protease at room temperature overnight. Proteins were TCA precipitated and digested as previously described (19Banks C.A.S. Thornton J.L. Eubanks C.G. Adams M.K. Miah S. Boanca G. Liu X. Katt M.L. Parmely T.J. Florens L. Washburn M.P. A structured workflow for mapping human Sin3 histone deacetylase complex interactions using Halo-MudPIT affinity-purification mass spectrometry.Mol. Cell. Proteomics. 2018; 17: 1432-1447Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Peptides were resolved on a 50 μm inner diameter microcapillary column containing 15 cm of 1.9 μm C18 resin (ESI Source Solutions, Woburn, MA). Peptides were identified with an Orbitrap Fusion™ Lumos™ mass spectrometer (Thermo Fisher Scientific) and data were acquired as previously described (20Banks C.A.S. Zhang Y. Miah S. Hao Y. Adams M.K. Wen Z. Thornton J.L. Florens L. Washburn M.P. Integrative modeling of a Sin3/HDAC complex sub-structure.Cell Rep. 2020; 31: 107516Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). To characterize protein interaction networks, a minimum of three biological replicates were acquired for each affinity purification MS (APMS) analysis. As a control, Flp-In™-293 cells expressing no transgenes were also analyzed. Acquired .RAW files were converted to .ms2 files using RAWDistiller (23Zhang Y. Wen Z. Washburn M.P. Florens L. Improving proteomics mass accuracy by dynamic offline lock mass.Anal. Chem. 2011; 83: 9344-9351Crossref PubMed Scopus (34) Google Scholar). ProLuCID v1.3.5 (24Xu T. Park S.K. Venable J.D. Wohlschlegel J.A. Diedrich J.K. Cociorva D. Lu B. Liao L. Hewel J. Han X. Wong C.C.L. Fonslow B. Delahunty C. Gao Y. Shah H. Yates J.R. ProLuCID: An improved SEQUEST-like algorithm with enhanced sensitivity and specificity.J. Proteomics. 2015; 129: 16-24Crossref PubMed Scopus (278) Google Scholar) was used to match spectra against a database (Genome Reference Consortium Human Build 38 patch release 13) containing 44,519 unique proteins, 426 of which were contaminant proteins. The database was shuffled for false discovery rate (FDR) estimation, producing a final database that contained 89,038 total sequences. The database was searched for fully tryptic peptides, allowing for a maximum of 3 internal cleavage sites and a minimum peptide length of 7 amino acids. Database searches were performed with a static modification of +57 Da for cysteine, a dynamic modification of +16 Da for methionine, and a mass tolerance of 800 ppm for precursor and fragment ions. DTASelect and Contrast (25Tabb D.L. McDonald W.H. Yates J.R. DTASelect and Contrast: tools for sssembling and comparing protein identifications from shotgun proteomics.J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1137) Google Scholar) were used to filter results and NSAF v7 (26Zhang Y. Wen Z. Washburn M.P. Florens L. Refinements to label free proteome quantitation: how to deal with peptides shared by multiple proteins.Anal. Chem. 2010; 82: 2272-2281Crossref PubMed Scopus (294) Google Scholar) was used to calculate label-free quantitative dNSAF values and generate final reports (supplemental Tables S2A–2B and S4A–4B). The spectral FDR mean ± S.D. for the 70 MudPIT runs was 0.337% ± 0.138%, the mean ± S.D. peptide FDR was 0.254% ± 0.122%, and the mean ± S.D. protein FDR was 0.917% ± 0.405%. For the analysis of SIN3A and SIN3B isoforms, the spectral FDR mean ± S.D. for the 20 MudPIT runs was 0.282% ± 0.133%, the mean ± S.D. peptide FDR was 0.272% ± 0.097%, and the mean ± S.D. protein FDR was 0.874% ± 0.329%. A DTASelect filter also established a minimum peptide length of 7 amino acids, and proteins that were subsets of others were removed using the parsimony option in Contrast. To identify cross-linked peptides, 5 technical replicates of SIN3B_2-HaloTag and 3 technical replicates for SIN3A-HaloTag were analyzed. Peptides were analyzed with an Orbitrap Fusion™ Lumos™ and data acquisition was performed as previously described (20Banks C.A.S. Zhang Y. Miah S. Hao Y. Adams M.K. Wen Z. Thornton J.L. Florens L. Washburn M.P. Integrative modeling of a Sin3/HDAC complex sub-structure.Cell Rep. 2020; 31: 107516Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Briefly, cross-linked peptides were identified using Proteome Discoverer v2.4 and the XlinkX module (27Liu F. Lössl P. Scheltema R. Viner R. Heck A.J.R. Optimized fragmentation schemes and data analysis strategies for proteome-wide cross-link identification.Nat. Commun. 2017; 8: 15473Crossref PubMed Scopus (148) Google Scholar). Acquired .RAW files were searched against a human proteome database (Genome Reference Consortium Human Build 38 patch release 13) containing 44,519 unique protein sequences, 426 of which were contaminant proteins. For XlinkX searches, the database was searched for fully tryptic peptides, allowing for a maximum of 2 missed cleavages and a minimum peptide length of 5 amino acids. Searches were performed with a static modification of +57.021 Da for cysteine and a dynamic modification of +15.995 Da for methionine. Precursor mass tolerance, FTMS fragment mass tolerance, and ITMS Fragment tolerance, were set to 10 ppm, 20 ppm, and 0.5 Da, respectively. Xlink Validator FDR threshold was set to 0.01. For Sequest HT searches, the database was searched for fully tryptic peptides, allowing for a maximum of 2 missed cleavages and a minimum peptide length of 6 amino acids. Searches were performed with a static modification of +57.021 Da for cysteine, a dynamic modification of +15.995 Da for methionine, a dynamic modification of +176.014 Da for lysine (water-quenched DSSO monoadduct), and a dynamic modification of + 279.08 Da for lysine (Tris-quenched DSSO monoadduct). Precursor mass tolerance and fragment mass tolerance were set to 10 ppm and 0.6 Da, respectively. Percolator target FDR (Strict) was set to 0.01. Identified cross-link spectrum matches are reported in supplemental Table S3. Data that has been previously described was included in our analyses and is summarized in supplemental Table S1. All MS data has been deposited into the MassIVE repository (http://massive.ucsd.edu). Data set identifiers are supplied in supplemental Table S1. To identify high-confidence interaction partners, QSPEC v1.3.5 (28Choi H. Kim S. Fermin D. Tsou C.C. Nesvizhskii A.I. QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics.J. Proteomics. 2015; 129: 121-126Crossref PubMed Scopus (37) Google Scholar) was used to calculate Z-statistic and log2 fold change values. Prey proteins that were not present in at least half of at least one bait protein purification (supplemental Table S2C, supplemental Table S4C) were excluded before QSPEC scoring (supplemental Tables S2D, S4D). QSPEC analysis was performed with a burn in value of 2000 and 10,000 iterations. To identify enriched proteins over negative AP controls, Z-statistic values of ≥ 3 and log2 fold change values ≥ 2 were selected as filter values. HDAC activity assays of transiently produced proteins were performed as described (29Adams M.K. Banks C.A.S. Miah S. Killer M. Washburn M.P. Purification and enzymatic assay of class I histone deacetylase enzymes.Methods Enzymol. 2019; 626: 23-40Crossref PubMed Scopus (1) Google Scholar). Briefly, ∼1 × 107 293T cells were plated in 150 mm dishes and cultured in 25 ml DMEM + 10% fetal bovine serum + 1 × GlutaMAX Supplement. 24 h after seeding, cells were transfected with 7.5 µg plasmid DNA, 7.5 µL Plus Reagent, and 50 µL LipofectAMINE LTX diluted in 6.6 ml OptiMEM. Cells were harvested after an additional 48 h of culture. Two mg of whole cell extract were added to 100 µL of washed Magne® HaloTag® Beads slurry and incubated at 4 °C for 2 h. Beads were washed 4 times with 1 ml cold TBS pH 7.4 + 0.05% Igepal CA-630 (Sigma Aldrich Corporation). Protein was eluted with 5 units AcTEV™ Protease (Thermo Fisher Scientific) in 100 µL of 50 mm Tris-HCl pH 8.0, 0.5 mm EDTA, 1 mm DTT for 16 h at 4 °C. Ten µL of the 100 µL purified protein was diluted with 32.5 µL TBS (25 mm Tris, 150 mm NaCl, 2 mm KCl, pH 7.4). Samples were supplemented with 2.5 µL of DMSO or 200 μm SAHA (Cayman Chemical Company) resuspended in DMSO for a final concentration of 10 μm SAHA. 5 µL of 1 mm Boc-Lys(Ac)-AMC (APExBIO Technology LLC, Houston, TX) was added to each reaction to a final concentration of 100 μm. The reactions, at a final volume of 50 µL, were performed at 37 °C for 1 h. Reactions were quenched with 2.5 µl of 200 μm SAHA and incubated at 37 °C for 5 min. Six µL of 50 mg/ml trypsin from porcine pancreas (Sigma Aldrich) was added to the reactions for a final concentration of 5 mg/ml. Reactions were incubated an additional 1 h at 37 °C. Fluorescence was measured with a SPECTRAmax GEMINI XS (Molecular Devices, San Jose, CA) using an excitation wavelength of 355, an emission wavelength of 460 nm, and a cutoff wavelength of 455 nm. Proteins were separated on polyacrylamide gels and transferred to Amersham Pharmacia Biotech™ Hybond™ 0.2 μm PVDF membranes (GE Healthcare Life Science, Marlborough, MA). Blots were probed with a 1:3000 dilution of rabbit-anti-SIN3A (#ab3479 Abcam, Cambridge, MA) or a 1:5000 dilution of mouse-anti-SIN3B (sc-13145x Santa Cruz Biotechnology, Dallas, TX). Membranes were then probed with a 1:10,000 dilution of IRDye® 680LT Goat-anti-Mouse (LI-COR, Lincoln, NE), IRDye® 800CW Goat-anti-Mouse (LI-COR), or a 1:10,000 dilution of IRDye® 800CW Goat-anti-Rabbit (LI-COR). Images were acquired with an Odyssey® CLx (LI-COR). A pairwise alignment of SIN3A and SIN3B_2 (supplemental Fig. S1) was generated using the EMBOSS-Needle algorithm (30Madeira F. Park Y.M. Lee J. Buso N. Gur T. Madhusoodanan N. Basutkar P. Tivey A.R.N. Potter S.C. Finn R.D. Lopez R. The EMBL-EBI search and sequence analysis tools APIs in 2019.Nucleic Acids Res. 2019; 47: W636-W641Crossref PubMed Scopus (2580) Google Scholar). An alignment of SIN3A (NP_001138829.1), SIN3B_1 (NP_056075.1), SIN3B_2 (NP_001284524.1), and SIN3B_3 (NP_001284526.1) in supplemental Fig. S2 was generated using ETE v3 (31Huerta-Cepas J. Serra F. Bork P. ETE 3: reconstruction, analysis, and visualization of phylogenomic data.Mol. Biol. Evol. 2016; 33: 1635-1638Crossref PubMed Scopus (880) Google Scholar) and ClustalO (32Sievers F. Wilm A. Dineen D. Gibson T.J. Karplus K. Li W. Lopez R. McWilliam H. Remmert M. Soding J. Thompson J.D. Higgins D.G. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.Mol. Syst. Biol. 2011; 7: 539Crossref PubMed Scopus (9093) Google Scholar). As an initial measure to characterize properties of human Sin3 complexes, we stably expressed SIN3A (NM_001145357.2, NP_001138829.1) and SIN3B isoform 2 (transcript NM_001297595.1, NP_001284524.1) as fusions with a
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