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• W2116007004 abstract "The chromatin-associated proteome (chromatome) regulates cellular gene expression by restricting access of transcriptional machinery to template DNA, and dynamic re-modeling of chromatin structure is required to regulate critical cell functions including growth and replication, DNA repair and recombination, and oncogenic transformation in progression to cancer. Central to the control of these processes is efficient regulation of the host cell cycle, which is maintained by rapid changes in chromatin conformation during normal cycle progression. A global overview of chromatin protein organization is therefore essential to fully understand cell cycle regulation, but the influence of the chromatome and chromatin binding topology on host cell cycle progression remains poorly defined. Here we used partial MNase digestion together with iTRAQ-based high-throughput quantitative proteomics to quantify chromatin-associated proteins during interphase progression. We identified a total of 481 proteins with high confidence that were involved in chromatin-dependent events including transcriptional regulation, chromatin re-organization, and DNA replication and repair, whereas the quantitative data revealed the temporal interactions of these proteins with chromatin during interphase progression. When combined with biochemical and functional assays, these data revealed a strikingly dynamic association of protein HP1BP3 with the chromatin complex during different stages of interphase, and uncovered a novel regulatory role for this molecule in transcriptional regulation. We report that HP1BP3 protein maintains heterochromatin integrity during G1–S progression and regulates the duration of G1 phase to critically influence cell proliferative capacity. The chromatin-associated proteome (chromatome) regulates cellular gene expression by restricting access of transcriptional machinery to template DNA, and dynamic re-modeling of chromatin structure is required to regulate critical cell functions including growth and replication, DNA repair and recombination, and oncogenic transformation in progression to cancer. Central to the control of these processes is efficient regulation of the host cell cycle, which is maintained by rapid changes in chromatin conformation during normal cycle progression. A global overview of chromatin protein organization is therefore essential to fully understand cell cycle regulation, but the influence of the chromatome and chromatin binding topology on host cell cycle progression remains poorly defined. Here we used partial MNase digestion together with iTRAQ-based high-throughput quantitative proteomics to quantify chromatin-associated proteins during interphase progression. We identified a total of 481 proteins with high confidence that were involved in chromatin-dependent events including transcriptional regulation, chromatin re-organization, and DNA replication and repair, whereas the quantitative data revealed the temporal interactions of these proteins with chromatin during interphase progression. When combined with biochemical and functional assays, these data revealed a strikingly dynamic association of protein HP1BP3 with the chromatin complex during different stages of interphase, and uncovered a novel regulatory role for this molecule in transcriptional regulation. We report that HP1BP3 protein maintains heterochromatin integrity during G1–S progression and regulates the duration of G1 phase to critically influence cell proliferative capacity. The eukaryotic cell cycle consists of two major consecutive events: duplication of the genome by DNA synthesis and distribution of the duplicated genome into daughter cells via mitosis. Between mitotic cycles, the cells enter an intermediate state known as interphase, which consists of a DNA synthesis step (S phase) flanked by periods of relative inactivity (gaps G1 and G2), during which the cells prepare to undergo mitosis. Numerous chromatin-dependent genetic events occur during interphase, those are required to regulate cell cycle progression, and successful completion of these events requires transient access to the DNA template. However, the influence of these dynamic changes in chromatin protein conformation on cell cycle progression remains poorly understood. Rapid access to the DNA template during interphase is achieved by maintaining chromatin in a highly dynamic state (1.Pombo A. Advances in imaging the interphase nucleus using thin cryosections.Histochem.Cell Biol. 2007; 128: 97-104Crossref PubMed Scopus (12) Google Scholar, 2.Bailis J.M. Forsburg S.L. It's all in the timing: linking S phase to chromatin structure and chromosome dynamics.Cell cycle. 2003; 2: 303-306Crossref PubMed Scopus (13) Google Scholar), and is required for the efficient transmission of genetic and epigenetic information into daughter cells via careful regulation of the host cell cycle (3.Probst A.V. Dunleavy E. Almouzni G. Epigenetic inheritance during the cell cycle.Nat. Rev. Mol. Cell Biol. 2009; 10: 192-206Crossref PubMed Scopus (580) Google Scholar, 4.Liu Q. Gong Z. The coupling of epigenome replication with DNA replication.Curr. Opin. Plant Biol. 2011; 14: 187-194Crossref PubMed Scopus (30) Google Scholar). Epigenetic modifications are critical mediators of host cell differentiation, which plays an important role in developmental biology (5.Gehring M. Huh J.H. Hsieh T.F. Penterman J. Choi Y. Harada J.J. Goldberg R.B. Fischer R.L. DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.Cell. 2006; 124: 495-506Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar) as well as in many of the un-programmed cellular events that drive pathological changes in cancer (6.Zhang C. Su Z.Y. Khor T.O. Shu L. Kong A.N. Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation.Biochem. Pharmacol. 2013; 85: 1398-1404Crossref PubMed Scopus (159) Google Scholar, 7.Ng M.H. Wong I.H. Lo K.W. DNA methylation changes and multiple myeloma.Leuk. Lymphoma. 1999; 34: 463-472Crossref PubMed Scopus (22) Google Scholar, 8.Halley-Stott R.P. Gurdon J.B. Epigenetic memory in the context of nuclear reprogramming and cancer.Brief Funct. Genomics. 2013; 12: 164-173Crossref PubMed Scopus (40) Google Scholar). Transmission of epigenetic information from mother cell to daughter cells (epigenetic inheritance) is mediated via DNA and histone modifications, histone variants, non-histone chromatin proteins, nuclear RNA, and changes in higher-order chromatin structure (9.Martin C. Zhang Y. Mechanisms of epigenetic inheritance.Curr. Opin. Cell Biol. 2007; 19: 266-272Crossref PubMed Scopus (180) Google Scholar, 10.Huang C. Zhang Z. Xu M. Li Y. Li Z. Ma Y. Cai T. Zhu B. H3.3-h4 tetramer splitting events feature cell-type specific enhancers.PLoS Genet. 2013; 9: e1003558Crossref PubMed Scopus (51) Google Scholar, 11.Wysocka J. Swigut T. Milne T.A. Dou Y. Zhang X. Burlingame A.L. Roeder R.G. Brivanlou A.H. Allis C.D. WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development.Cell. 2005; 121: 859-872Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 12.Ahmad K. Henikoff S. The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly.Mol. Cell. 2002; 9: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar, 13.Vermeulen M. Eberl H.C. Matarese F. Marks H. Denissov S. Butter F. Lee K.K. Olsen J.V. Hyman A.A. Stunnenberg H.G. Mann M. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.Cell. 2010; 142: 967-980Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). During interphase, chromatin is structured in two distinct forms; euchromatin (loosely compacted active regions) and heterochromatin (highly compacted silent regions) that are continuously interchanged according to requirements for access to the DNA template. Euchromatin is essential for gene transcription and normal cell function between replicative cycles (14.Yong K.J. Milenic D.E. Baidoo K.E. Brechbiel M.W. (212)Pb-radioimmunotherapy induces G(2) cell-cycle arrest and delays DNA damage repair in tumor xenografts in a model for disseminated intraperitoneal disease.Mol. Cancer Ther. 2012; 11: 639-648Crossref PubMed Scopus (38) Google Scholar, 15.Sinha M. Peterson C.L. Chromatin dynamics during repair of chromosomal DNA double-strand breaks.Epigenomics. 2009; 1: 371-385Crossref PubMed Google Scholar, 16.Mosesso P. Palitti F. Pepe G. Pinero J. Bellacima R. Ahnstrom G. Natarajan A.T. Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes.Mutat. Res. 2010; 701: 86-91Crossref PubMed Scopus (17) Google Scholar, 17.Audit B. Zaghloul L. Vaillant C. Chevereau G. d'Aubenton-Carafa Y. Thermes C. Arneodo A. Open chromatin encoded in DNA sequence is the signature of ‘master’ replication origins in human cells.Nucleic Acids Res. 2009; 37: 6064-6075Crossref PubMed Scopus (48) Google Scholar), whereas heterochromatin is vital to prevent aberrant DNA access that could disrupt the cell cycle and impede genome transfer into progeny cells (18.Mora-Bermudez F. Ellenberg J. Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy.Methods. 2007; 41: 158-167Crossref PubMed Scopus (45) Google Scholar, 19.Hartl P. Gottesfeld J. Forbes D.J. Mitotic repression of transcription in vitro.J. Cell Biol. 1993; 120: 613-624Crossref PubMed Scopus (82) Google Scholar, 20.Zhang X. Yu Q. Olsen L. Bi X. Functions of protosilencers in the formation and maintenance of heterochromatin in Saccharomyces cerevisiae.PloS ONE. 2012; 7: e37092Crossref PubMed Scopus (4) Google Scholar, 21.Aird K.M. Zhang R. Detection of senescence-associated heterochromatin foci (SAHF).Methods Mol. Biol. 2013; 965: 185-196Crossref PubMed Scopus (85) Google Scholar, 22.Tu Z. Aird K. Zhang R. Chromatin remodeling, BRCA1, SAHF, and cellular senescence.Cell cycle. 2013; 12: 1653-1654Crossref PubMed Scopus (10) Google Scholar). Accordingly, imbalance in the chromatin structure-dependent regulatory system de-regulates the cell cycle and promotes carcinogenesis (23.Jespersen C. Soragni E. James Chou C. Arora P.S. Dervan P.B. Gottesfeld J.M. Chromatin structure determines accessibility of a hairpin polyamide-chlorambucil conjugate at histone H4 genes in pancreatic cancer cells.Bioorg. Med. Chem. Let. 2012; 22: 4068-4071Crossref PubMed Scopus (15) Google Scholar, 24.Di Paola D. Rampakakis E. Chan M.K. Zannis-Hadjopoulos M. Differential chromatin structure encompassing replication origins in transformed and normal cells.Genes Cancer. 2012; 3: 152-176Crossref PubMed Scopus (4) Google Scholar). Chromatin conformation is regulated by protein-DNA and protein-protein interactions that are influenced by structural modification of the component histones (25.Eberl H.C. Spruijt C.G. Kelstrup C.D. Vermeulen M. Mann M. A map of general and specialized chromatin readers in mouse tissues generated by label-free interaction proteomics.Mol. Cell. 2013; 49: 368-378Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and DNA sequences (26.Dulac C. Brain function and chromatin plasticity.Nature. 2010; 465: 728-735Crossref PubMed Scopus (201) Google Scholar, 27.Chadwick B.P. Willard H.F. Cell cycle-dependent localization of macroH2A in chromatin of the inactive X chromosome.J. Cell Biol. 2002; 157: 1113-1123Crossref PubMed Scopus (97) Google Scholar, 28.Izuta H. Ikeno M. Suzuki N. Tomonaga T. Nozaki N. Obuse C. Kisu Y. Goshima N. Nomura F. Nomura N. Yoda K. Comprehensive analysis of the ICEN (Interphase Centromere Complex) components enriched in the CENP-A chromatin of human cells.Genes Cells. 2006; 11: 673-684Crossref PubMed Scopus (150) Google Scholar). However, the contribution of non-histone proteins to control chromatin biology is poorly understood. Recent data indicate that non-histone proteins including RNA polymerase III, topoisomerase II α, heterochromatin protein 1, and high-mobility group nucleosome-binding proteins (HMGNs) critically influence chromatin structural dynamics (29.Cherukuri S. Hock R. Ueda T. Catez F. Rochman M. Bustin M. Cell cycle-dependent binding of HMGN proteins to chromatin.Mol. Biol. Cell. 2008; 19: 1816-1824Crossref PubMed Scopus (31) Google Scholar, 30.Agostinho M. Rino J. Braga J. Ferreira F. Steffensen S. Ferreira J. Human topoisomerase IIalpha: targeting to subchromosomal sites of activity during interphase and mitosis.Mol. Biol. Cell. 2004; 15: 2388-2400Crossref PubMed Google Scholar, 31.Iwasaki O. Noma K. Global genome organization mediated by RNA polymerase III-transcribed genes in fission yeast.Gene. 2012; 493: 195-200Crossref PubMed Scopus (17) Google Scholar, 32.Minc E. Courvalin J.C. Buendia B. HP1gamma associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes.Cytogenet. Cell Genet. 2000; 90: 279-284Crossref PubMed Scopus (127) Google Scholar, 33.Hayakawa T. Haraguchi T. Masumoto H. Hiraoka Y. Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase.J. Cell Sci. 2003; 116: 3327-3338Crossref PubMed Scopus (113) Google Scholar), but current data are insufficient to fully establish the role played by chromatin-associated proteins in the control of the host cell cycle. Data describing the chromatin-associated proteome (chromatome) during cell division have been reported for C. Elegans (34.Chu D.S. Liu H. Nix P. Wu T.F. Ralston E.J. Yates 3rd, J.R. Meyer B.J. Sperm chromatin proteomics identifies evolutionarily conserved fertility factors.Nature. 2006; 443: 101-105Crossref PubMed Scopus (149) Google Scholar), yeast (35.Kubota T. Stead D.A. Hiraga S. ten Have S. Donaldson A.D. Quantitative proteomic analysis of yeast DNA replication proteins.Methods. 2012; 57: 196-202Crossref PubMed Scopus (15) Google Scholar), and human cells (36.Uchiyama S. Kobayashi S. Takata H. Ishihara T. Hori N. Higashi T. Hayashihara K. Sone T. Higo D. Nirasawa T. Takao T. Matsunaga S. Fukui K. Proteome analysis of human metaphase chromosomes.J. Biol. Chem. 2005; 280: 16994-17004Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), whereas other investigators have carried out proteomic analyses of the DNA damage response complex (37.Choi S. Srivas R. Fu K.Y. Hood B.L. Dost B. Gibson G.A. Watkins S.C. Van Houten B. Bandeira N. Conrads T.P. Ideker T. Bakkenist C.J. Quantitative proteomics reveal ATM kinase-dependent exchange in DNA damage response complexes.J. Proteome Res. 2012; 11: 4983-4991Crossref PubMed Scopus (24) Google Scholar) and post-meiotic genome in mice (38.Rousseaux S. Khochbin S. Combined proteomic and in silico approaches to decipher post-meiotic male genome reprogramming in mice.Syst. Biol. Reprod. Med. 2012; 58: 191-196Crossref PubMed Scopus (13) Google Scholar). However, detailed profiling of chromatome dynamics during cell cycle progression has not previously been conducted. In the current report, we extracted chromatin from cells synchronized in different stages of interphase and used partial MNase digestion together with an iTRAQ-based quantitative proteomic approach to delineate chromatome components and topology during normal cell cycle progression. We provide valuable new data on the dynamics of chromatin re-modeling during cell cycle progression that may help to clarify how disruption of chromatome configuration promotes pathological changes in diseases such as cancer. We further report that novel chromatin protein HP1BP3 maintains heterochromatin integrity during G1–S progression in order to regulate the duration of G1 phase and thereby control cell proliferation. Our approach of constructing a global picture of chromatin-associated protein dynamics during interphase progression may allow future studies to better determine the influence of chromatin biology on other cellular processes and disease pathogenesis. All reagents were purchased from Sigma-Aldrich unless otherwise indicated (Sigma-Aldrich, WI). Antibodies against α-tubulin (sc-5286), GAPDH (sc-32233), Ku-70 (sc-17789), and Ku-80 (sc-5280) were from Santa Cruz Biotechnology, Santa Cruz, CA. Anti-histone H2A (ab13923), histone H4 (ab10158), and HP1BP3 (ab98894) antibodies were purchased from Abcam, UK. The anti-actin antibody (MAB1501) was purchased from Millipore, USA. Mono-methyl histone H3 (Lys4) (D1A9) XP (5326P) and Di-methyl histone H3 (Lys4) (C64G9) Rabbit mAb (#9725P) were procured from Cell Signaling Technology, Danvers, MA. The 293F cells (Invitrogen, Carlsbad, CA) were cultured and maintained in Free-Style 293 Expression medium (GIBCOTM, Invitrogen, USA) according to the protocol provided. 293F cells were easily cultured in bulk quantity in a single flask, which could reduce experimental variation. Viable cells (3 × 105/ml) were seeded into sterile 125 ml vented PETG flasks (NALGENE) and incubated at 37 °C in a shaking incubator with 5% CO2 at 120rpm. Cells were synchronized in G1/S phase by double thymidine treatment (exposure to 2.5 mm thymidine for 18h, release from block for 8h, then a second exposure to 2.5 mm thymidine for 18 h). G2/M phase synchronization was achieved by treating the cells with 2.5 mm thymidine for 24 h and then 3 h release prior to addition of 200 ng/ml nocodazole for 16h. G0/G1 synchronization was achieved by releasing cells for 6h after the G2/M blocking step. Synchronization experiment was performed in a triplicate. Cells form all three replicates pooled together and used for the subsequent chromatin extraction. Viable cells (2 × 106) were washed with ice cold PBS and fixed with −20 °C ethanol. The fixed cells were then stained with propidium iodide (0.5 mg/ml) for 15min at 37 °C. Cellular DNA content was measured by flow cytometer (BD FACSCalibur™, BD Biosciences, USA) and analyzed using CELLQUEST software (BD FACSCalibur™, BD Biosciences, USA). Chromatin isolation was performed according to the method published by Dutta et al. (39.Dutta B. Adav S.S. Koh C.G. Lim S.K. Meshorer E. Sze S.K. Elucidating the temporal dynamics of chromatin-associated protein release upon DNA digestion by quantitative proteomic approach.J. Proteomics. 2012; 75: 5493-5506Crossref PubMed Scopus (14) Google Scholar) with minor modification. Briefly, 7 × 107 cells were suspended in nuclei extraction buffer A (10 mm HEPES pH 7.5, 10 mm KCl, 1 mm MgCl2, 0.34 m sucrose, 0.1% triton X-100, 1 mm DTT, and protease inhibitor mixture from Roche Diagnostics, Mannheim, Germany) and were kept on ice for 30min prior to pestle homogenization. The homogenate was centrifuged at 2000 × g for 3min at 4 °C and the supernatant discarded. The cell pellet was then re-suspended in nuclei extraction buffer B (10 mm HEPES pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 0.34 m sucrose, 0.1% Nonidet P-40, 1 mm DTT, and protease inhibitor mixture) and then pestle homogenized for a second time. The homogenate was loaded onto a 2.1 m sucrose gradient (10 mm HEPES pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 2.1 m sucrose, 1 mm DTT, and protease inhibitor mixture) prior to ultra-centrifugation at 150,000 × g for 3h at 4 °C using a SW41 rotor with the Optima™ l-100XP Ultra apparatus (Beckman Coulter, USA). After ultracentrifugation, the supernatant was removed and the chromatin pellet collected from bottom of the tube. The chromatin pellet was washed with wash buffer (10 mm HEPES pH 7.5, 1 mm DTT, and protease inhibitor mixture) and collected by centrifugation at 20,000 × g for 45min at 4 °C. The purified chromatin pellet was then suspended in 50 μl MNase digestion buffer (10 mm HEPES pH 7.5, 2.5 mm CaCl2, and 2.5 mm MgCl2) before 20U MNase was added and the digestion mixture incubated for 60min at 37 °C in a water bath with occasional vortexing. Addition of 1 mm EDTA was used to stop the digestion before the supernatant fraction (fraction S) was collected by centrifugation at 20,000 × g at 4 °C for 30min. The remaining undigested pellet (fraction P) was dissolved in 2% SDS solution. The protein contents of the supernatant and pellet fractions were subsequently measured using 2-D Quant Kits (Amersham Biosciences, USA). A total mass of 75 μg protein from each sample was loaded into 12.5% SDS-PAGE gels for electrophoresis at 80V for 15min. Each sample lane was cut into small pieces and washed with 25 mm triethylammonium bicarbonate (TEAB) in 75% ACN. The gel pieces were then dehydrated with 100% ACN and vacuum dried. Reduction was carried out using 5 mm Tris 2-carboxyethyl phosphine hydrochloride (TCEP) in 25 mm TEAB buffer at 60 °C for 30min, followed by alkylation with 10 mm methyl methanethiosulfonate (MMTS) in 25 mm TEAB buffer at room temperature for 45min. TCEP and MMTS were removed by performing alternate washes with 25 mm TEAB buffer and 25 mm TEAB in 75% ACN. The gel pieces were then dehydrated and dried for a second time. Sequencing-grade modified trypsin solution (Promega Corporation, Madison, WI) in 25 mm TEAB (10 ng/ml) was used for overnight digestion at 37 °C. Tryptic peptides were extracted using 50% ACN and 5% acetic acid before being dried via vacuum centrifugation (Eppendorf). Labeling was performed using iTRAQ reagent multiplex kits according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). Supernatant fractions G1-S, G1/S-S, and G2/M-S and pellet fractions G1-P, G1/S-P, and G2/M-P were labeled with 113, 114, 115, 117, 118, and 119 isobars accordingly. The iTRAQ-labeled peptides were pooled together and desalted using Sep-Pak C18 Vac cartridges (Waters, Milford, MA) before being vacuum-centrifuged to dryness. iTRAQ-labeled peptides were fractionated by ERLIC as described by Hao et al. (40.Hao P. Qian J. Ren Y. Sze S.K. Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) versus strong cation exchange (SCX) for fractionation of iTRAQ-labeled peptides.J. Proteome Res. 2011; 10: 5568-5574Crossref PubMed Scopus (32) Google Scholar) with minor modifications. Briefly, labeled peptides were reconstituted in 200 μl sample loading buffer (10 mm ammonium acetate in 85% ACN and 1% Formic acid) and fractionated using PolyWAX LP™ 20 columns (4.6 × 200 mm, 5 μm particle size, 300Å pore size) (PolyLC, Columbia, MD, USA) on a HPLC unit (Prominence™, Shimadzu, Kyoto, Japan) at flow rate of 1 ml/min. The HPLC gradient comprised 100% buffer A (85% ACN and 0.1% acetic acid) for 5min; 0–28% buffer B (30% ACN and 0.1% formic acid) for 40min; then 28%-100% buffer B for 5min, followed by 100% buffer B for 10min. The chromatograms were recorded at 260 nm. A total of 52 fractions were collected within the 60min time period. The collected fractions were combined into 28 fractions according to their chromatogram and then concentrated to dryness using vacuum centrifugation. The ERLIC-fractionated, iTRAQ-labeled peptides were reconstituted in HPLC-grade water containing 0.1% formic acid for LC-MS/MS analysis. The mass spectroscopy analysis was performed using a Q-STAR Elite mass spectrometer coupled with online nano-flow HPLC system (Applied Biosystems; MDS-Sciex, Foster City, CA). The peptides were separated using nano-bored C18 columns with a picofrit nanospray tip (75 μm ID × 15 cm, 5 μm particles) (New Objectives, Wubrun, MA).The flow-rate was maintained at 300 nl/min throughout and all LC-MS analysis was performed in triplicate. All MS data were acquired in positive ion mode with a mass range of 300–2000 m/z. Peptides with charge of +2 to +4 were selected for MS/MS. The top three most abundant peptide ions above a five count threshold were selected for MS/MS and dynamically excluded for 30s with 30mDa mass tolerance. Smart information-dependent acquisition (IDA) was activated with automatic collision energy and automatic MS/MS accumulation. The fragment intensity multiplier was set at 20 and maximum accumulation time was set at 2 s. The peak areas of the iTRAQ reporter ions reflected the relative abundance of the proteins in the samples. Data acquisition was performed using Analyst QS 2.0 software (Applied Biosystems/MDS SCIEX). The identification and quantification of proteins was performed using Protein Pilot 3 Software (Applied Biosystems). The Paragon algorithm in the Protein Pilot software was used for the peptide identification, and further processing was conducted by ProGroup algorithm (with isoform-specific quantification). The defined parameters were as follows: (1) Sample Type, iTRAQ 8-plex (Peptide Labeled); (2) Cysteine alkylation, MMTS; (3) Digestion, Trypsin; (4) Instrument, QSTAR Elite ESI; (5) Special factors, None; (6) Species, None; (7) Specify Processing, Quantities; (8) ID Focus, biological modifications, amino acid substitutions; (9) Database, The UniProt Knowledgebase (UniProtKB) human protein database (downloaded on 12 March 2010, 95,624 sequences and 36,307,192 residues) and its reversed complement were combined and used for the searches, and the corresponding reverse sequence was used for false discovery rate (FDR) estimation; and (10) Search effort, thorough. The search parameters set as a default were: (1) mass tolerance of 0.2Da for both precursor and fragment mass; and (2) maximum considerable missed and/or nonspecific cleavages number is 2. The peptide for quantification was automatically selected by ProGroup algorithm with criteria: (1) the peptide was usable for quantitation, that is, the iTRAQ reporter area is not zero; (2) the peptide was identified with good confidence; and (3) the peptide was not shared with another protein identified with higher confidence to calculate the reporter peak area, error factor (EF) and p value. Protein pilot calculate the p value based upon degrees of freedom for the average ratio and t value [t = (Weighted Average of Log Ratios- Log Bias) ÷ Standard Error of the Weighted Average of Log Ratios]. The p value evaluated the result based on the certainty of the change, not by the magnitude of the change. The resulting data set was automatically bias-corrected to eliminate variation because of potential unequal mixing while combining the different samples. We used Protein knowledgebase (UniProtKB) database for classification and functional annotation of enlisted proteins. We combined the iTRAQ ratios of matching pellet and supernatant fractions were used to determine their overall chromatin association. Online Gene Pattern software (http://genepattern.broadinstitute.org) was used for cluster analysis. Hierarchical clustering and Pearson correlation methods were applied for the clustering. Cell lysates were prepared by dissolving frozen cells via sonication in lysis buffer (1% SDS in 10 mm tris-HCl pH 7.4) and protein concentration was measured using the Bradford method. For whole chromatin samples, chromatin extracts were sonicated and dissolved in 2.5% SDS in 10 mm tris-HCl (pH 7.4) and sample protein content was measured using 2-D Quant Kits. Equal amounts of protein from each sample were used for Western blot analysis. Protein samples were resolved on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. Immunoblotting was performed using antiprotein antibodies together with the ECL system for detection (Invitrogen). HP1BP3 inhibition was achieved by shRNA-mediated knockdown using the Thermo Scientific Open Biosystems RNA intro GIPZ Lentiviral shRNAmir Starter Kit. (Catalogue #: RHS4287). All pGIPZ clones were grown at 37 °C in 100 μg/ml ampicillin containing 2X LB broth (low salt) media. Plasmids were extracted using plasmid extraction kits (Axygen, USA) and cell transfection with GIPZ Lentiviral shRNAmir (either non-silencing or HP1BP3-specific) was performed using TurboFect Transfection Reagent (Thermo Scientific) according to the manufacturer's protocol. A total of 4 μg GIPZ Lentiviral shRNAmir DNA and 6 μl TurboFectin reagent was used for each transfection. Transfected cells were incubated in DMEM supplemented with 10% FBS at 37 °C with 5% CO2 for 48 h, and transgene expression was determined by detection of GFP expression. After 48 h, the transfected cells were trypsinized and re-suspended in DMEM containing 2 μg/ml puromycin and 10% FBS for selection of stable transfectants. Cells were cultured in puromycin-containing medium for 15 days thereafter and the medium was occasionally refreshed as required. After 15d, each colony was cultured separately and the efficiency of HP1BP3 knock-down was determined by Western blot using an anti-HP1BP3 antibody. The colony that exhibited the most efficient HP1BP3 inhibition was then used as a cell source for all subsequent experiments. Cellular chromatin was extracted according to the above protocol and then suspended in MNase digestion buffer (50 mm tris-Cl pH 7.9; 5 mm CaCl2). Chromatin suspensions were then divided into three equal volumes and increasing quantities of MNase were added to each (0, 5, and 10 U). Digestion was performed at 37 °C for 10 min and the reaction was terminated by addition of an equal volume of 2X TNESK solution (20 mm tris-Cl pH 7.4; 200 mm NaCl; 2 mm EDTA; 1% SDS; and 0.2 mg/ml protein kinase K) with overnight incubation at 37 °C. DNA fragments were extracted using Phenol-chloroform and DNA content was measured by NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). Equal amounts of DNA from each sample were then loaded and resolved on 1% agarose gels for analysis. Quantitative proteomic profiling was performed using iTRAQ technology. All three biological replicates were pooled and the cells lysed by sonication. Equal amounts of lysate from mock- and HP1BP3-depleted cells were digested and labeled with 114 and 115 isobars of 4-plex iTRAQ reagent respectively, and then fractionated as described above. The LC-MS/MS analysis was performed using a Q Exactive mass spectrometer coupled with o" @default.
• W2116007004 created "2016-06-24" @default.
• W2116007004 creator A5011537784 @default.
• W2116007004 creator A5022175957 @default.
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• W2116007004 date "2014-09-01" @default.
• W2116007004 modified "2023-10-18" @default.
• W2116007004 title "Profiling of the Chromatin-associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression" @default.
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• W2116007004 doi "https://doi.org/10.1074/mcp.m113.034975" @default.
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