Multidimensional Proteomics Reveals a Role of UHRF2 in the Regulation of Epithelial-Mesenchymal Transition (EMT)
2016; Elsevier BV; Volume: 15; Issue: 7 Linguagem: Inglês
10.1074/mcp.m115.057448
ISSN1535-9484
AutoresMi Lai, Lizhu Liang, Jiwei Chen, Naiqi Qiu, Sai Ge, Shuhui Ji, Tieliu Shi, Bei Zhen, Mingwei Liu, Chen Ding, Yì Wáng, Jun Qin,
Tópico(s)RNA modifications and cancer
ResumoUHRF1 is best known for its positive role in the maintenance of DNMT1-mediated DNA methylation and is implicated in a variety of tumor processes. In this paper, we provided evidence to demonstrate a role of UHRF2 in cell motility and invasion through the regulation of the epithelial-mesenchymal transition (EMT) process by acting as a transcriptional co-regulator of the EMT-transcription factors (TFs). We ectopically expressed UHRF2 in gastric cancer cell lines and performed multidimensional proteomics analyses. Proteome profiling analysis suggested a role of UHRF2 in repression of cell-cell adhesion; analysis of proteome-wide TF DNA binding activities revealed the up-regulation of many EMT-TFs in UHRF2-overexpressing cells. These data suggest that UHRF2 is a regulator of cell motility and the EMT program. Indeed, cell invasion experiments demonstrated that silencing of UHRF2 in aggressive cells impaired their abilities of migration and invasion in vitro. Further ChIP-seq identified UHRF2 genomic binding motifs that coincide with several TF binding motifs including EMT-TFs, and the binding of UHRF2 to CDH1 promoter was validated by ChIP-qPCR. Moreover, the interactome analysis with IP-MS uncovered the interaction of UHRF2 with TFs including TCF7L2 and several protein complexes that regulate chromatin remodeling and histone modifications, suggesting that UHRF2 is a transcription co-regulator for TFs such as TCF7L2 to regulate the EMT process. Taken together, our study identified a role of UHRF2 in EMT and tumor metastasis and demonstrated an effective approach to obtain clues of UHRF2 function without prior knowledge through combining evidence from multidimensional proteomics analyses. UHRF1 is best known for its positive role in the maintenance of DNMT1-mediated DNA methylation and is implicated in a variety of tumor processes. In this paper, we provided evidence to demonstrate a role of UHRF2 in cell motility and invasion through the regulation of the epithelial-mesenchymal transition (EMT) process by acting as a transcriptional co-regulator of the EMT-transcription factors (TFs). We ectopically expressed UHRF2 in gastric cancer cell lines and performed multidimensional proteomics analyses. Proteome profiling analysis suggested a role of UHRF2 in repression of cell-cell adhesion; analysis of proteome-wide TF DNA binding activities revealed the up-regulation of many EMT-TFs in UHRF2-overexpressing cells. These data suggest that UHRF2 is a regulator of cell motility and the EMT program. Indeed, cell invasion experiments demonstrated that silencing of UHRF2 in aggressive cells impaired their abilities of migration and invasion in vitro. Further ChIP-seq identified UHRF2 genomic binding motifs that coincide with several TF binding motifs including EMT-TFs, and the binding of UHRF2 to CDH1 promoter was validated by ChIP-qPCR. Moreover, the interactome analysis with IP-MS uncovered the interaction of UHRF2 with TFs including TCF7L2 and several protein complexes that regulate chromatin remodeling and histone modifications, suggesting that UHRF2 is a transcription co-regulator for TFs such as TCF7L2 to regulate the EMT process. Taken together, our study identified a role of UHRF2 in EMT and tumor metastasis and demonstrated an effective approach to obtain clues of UHRF2 function without prior knowledge through combining evidence from multidimensional proteomics analyses. UHRF (ubiquitin-like, containing PHD and RING finger domains, inverted CCAAT Box-Binding Protein of 90 kDa) family contains four members characterized with multiple domains in structure (1Bronner C. Achour M. Arima Y. Chataigneau T. Saya H. 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Metastasis of cancer cells is a complex process that is partly regulated by activation of epithelial to mesenchymal transition (EMT) 1The abbreviations used are:EMTEpithelial to mesenchymal transitionWCEwhole cell extractsNEnucleus extractsTFREtranscription factor response elementssRPmanual RPLC-MS/MSLiquid chromatography - tandem mass spectrometryFDRfalse discovery rateiBAQintensity based absolute quantificationFOTfraction of totalLFQlabel-free quantificationTFstranscription factorsCoRscoregulatorsGOgene ontologygRNAguide RNAq-PCRquantitative real time PCRChIPchromatin immunoprecipitation. to acquire the ability to invade and metastasize (41Spano D. Heck C. De Antonellis P. Christofori G. Zollo M. Molecular networks that regulate cancer metastasis.Semin. Cancer Biol. 2012; 22: 234-249Crossref PubMed Scopus (275) Google Scholar). During EMT, epithelial cells lose cell-cell contacts and cell polarity, and acquire mesenchymal-like characteristics with increased ability of migration and invasion. EMT is orchestrated by transcription factor cascades that regulate the expression of proteins involved in cell-cell contacts, cell polarity, cytoskeleton structure and extracellular matrix degradation. For instance, EMT-TFs repress one of the key epithelial genes E-cadherin through binding the promoter region of CDH1 directly or indirectly. The reported key EMT-TFs include SNAIL1/2, TWIST1/2, ZEB1/2, TCF3 and FOXC2 (42Lamouille S. Xu J. Derynck R. Molecular mechanisms of epithelial-mesenchymal transition.Nature reviews. Mol. Cell Biol. 2014; 15: 178-196Crossref PubMed Scopus (5230) Google Scholar, 43Puisieux A. Brabletz T. Caramel J. Oncogenic roles of EMT-inducing transcription factors.Nat. Cell Biol. 2014; 16: 488-494Crossref PubMed Scopus (707) Google Scholar, 44De Craene B. Berx G. 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MS-based proteomics is a powerful approach for large scale protein analysis in biological research (46Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5585) Google Scholar, 47Cravatt B.F. Simon G.M. Yates Iii, J.R. The biological impact of mass-spectrometry-based proteomics.Nature. 2007; 450: 991-1000Crossref PubMed Scopus (571) Google Scholar). Our lab has developed a fast, label-free quantification workflow (Fast-quan) for protein identification, in which 7,000 proteins can be identified and quantified with 12 h of MS running time (48Ding C. Jiang J. Wei J. Liu W. Zhang W. Liu M. Fu T. Lu T. Song L. Ying W. Chang C. Zhang Y. Ma J. Wei L. Malovannaya A. Jia L. Zhen B. Wang Y. He F. Qian X. Qin J. A fast workflow for identification and quantification of proteomes.Mol. Cell. Proteomics. 2013; 12: 2370-2380Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). This has enabled analysis of multiple samples. We also developed a concatenated tandem array of transcription factor response elements (catTFRE) pull-down assay that allows for enrichment and identification of endogenous transcription factors (TFs) (49Ding C. Chan D.W. Liu W. Liu M. Li D. Song L. Li C. Jin J. Malovannaya A. Jung S.Y. Zhen B. Wang Y. Qin J. Proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 6771-6776Crossref PubMed Scopus (81) Google Scholar). The combination of measuring changes in DNA binding activity of TFs and proteome-wide profiling of protein abundance allows us to correlate TF activity with target genes in response to exogenous stimulation. Thus, the proteome-wide identification of activated TFs when cells are perturbed can provide important biological clues about the mechanisms and signal transduction pathways. Current UHRF researches focused on how UHRF proteins impact genome DNA methylation. This direction is important in studying cancer initiation when changes in UHRF proteins can reprogram the epigenome. It is entirely not clear whether and what roles UHRF2 may play when cells become cancerous. We thus ectopically expressed UHRF2 in gastric cancer cell lines and performed multidimensional proteomics analyses to obtain clues for UHRF2 functions in a consistent manner. The MS profiling revealed down-regulation of a number of epithelial markers including CDH1, JUP, TJP1, DSG2, INADL, CXADR, SPINT1, and TJP2. The catTFRE-MS analysis also demonstrated up-regulation of multiple key transcription factors involved in EMT, including TWIST2, FOXC2, and TCF family of transcription factors. Furthermore, we demonstrated that silencing UHRF2 in gastric cancer cells could inhibit the ability of cell migration and invasion in vitro. Together, these results suggested that UHRF2 play a role in tumor metastasis. SGC7901, MKN74, N87, and MKN45 human gastric cancer cell lines were cultured in DMEM supplemented with 10% FBS (fetal bovine serum) (Gibco, Carlsbad, CA), 1% Penicillin-Streptomycin (Sigma-Aldrich, St. Louis, MO) and incubated at 37 °C and 5% CO2. Antibody specific to UHRF2 was generated against a recombinant GST-tagged UHRF2 (N-terminal 351aa) fragment. Specific antibody was purified from serum of immunized rabbit with His-tagged-UHRF2 protein. The specific recognition of endogenous UHRF2 by the antibody was confirmed by IP-MS. The human UHRF2 coding sequence was amplified by PCR from cDNA libraries. The amplified fragment was cloned into the pENTR-vector. The lentiviral vector containing UHRF2 cDNA was constructed by recombination of pHAGE-EF-ZsG-DEST with pENTR. The sequence was verified by DNA sequencing. Lentivirus supernatants were collected 48 h after transfecting pHAGE-EF-UHRF2-ZsG-DEST with the packaging vectors pMD2.G and psPAX2 into 293T cells using lipofectamine 2000 reagent (Invitrogen). The lentivirus packaged with the empty pHAGE-EF-ZsG-DEST vector was used as control. The concentrated and purified lentivirus from supernatants were used to infect cells with 8 μg/ml Polybrene to generated control or UHRF2-expressing stable cells of SGC7901, MKN74, N87, and MKN45. The expression of UHRF2 in different cell lines was analyzed by Western blot. CatTFRE was done as previously described (49Ding C. Chan D.W. Liu W. Liu M. Li D. Song L. Li C. Jin J. Malovannaya A. Jung S.Y. Zhen B. Wang Y. Qin J. Proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 6771-6776Crossref PubMed Scopus (81) Google Scholar). Briefly, the catTFRE DNA was prepared by PCR amplification with biotinylated primers. Nuclear extracts (NEs) were prepared with NE-PER nuclear extraction reagents (Thermo Fisher Scientific, Boston, MA). Two milligram of control or OE NEs was used to incubate with pre-immobilized biotinylated catTFRE on Dynabeads (Dynabeads M-280 Streptavidin, Invitrogen, Carlsbad, CA). The mixture was supplemented with EDTA to a final concentration of 1 mm and adjusted with NaCl to 200–250 mm total salt concentration and incubated for 2h at 4 °C. The supernatant was discarded and Dynabeads were washed with NETN [100 mm NaCl, 20 mm Tris-Cl, 0.5 mm EDTA, and 0.5% (v/v) Nonidet P-40] twice followed by PBS twice. Beads were added to 20 μl 2× loading buffer and incubated in 95 °C for 5 min. The supernatant was put on SDS-PAGE for separation. SDS-PAGE gels were stained with Coomassie Blue R-250 to visualize the protein bands and each lane was cut into 12 gel slices. In-gel trypsin digestion was performed. After incubation with rotation for overnight at 37 °C, the digested products were extracted with acetonitrile (ACN) and dried in vacuum. Whole cellular lysates were extracted using 8 m urea. MKN74 control and UHRF2-OE cells (MKN74 is a gastric cancer cell line with low invasiveness, which expresses low level of UHRF2 and high level of CDH1 on its surface) were lysed with 8 m urea containing protease inhibitor PMSF for 30min at 4 °C. The lysate was centrifuged at 24,000 × g and the supernatant was collected as whole cell extracts (WCE). Protein concentration was determined by BCA assay. Twenty micrograms of control and OE proteins were digested with trypsin. Tryptic peptides were separated on a C18 column with acetonitrile of different percentage as 6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, and 35%. Nine separations were combined to six fractions and dried in vacuum. Peptides were stored at −80 °C until re-dissolved for MS analysis. NEs were prepared with NE-PER Kit (Thermo) from control and UHRF2-OE cells of N87, MKN45, SGC7901, and MKN74. Equal amounts (1 mg∼10 mg) of NEs from control and OE cells were incubated with 5 μg UHRF2 antibody. The incubation solution was adjusted with NaCl to 200 mm total salt concentration and incubated at 4 °C for overnight. After the addition of 30 μl protein A/G-Sepharose for each IP reaction and incubation for another 2h, the immunoprecipitate was washed with NETN twice followed by PBS buffer twice. The Sepharose beads were re-suspended in 20 μl 2× loading buffer and incubated at 95 °C for 5 min. The supernatant was resolved by 10% SDS-PAGE and in-gel trypsin digestion was performed after the gel was sliced. Dried peptide samples were re-dissolved in solvent A (0.1% formic acid in water). Liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis was performed with Q-Exactive Plus or FUSION mass spectrometer (Thermo) equipped with an online Easy-nLC 1000 nano-HPLC system (Thermo). The injected peptides were separated on a reversed phase nano-HPLC C18 column (Pre-column: 5 μm, 300 Å, 2 cm × 100 μm ID; analytical column: 3 μm, 120 Å, 15 cm × 75 μm ID) at a flow rate of 350 nl/min with a 75-min gradient of 3 to 30% solvent B (0.1% formic acid in acetonitrile). For the detection with FUSION mass spectrometry, a precursor scan was measured in the Orbitrap by scanning from m/z 300–1400 with a resolution of 120,000. Ions selected under top-speed mode were isolated in Quadrupole and fragmented by higher energy collision-induced dissociation (HCD) with normalized collision energy of 35%, then measured in the linear ion trap. Typical mass spectrometric conditions were: AGC targets were 5e5 ions for Orbitrap scans and 5e3 for MS/MS scans; dynamic exclusion was employed for 18 s. For the Q-Exactive Plus, the instrument was operated in the data-dependent acquisition mode with a resolution of 70,000 at full scan mode and 17,500 at MS/MS mode. The full scan was processed in the Orbitrap from m/z 300–1400, the top 20 most intense ions in each scan were automatically selected for HCD fragmentation with normalized collision energy of 27% and measured in Orbitrap. Typical mass spectrometric conditions were: AGC targets were 3e6 ions for full scans and 5e4 for MS/MS scans; dynamic exclusion was employed for 18 s. The acquired MS/MS spectra were searched by Mascot 2.3 (Matrix Science Inc, MA) implemented on Proteome Discoverer 1.4 (Thermo) against the human National Center for Biotechnology Information (NCBI) RefSeq protein databases (updated on April 7, 2013, 32,015 protein entries). The parameter settings were: the mass tolerances were 20 ppm for precursor and 50mmu for product ions from Q-Exactive Plus and 20 ppm for precursor and 0.5 Da for product ions from FUSION respectively; two missed cleavages were allowed; the fixed modification was set as carbamidomethyl (C), dynamic modifications were protein acetyl (protein N-term), oxidation(M) for profiling data; dynamic modification for catTFRE were phosphor (Y), phosphor (ST), deStreak (
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