Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/345Elie_A39_B2_N3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/345Elie_A410_B2_N3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/343Elie_39_B2_N2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/345Elie_A17_B2_N3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/338Elie_BioID2_39.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/338Elie_BioID2_28.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/343Elie_28_B2_N2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/338Elie_BioID2_511.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/343Elie_410_B2_N2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/338Elie_BioID2_17.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/343Elie_17_B2_N2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/338Elie_BioID2_612.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/345Elie_A28_B2_N3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/343Elie_612_B2_N2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/345Elie_A612_B2_N3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/338Elie_BioID2_410.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/343Elie_511_B2_N2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/345Elie_A511_B2_N3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/04/PXD012146/txt.zipprimaryOK200fm.boisvert@usherbrooke.caFrancois-Michel BoisvertAffinity purification coupled with mass spectrometry proteomicsMass SpectrometryShotgun proteomicsColon CancerNot availableTranscriptionProteomicsHnf4αIsoformsColorectal cancerSilacNuclear receptorshttp://www.ebi.ac.uk/pride/archive/projects/PXD012146Epithelial CellCell CultureColonReduction, alkylation and digestion of proteins All buffers used from this stage were prepared with MS grade water. The protein reduction step was carried out by incubating the beads in 100 μl of 20 mM ammonium bicarbonate buffer containing 10 mM DTT (Thermo Fisher Scientific, Waltham, USA) with stirring (1250 rpm) for 30 minutes at 60 °C. The alkylation of the proteins was carried out by adding another volume of 100 μl of 20 mM ammonium bicarbonate buffer, and then supplementing to 15 mM IAA (Sigma-Aldrich, Saint-Louis, United States) final before stirring for 1 hour at room temperature away from light. The IAA was then neutralized by completing 15mM DTT and stirring for 10 minutes at 37 ° C. The proteins were digested by adding 1 μg Pierce MS-grade trypsin (Thermo Fisher Scientific, Waltham, USA) and incubated overnight at 37 ° C with shaking. Purification and desalting of the peptides on C18 columns Digestion was stopped by adding 1% formic acid (FA) (Thermo Fisher Scientific, Waltham, USA) to a total volume of 200 μl, followed by stirring for 5 minutes at room temperature. The beads were spun at 6000 x g for 3 minutes before harvesting the supernatant and transferring to a new low binding microtube. The beads were resuspended in 200 .mu.l of buffer containing 60% acetonitrile (ACN) (Sigma-Aldrich, St. Louis, USA) and 0.1% FA, and then stirred for 5 minutes at room temperature. The supernatant was harvested and combined with that obtained previously. These samples were concentrated by a centrifugal evaporator at 65 ° C until complete drying (approximately 2 hours), and resuspended in 30 μl of 0.1% trifluoroacetic acid (TFA) buffer (Sigma-Aldrich, St. Louis, USA). United). The peptides were purified with ZipTip micropipette tips of 10 μl format containing a C18 column (EMD Millipore, Burlington, USA). The ZipTip was first moistened by suctioning 10 μl of 100% ACN solution three times, then equilibrated by suctioning 10 μl of 0.1% TFA buffer three times. Each sample of peptides was passed on the balanced ZipTip by succeeding up-and-downs of 10 μl of the sample 10 times. This step was performed three times in order to pass the entire sample on the column. The ZipTip was then washed with 10 μl of 0.1% TFA buffer three times. The elution of the peptides was made in a new low-binding microtube, 10 times with a volume of 10 μl of buffer 50% ACN and 0.1% FA. This step was carried out three times to obtain a final volume of 30 μl. The peptides were concentrated by centrifugal evaporator at 65 ° C. until complete drying (approximately 30 minutes) and then resuspended in 25 μl of 1% FA buffer. Peptides were assayed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA) reading the absorbance at 205 nm. The peptides were then transferred to a glass vial (Thermo Fisher Scientific, Waltham, USA) and stored at -20 ° C until analyzed by mass spectrometry.PrideSILACbiotinylated residueiodoacetamide derivatized residuemonohydroxylated residue6x(13)C labeled residueacetylated residueLC-MS/MS analysis Trypsin digested peptides were separated using a Dionex Ultimate 3000 nanoHPLC system. 10 μl of sample (a total of 2 μg) in 1% (vol/vol) formic acid was loaded with a constant flow of 4 μl/min onto an Acclaim PepMap100 C18 column (0.3 mm id x 5 mm, Dionex Corporation). After trap enrichment peptides were eluted off onto an EasySpray PepMap C18 nano column (75 μm x 50 cm, Dionex Corporation) with a linear gradient of 5-35% solvent B (90% acetonitrile with 0.1% formic acid) over 240 minutes with a constant flow of 200 nl/min. The HPLC system was coupled to an OrbiTrap QExactive mass spectrometer (Thermo Fisher Scientific Inc) via an EasySpray source. The spray voltage was set to 2.0 kV and the temperature of the column was set to 40 oC. Full scan MS survey spectra (m/z 350-1600) in profile mode were acquired in the Orbitrap with a resolution of 70,000 after accumulation of 1,000,000 ions. The ten most intense peptide ions from the preview scan in the Orbitrap were fragmented by collision induced dissociation (normalised collision energy 35% and resolution of 17,500) after the accumulation of 50,000 ions. Maximal filling times were 250 ms for the full scans and 60 ms for the MS/MS scans. Precursor ion charge state screening was enabled and all unassigned charge states as well as singly, 7 and 8 charged species were rejected. The dynamic exclusion list was restricted to a maximum of 500 entries with a maximum retention period of 40 seconds and a relative mass window of 10 ppm. The lock mass option was enabled for survey scans to improve mass accuracy. Data were acquired using the Xcalibur software. Protein identification by MaxQuant analysis The raw files were analyzed using the MaxQuant version 1.6.2.2 software (Cox and Mann, 2008) and the Uniprot human database (16/07/2013). Analyzes were initially performed separately for each isoform to obtain enrichment ratios for the complete sequence of each. A common analysis was then performed to integrate all raw MS / MS analysis files. The MaxQuant software default settings were used, except for the following parameters: multiplicity of 3 SILAC media (R0K0, R6K4 and R10K8), identification values "PSM FDR", "Protein FDR" and "Site decoy fraction" 0.05, minimum ratio count of 1 and selection of the "Re-quantify" option. Following the analysis, the results were sorted according to several parameters. Proteins positive for at least one of the Reverse, Only identified by site and Potential contaminants categories were eliminated, as well as proteins identified from a single peptide. The ratios identified in only one of the three replicas for each experiment were eliminated. The ratios identified in two of the three replicas were eliminated when they were considered to be too divergent, ie when the standard deviation was greater than the average of the two enrichment ratios. Outliers for the ratios measured in the three replicas were detected using the Grubbs test at a value of α = 0.05 and then eliminated. Following this sorting, proteins for which no ratio was calculated in at least one experiment were removed. Gene ontology enrichment analyzes were performed using the Panther 13.1 tool (Mi, Dong, Muruganujan, Gaudet, Lewis, and Thomas, 2010). Heatmap visualizations were created using the Morpheus software (Gould, 2018).ProteomicsFrancois-Michel BoisvertQ ExactiveDepartment of Anatomy and Cell Biology, Université de Sherbrooke, CanadaPARTIALHomo Sapiens (human)fm.boisvert@usherbrooke.ca32123031 Lambert É, Babeu JP, Simoneau J, Raisch J, Lavergne L, Lévesque D, Jolibois É, Avino M, Scott MS, Boudreau F, Boisvert FM. Human Hepatocyte Nuclear Factor 4-α encodes isoforms with distinct transcriptional functions. Mol Cell Proteomics. 2020 10.1074/mcp.ra119.001909BiomedicalUniversité de SherbrookeCanadaHNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.Human Hepatocyte Nuclear Factor 4-α Encodes Isoforms with Distinct Transcriptional Functions.Lambert Élie É, Babeu Jean-Philippe JP, Simoneau Joël J, Raisch Jennifer J, Lavergne Laurie L, Lévesque Dominique D, Jolibois Émilie É, Avino Mariano M, Scott Michelle S MS, Boudreau François F, Boisvert Francois-Michel FMsmall, Transcriptome, Activity, determination, Transcriptome Profile, Effects, Expression Profiles, Longterm Effect, Gene Expression Profile, total expressed protein, Gene, Profiles, function, Factor, Development, Transcription Factor, Long Term, Transcripts, Splicings, element, RNA Splicings, Gene Expression, period, Alternative, Splicing, Alternative RNA Splicings, reduced, subnumerary, Expression Signatures, responsivity, Gene Expression Signatures, Long Term Effects, Consensus, chemical analysis, Alternative RNA Splicing, atomo, atomus, Gene Expression Signature, tiny, atome, Expression Profile, present in fewer numbers in organism, Effect, Alternate Splicing, Nested Transcript, Transcriptome Profiles, reactivity, Nested Transcripts, Transcription, Element, Factors, Nested, Gene Expressions, underdeveloped, Longterm, exonic region, Profile, atoms, RNA Splicing, hypoplasia, Long-Term, decreased number, Expressions, Signatures, Longterm Effects, Alternate, Transcript, Alternative Splicings, decreased, Expression Signature, Gene Expression Profiles, anatomical unit, data., body organ, Alternative RNA, Transcriptomes, Long-Term Effect, Consensus Development, Expression, assay, response, Signature, associated, elements, Proteomes, General activity, time, Long-Term Effects, Alternate Splicings, atomsodium salt, ammonium formate, 13C-labeled, cox4, cadmium salt, MeCN, ion, Raw, Irip, magnesium formate, X-50, zinc salt, NAALAdase, Solvent, RP11-508D10.1, Long Term, 5730420M11Rik, Polypeptides, NAALADase I, protein polypeptide chains, FOLH, cobalt(II) formate dihydrate, morpheus, CH3-C#N, B1, C530001K22Rik, 3, 4, Software Engineering, 9, WMS, average, somitogenic mesoderm, SET, Divorced, type 5 acid phosphatase, Membrane glutamate carboxypeptidase, COXIV, phosphatase 2A inhibitor I2PP2A, Divorces, proteins, COXA_DROME, HPLC, AI047805, DmelCG4299, set, T-cell antigen Gp39, 15-disilaoctadecane, column, Cox4, sample, D1, n, 1.3.3.3, TNFSF5, z, COX-VA, nickel salt, SGS, LC-MS2, cobalt (+2) salt, unsegmented mesenchyme, Tnfrsf5, Longterm Effect, Normalcy, SH2-B PH domain-containing signaling mediator 1, not genetically inherited, Software Tools, hCD40L, ACMICD, Computer Applications, sodium (4:1:1) salt, 11-tetrathia-4, count, magnesium salt, Dm1, AL024441, Computer Applications Software, IMD3, Ontology Projects, NADH-cytochrome b5 reductase 3 soluble form, Ontology, Software Applications, Humb5R2, HLA-DR-associated protein II, maintenance of localization, Projects, DI-2, Coprogen oxidase, Source Software, I-2Dm, FGCP, formate, NKTL, proportionality, pooled, LEW/Crl, cromium (+3), rate, inhibitor of granzyme A-activated DNase, beta Trypsin, human, I-2PP1, Pro-rich, Applications, TAF-IBETA, liquid chromatography-tandem mass spectroscopy, ammonium (4:1) salt, TAF-Ibeta, methyl cyanide, lead (+2) salt, Computer Software Applications, nickel formate dihydrate, lead formate, Effects, region or site annotation, Ly62, fractured, TAPK, aluminum salt, precursor, thomson, protein-containing complex, TR-AP, body system, Gene Ontology Project, CG7826, Gene Ontology, mass-to-charge ratio, period, CG10664, LEW/CrlBR, IV, LC-MSMS, GP39, CG7835, Gene Products, CG42273, system, Application, 2018., gp39, CD154, Normalities, Open Source Softwares, positional, proportion, anatomical systems, GKLP, CATC4, thallium (+1) salt, Longterm, Software Application, beta-Trypsin, rubidium salt, Open Source Software, 2pp2a, Long-Term, methanoic acid, ions, man, CG10574, Computer Software Application, PCTAIRE2-binding protein, MGC:45012, 2PP2A, Tools, COX, dSET, dSet, peptidos, species, PTHB1, constant, NTKL, 1-(14)C-labeled, N-terminal cytochrome b5 and cytochrome b5 oxidoreductase domain-containing protein, Proteins, split, potassium formate, copper (+2) salt, ACP5, acetonitrile, SPENCDI, Tool, polypeptide, Experiment, Temperatures, native protein, I-2PP2A, C18, cupric formate, chemical analysis, Dm I-2, Long Term Effects, hemorrhaged, MFS1, segmental plate, aluminum formate, Sh2bpsm1, AU020952, torn, Ontology Project, lithium formate, Col4a-1, storage, Separations, ethanenitrile, Cytochrome b5 reductase, b5R.1, b5R.2, primary structure of sequence macromolecule, Longterm Effects, Folate hydrolase 1, ME-IV, Computer Programs, Gene Proteins, CG14724, Applications Softwares, binding_or_interaction_site, quotient, liquid chromatography tandem mass spectrometry, TRACP, sequestering, LEW, liquid chromatography tandem mass spectroscopy, SH2-Bb, IPP2A2, Bru, Gene Ontology Projects, NCMe, determination, selection process, zinc formate, lead salt, protein, 10, binding site, peptide, Tnfsf5, 15, ammonium tetraformate, peptido, ClvPrd, Flavohemoprotein b5|b5R, Project, Min, 3.4.17.21, DmelCG10664, protein aggregate, Effect, Computer Program, DmelCG42273, T-BAM, Svc, SH2-B, LCMSMS, tartrate-resistant acid ATPase, peptides, TAF-I, cb5|cb5R, NPIP, Open, min, Computer Programs and Programming, mAPC, NAALAD1, copper, SCAN, Folylpoly-gamma-glutamate carboxypeptidase, retention, 1.6.2.2, TRAP3, IGAAD, lithium salt, Tudor repeat associator with PCTAIRE-2, DmelCG10574, MDC-01-40, ppm, T5ap, MGC - 45012, GPHYSD2, ammonium (2:1) salt, Long-Term Effects, B5R, ratio, CD40L, phapii, NPIPA, T5E21.12, LC-MS/MS, Cd40l, Dmel_CG7826, StF-IT-1, Th, fragmented, Source Softwares, results, Programs, Program, Ionen, VA, PSM, Psm, Computer Applications Softwares, TNF-related activation protein, Softwares, COX IV, presumptive somite mesoderm, Prostate-specific membrane antigen, nickel (+2) salt, Ly-62, Dmel_CG7835, region, 15-tetraethoxy-, T5E21_12, Mnb, MNB, parent ion, Gene Ontologies, ammonium salt, Pteroylpoly-gamma-glutamate carboxypeptidase, positional polypeptide feature, Ontologies, Va, cracked, COX IV-1, cobaltous formate, AW743063, common, CG4299, MASS, AW124434, AI326936, organ system, Health, precursor ion, TrATPase, copper salt, IGM, PSMA, CD40LG, Polypeptide, DmelCG14724, i2pp2a, time, Cox4a, SH2 domain-containing protein 1B, human being, TRAP, p50, cesium salt, unsegmented paraxial mesoderm, FBN, Gene, 2-(1-benzyl-4-methylpyrazol-3-yloxy)-2-methylpropionic acid, Computer, somitomeric mesoderm, GCPII, b5+b5R, PHAPII, LC-MS-MS, formic acid, potassium salt, template-activating factor I, polypeptide chain, mixt. with silica, ECTOL1, 14C-labeled, NAD(P)H - quinone oxidoreductase type 3 polypeptide A2, N-acetylated-alpha-linked acidic dipeptidase I, mKIAA1299, Del(8)44H, CD40-L, Separated, SH2B, Coproporphyrinogenase, iones, ipp2a2, uniform, mGCP, calcium formate, OCTD, 10^[-6], TEIF, Trap, taf-ibeta, HIGM1, site, cromium (+3) salt, Long-Term Effect, sodium formate, NADH-cytochrome b5 reductase 3 membrane-bound form, C78062, Applications Software, P105, Open Source, DYRK1, data, Computer Software, nickel formate, 3H-labeled, protein complex, PCTAIRE2BP, igaad, strontium formate, Bp50, strontium salt, Peptide, GCP2, Glutamate carboxypeptidase II, Diaphorase-1, LC/MS/MS, Software Tool, Cell growth-inhibiting gene 27 protein, natural protein, 16-Dioxa-8, Protein, I2PP2A, sequence, IV-1, Dyrk1, connected anatomical system, Software, ACETONITRILE, cyanomethane, WMS2, Data Base, Separation, 10^[-9], PH and SH2 domain-containing signaling mediator, Normality, INSDC_feature:misc_binding, CC1, Engineering, tend, Tripcellim, sample population, chromic formate, Protein Gene Products, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, Ion, SSKS, AI425885, calcium salt, Peptid, assay, AA407739alpha-Ada, anon-EST:fe2E2, alpha-Spectrin, BamF, l(3)alpha-Spec, AP-2, BamC, 3A9, Hepatocyte Nuclear Factor 4 beta, DmelCG1977, alpha-Spect, alpha-Spe, HNF4, ham, Transcription Factor, alpha-Spc, CG31654, l(2)SH2 0460, Nuclear Receptor Subfamily 2 Group A, ada, fs(3)neo61, CG10422, alphaSpec, l(3)04276, alpha-ada, D-Ada, klo, cg4260, alpha-adaptin, MENE(2L)-A, Hepatocyte Nuclear Factor 4 alpha, alpha-Sp, dAP-2a, DmelCG10422, CG1977, alphaSp, spectrin, alpha-spec, l(2)06694, MENE (2L)-A, Hepatocyte Nuclear Factor Forkhead Homolog 4, D-alphaAda, mating_type_alpha, CG4260, FCP-B, dre3, Hepatocyte Nuclear Factor 4-beta, alpha, AP-2alpha, Hepatocyte Nuclear Factor 4-gamma, Alpha-Adaptin, Hepatocyte Nuclear Factor 4 gamma, alpha-spectrin, DmelCG4260, AP2, Nuclear Receptor Subfamily 2, Hepatocyte Nuclear Factor-Forkhead Homolog 4, HNF4 Transcription Factor, Member 2, l(3)62Bd, Bam-C, BAM, Member 1, alpha mating type (yeast), Bam, Hepatocyte Nuclear Factor 4-alpha, l(2)SH0460, alfa-Spec, Group A, l(3)dre3., Specdistinct., Hepatocyte Nuclear Factor Forkhead Homolog 4, Nuclear Receptor Subfamily 2, Hepatocyte Nuclear Factor-Forkhead Homolog 4, human being, HNF4 Transcription Factor, Member 2, Hepatocyte Nuclear Factor 4 beta, Member 1, Hepatocyte Nuclear Factor 4-alpha, Hepatocyte Nuclear Factor 4 alpha, HNF4, Group A, Hepatocyte Nuclear Factor 4-beta, Hepatocyte Nuclear Factor 4-gamma, man, Transcription Factor, Hepatocyte Nuclear Factor 4 gamma, human, Nuclear Receptor Subfamily 2 Group Asodium salt, Sta, STA, ammonium formate, 13C-labeled, cadmium salt, MeCN, Meth, NCMe, zinc formate, lead salt, Visible Light, protein, 2-(indol-3-yl)ethanoic acid, magnesium formate, monosomy 2q32, zinc salt, Alkylations, peptide, Trifluoroacetate, Polypeptides, protein polypeptide chains, ammonium tetraformate, IAA, peptido, BANF, cobalt(II) formate dihydrate, GRP1, CH3-C#N, Grp1, hnu, B1, Analysis, protein aggregate, monosomy 2q32q33, foton, Mass Spectrum Analysis, me75, C79325, peptides, Analyses, PTPSTEP, 2610036I19Rik, 2610510L13Rik, proteins, EDMD, copper, D17Mit170, T1, fSAP152, lithium salt, chromosome 2q32-q33 deletion syndrome, h, column, Ice, l(2)k08110, ACN, D1, Acn, trifluoro-, sample, monosomy 2q32-q33, stage, ammonium (2:1) salt, GPH, Saint, gamma, nickel salt, Radiation, cobalt (+2) salt, Light, Tl3, Tl2, Spectrum Analysis, Spectroscopy, glass, (Indol-3-yl)acetate, sodium (4:1:1) salt, LIGHT, magnesium salt, SATB2-associated syndrome, 3.1.3.48, Del(2)(q32), IES, nickel (+2) salt, Acetic acid, Hydrogen Oxide, Crystal, Visible Radiations, Acid, ammonium salt, Visible Radiation, 2q32q33 microdeletion syndrome, HVEML, Step, CG11628, Xylella fastidiosa str. Temecula1, formate, Trifluoroacetic, cobaltous formate, Spectrometry, cromium (+3), Del(2)(q32q33), Striatum-enriched protein-tyrosine phosphatase, beta Trypsin, Speed, Indole-3-acetic acid, light quantum, heteroauxin, Cesium, STEP, copper salt, ammonium (4:1) salt, Polypeptide, methyl cyanide, lead (+2) salt, GRP1/cytohesin 1, nickel formate dihydrate, lead formate, developmental stage, aluminum salt, cesium salt, Pierce, Gene, ACINUS, Spectrum Analyses, protein-containing complex, Chalk, Buffer, Ly113, formic acid, CG11633, cytohesin/GRP1, SATB2 syndrome, potassium salt, polypeptide chain, LEMD5, glass syndrome, 14C-labeled, Tina, Gene Products, Mass, light, Low, Mass Spectroscopy, Cesium Trifluoroacetate, Glass, Acinus, thallium (+1) salt, Lichtquant, ligand, beta-Trypsin, rubidium salt, l(2)SH2 0323, Indoleacetic acid, Visible, calcium formate, methanoic acid, Solution, Xylella fastidiosa (strain Temecula1 / ATCC 700964), acinusL, Neural-specific protein-tyrosine phosphatase, (indol-3-yl)acetic acid, acinusS, photon, peptidos, cromium (+3) salt, TR2, sodium formate, PTHB1, 1H-indol-3-ylacetic acid, 2q32-q33 microdeletion syndrome, cou, nickel formate, 1-(14)C-labeled, 3H-labeled, 2q32q33 microdeletion syndromes, protein complex, Proteins, strontium formate, stepk, potassium formate, copper (+2) salt, strontium salt, acetonitrile, buffer, CD258, Peptide, mKIAA0670, polypeptide, Lr, native protein, Temperatures, natural protein, C18, cupric formate, Protein, aluminum formate, l(2)SH0323, ACETONITRILE, Mass Spectrum Analyses, cyanomethane, CYH1, Radiations, Mass Spectrum, AW550900, SAS, lithium formate, HVEM-L, Photoradiation, 3-Indolylessigsaeure, Tripcellim, VIaL, ethanenitrile, chromic formate, sample population, DmelCG11628, LTg, Protein Gene Products, Gene Proteins, Trypure, Photoradiations, calcium salt, Bra, Mass., Peptid, SCHbiochemical pathways, transcription factor, Forms, T-cell leukemia, Ribonucleic, Metabolic Process, Materials, single-organism developmental process, HCT116, Activity, determination, AGL4, Metabolic Concepts, Mbp1, Long Term, Transcripts, element, Alternative, E130305N23Rik, RNA polymerase II distal enhancer sequence-specific DNA binding transcription factor activity, responsivity, Concepts, Analysis, Metabolism Concept, Phenomenon, myd, Effect, present in fewer numbers in organism, Non Polyadenylated, RNA Gene Products, Mass Spectrum Analysis, Gene Expressions, Analyses, catabolism, long, RNA Splicing, hypoplasia, F10N7_150, Mbp-1, metabolic process resulting in cell growth, number of, RNA polymerase II core promoter proximal region sequence-specific DNA binding transcription factor activity, Transcript, DNA-dependent, decreased, data., AGAMOUS-like 4, biotransformation, zinc ion regulated proximal promoter sequence-specific DNA binding, Consensus Development, sequence-specific transcription regulatory region DNA binding RNA polymerase II transcription factor recruiting transcription factor activity, associated, transcription from bacterial-type RNA polymerase promoter, Catabolism, Long-Term Effects, Alternate Splicings, IRF-2-binding protein 2, TRANSCRIPTION FACTOR, RNA polymerase II distal enhancer sequence-specific binding, RNA polymerase II core promoter proximal region sequence-specific binding, DNA-dependent transcription, Process, ribose nucleic acid, gyltl1b-b, metabolism resulting in cell growth, ribonucleic acids, Longterm Effect, extra or missing physical or functional parts, metal ion regulated sequence-specific DNA binding RNA polymerase II transcription factor activity, Spectrum Analysis, Spectroscopy, Splicing, Alternative RNA Splicings, MDDGA6, Ribonukleinsaeure, mKIAA0609, Genetic Materials, pentosenucleic acids, secretion, atomo, Ribonucleic acids, sequence-specific distal enhancer binding RNA polymerase II transcription factor activity, atome, Genetic Material, Alternate Splicing, F14P3.4, fg, Acid, Element, Factors, gyltl1b, HCT-116, atoms, mdc1d, F14P3_4, Spectrometry, expanded, Transcription factor, MDC1D, Alternative Splicings, enr, enlarged, Material, bacterial transcription, copper ion regulated core promoter proximal region sequence-specific DNA binding RNA polymerase II transcription factor activity, Cistron, metal ion regulated sequence-specific DNA binding, Proteomes, time, metal ion regulated proximal promoter sequence-specific DNA binding, big, transcription, Effects, Processes, number, Gene, SEPALLATA 2, Spectrum Analyses, Development, Metabolic Processes, Transcription Factor, presence, froggy, Gyltl1a, period, large, zinc ion regulated core promoter proximal region sequence-specific DNA binding, reduced, subnumerary, Metabolism, Consensus, Alternative RNA Splicing, Gene Products, Mass, sequence-specific DNA binding, tiny, Metabolism Phenomena, IRF-2BP2, Mass Spectroscopy, study, reactivity, Nested Transcripts, Transcription, Genetic, Longterm, MDDGB6, Metabolic Concept, Long-Term, decreased number, Expressions, Non-Polyadenylated RNA, BPFD#36, RNA polymerase II proximal promoter sequence-specific DNA binding, anatomical unit, copper ion regulated proximal promoter sequence-specific DNA binding, great, body organ, has or lacks parts of type, Long-Term Effect, Expression, elements, Differentiation, atom, small, cellular transcription, RNA, degradation, total expressed protein, zinc ion regulated core promoter proximal region sequence-specific DNA binding RNA polymerase II transcription factor activity, homeobox 1, RNS, function, Factor, Cistrons, Cell, Splicings, Concept, Metabolic Phenomena, transcription factor activity, whole transcriptome, mereological quality, development, RNA Splicings, Metabolism Concepts, count in organism, yeast nucleic acid, Long Term Effects, chemical analysis, Phenomena, RNA polymerase II transcription factor activity, atomus, metabolism, metal ion regulated core promoter proximal region sequence-specific DNA binding RNA polymerase II transcription factor activity, KNOTTED1-like homeobox gene 5, Mass Spectrum Analyses, Nested Transcript, Differentiations, Metabolic Phenomenon, ribonucleic acid, Mass Spectrum, multicellular organism metabolic process, Nested, biodegradation, Metabolic, underdeveloped, Non Polyadenylated RNA, Non-Polyadenylated, DNA-templated, Cell Differentiations, Ribonucleic Acid, sequence-specific DNA binding RNA polymerase II transcription factor activity, metal ion regulated core promoter proximal region sequence-specific binding, copper ion regulated core promoter proximal region sequence-specific binding, F10N7.150, Longterm Effects, Alternate, single-organism metabolic process, HCT 116, cardinality, Alternative RNA, assay, quantitative, response, General activity, Anabolism, sequence-specific transcription regulatory region DNA binding, presence or absence in organismfalseFunctions of the 12 different isoforms of the hepatocyte nuclear factor 4 alpha (HNF4α)HNF4α is a nuclear receptor regulating the transcription of genes involved mainly in development, cell differentiation and metabolism. Opposite functions for the two classes of P1 and P2 isoforms of HNF4α have recently been highlighted. These classes include 12 variants of HNF4α that can be expressed by the use of two promoters and by alternative splicing. Until now, the characterization of this transcription factor has ignored this diversity and has remained confined to the study of a fraction of the isoforms. We therefore wanted to clarify the situation by specifically characterizing the transcriptional functions of the 12 isoforms of HNF4α. We have generated for this purpose stable lines expressing each isoform of HNF4α in HCT 116 cells. We analyzed the whole transcriptome associated with each isoform by sequencing RNA, as well as their proteome by a BioID approach coupled to quantitative mass spectrometry. We noted major differences in the transcriptional function of the 12 isoforms. The α4, α5 and α6 isoforms have been characterized for the first time, and show a greatly reduced transcriptional potential. We have shown that these isoforms are unable to recognize the consensus response element of HNF4α. The α1 and α2 isoforms are the most potent regulators of gene expression, while the α3 isoform exhibits significantly reduced activity. Several transcription factors and coregulators have been identified as potential specific partners for certain HFH4α isoforms. The IRF-2BP2 co-repressor interacts specifically with isoforms which include the long form of the F domain of HNF4α. This specific interaction could explain the large number of genes modulated negatively by α1 and α2 compared to α3. The analysis integrating the vast amount of transcriptomic and proteomic data allows the identification of transcriptional regulatory mechanisms specific to certain isoforms, demonstrating the importance of considering all isoforms which can have diverse functions.2020-04-172018-12-21PXD01214644544641524991755931173880433309286643235983906288705119459932716024619624619716032935811363589453275838511879475723071351225117750585557146835364916738721589103062915929158364151584938858033271591002263888691513558847518704359309451464796928771332164133280381034304365458333985808188946214320591182590632957174934693812472725632273219286728139545132949140816918045416193453745713903634530114311402064119810871355477142450732644210435551274432149671428316669455972910039237514615754647842242208964139231744712701390114319338293550823074111481194669489693529372512646907275802405507216599366581216595294128213312832965431282128033952321818431832920435315228112454357554787575928459330797253623114367331159556444214321381849223948824261914548140479109697622615515311179573347205968798662330980058884558626528173630943179698936160488566416242567825317513320496303555432046565050632633NCBITaxon:1313456586068860769938942468879554535120148875197959216129410016023526420328909694569410167631076228531643518543121574577760445246794144242324113NCBITaxon:177321409292979310239101178709NCBITaxon:405597499079913652611227349821717018736745101161035856915693132842626587210360983341336795108944866330315819NCBITaxon:4932992588391000589873299404458611535711471612249113430531156312017554421932993587263178723666987246426151345887274215726787254762422314146115104160791305546891118006655648412552285621988221361629976798251021699823117467389764116761080772326424115574468944447415367767446859600393765209611822635735784582730688086363266689NCBITaxon:269704910312NCBITaxon:1389838192011241368108931997596123077319747946522832917619662776689605960615729513722117110125673712334351052311009040544931799863878343061510429863578840193733496155087711982159739757487822971972154078211908206189985076480256318418985623951655190264946454635286746360NCBITaxon:110719646375939288357461287460113450664912538458814594319722152638NCBITaxon:615797787237953440812633803945264114595361235178616722724323074691123869978540979541NCBITaxon:29544243277627912924970361251804264307476795961532848123136254264282573131504752697961029938865990346941713443386263475154155403632997966287732396440426289930629548657558462823823522862871736231399463601068842043601043994796858355847954440426071017316780781440772NCBITaxon:619184023171993128118361003610502318557919080218787837499070384766416922596345910659212257862607046183260705334542260707NCBITaxon:780NCBITaxon:1502451516658457885318702944271211586910371647209285747078124966842528619212638541094343197383379192875NCBITaxon:103592829126806395602959870448300641802242752266437310555242354432243081433614690081502575412983964271191364516996328104121697927262327262430085212389936436801097791356223347471821314269704958331361858030NCBITaxon:5693826882976052779638185833424090611477871274414153681639588858130767494022432640817212816140559204254687613837081274423434271285099011912744201274426519534911370249240543734131641660817562615282352567506028532189517731193501846451450511492412153239564818822970915800257125614801549031384736031423166385962859631015101450520493227591428693732664179332648373153NCBITaxon:96152972210976771412621121114493760833341911079276283332100658123756126662555592924847914869177492003600946143534073120325835247225944751750375451367830649908515151438992NCBITaxon:1155237453747321230310000-0001-8882-8619