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• W3010322897 abstract "HNF4α 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. HNF4α 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. Nuclear receptors (NR) 1The abbreviations used are:NRNuclear receptorsDBDDNA-binding domainLBDligand binding domainHFN 4αHepatocyte Nuclear Factor 4 alphaDR1Direct Repeat 1. 1The abbreviations used are:NRNuclear receptorsDBDDNA-binding domainLBDligand binding domainHFN 4αHepatocyte Nuclear Factor 4 alphaDR1Direct Repeat 1. represent a class of transcription factors that encompasses 48 proteins in humans (1Zhang Z. Burch P.E. Cooney A.J. Lanz R.B. Pereira F.A. Wu J. Gibbs R.A. Weinstock G. Wheeler D.A. Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution from the rat genome.Genome Res. 2004; 14: 580-590Crossref PubMed Scopus (171) Google Scholar). The nomenclature surrounding the superfamily of NR, based on their phylogeny, consists of six subfamilies comprised of several groups (Nuclear Receptors Nomenclature Committee, 1999). NR share a structural organization of five to six distinct regions designated from A to F (2Robinson-Rechavi M. Escriva Garcia H. Laudet V. The nuclear receptor superfamily.J. Cell Sci. 2003; 116: 585-586Crossref PubMed Scopus (367) Google Scholar). The A/B region, located at the N-terminal end, is highly variable between the various NR. It typically contains an AF-1 transactivation region (Activation Function) which is active independently of ligand binding and allows the interaction of the receptor with various coregulators and other transcription factors (3Lavery D.N. McEwan I.J. Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations.Biochem. J. 2005; 391: 449-464Crossref PubMed Scopus (153) Google Scholar). The C domain, or DNA-binding domain (DBD), is the most conserved among NR. It allows the recognition of specific DNA response elements via two cysteine-rich zinc finger motifs (C-X2-C-X13-C-X2-C and C-X5-C-X9-C-X2-C) (2Robinson-Rechavi M. Escriva Garcia H. Laudet V. The nuclear receptor superfamily.J. Cell Sci. 2003; 116: 585-586Crossref PubMed Scopus (367) Google Scholar). These response elements are composed of repeated or inverted hexameric DNA sequences and separated by linkers varying from one to five nucleotides in length (4Khorasanizadeh S. Rastinejad F. Nuclear-receptor interactions on DNA-response elements.Trends Biochem. Sci. 2001; 26: 384-390Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The D region, also called hinge region, is less conserved and its main function is to facilitate free rotation between the DBD and the ligand binding domain (LBD). A nuclear localization signal (NLS) contained in this region participates in the regulation of the subcellular distribution of NR (5Germain P. Staels B. Dacquet C. Spedding M. Laudet V. Overview of nomenclature of nuclear receptors.Pharmacol. Rev. 2006; 58: 685-704Crossref PubMed Scopus (460) Google Scholar). The E domain, or LBD, consists of a hydrophobic pouch for binding a multitude of small lipophilic molecules such as steroid hormones, phospholipids, fatty acids and xenobiotics (6Pawlak M. Lefebvre P. Staels B. General molecular biology and architecture of nuclear receptors.Curr. Top Med. Chem. 2012; 12: 486-504Crossref PubMed Scopus (90) Google Scholar). Some NR for which no ligand has yet been identified are considered orphan receptors (7Giguere V. Orphan nuclear receptors: from gene to function.Endocr. Rev. 1999; 20: 689-725Crossref PubMed Scopus (714) Google Scholar). A second AF-2 activator region is in the LBD. In contrast to the AF-1 region, the activity of the AF-2 region is dependent on ligand binding to LBD. This induces a conformational change in the LBD, generating a pocket that can interact with the LXXLL motif present on a host of transcriptional coactivators (8Heery D.M. Kalkhoven E. Hoare S. Parker M.G. A signature motif in transcriptional co-activators mediates binding to nuclear receptors.Nature. 1997; 387: 733-736Crossref PubMed Scopus (1772) Google Scholar). The LBD, like the DBD, contains an important interface for receptor dimerization. Finally, at the C terminus of the NR is the F domain. Because of its highly variable sequence, the exact function of the F domain remains to be established and several NR have no F domain (9Patel S.R. Skafar D.F. Modulation of nuclear receptor activity by the F domain.Mol. Cell. Endocrinol. 2015; 418: 298-305Crossref PubMed Scopus (23) Google Scholar). Nevertheless, the deletion of this domain in those receptors bearing the domain have revealed its importance in certain instances in connection with various functions such as dimerization, activation and interaction with different coregulators (9Patel S.R. Skafar D.F. Modulation of nuclear receptor activity by the F domain.Mol. Cell. Endocrinol. 2015; 418: 298-305Crossref PubMed Scopus (23) Google Scholar). Nuclear receptors DNA-binding domain ligand binding domain Hepatocyte Nuclear Factor 4 alpha Direct Repeat 1. Nuclear receptors DNA-binding domain ligand binding domain Hepatocyte Nuclear Factor 4 alpha Direct Repeat 1. HNF4α (Hepatocyte Nuclear Factor 4 alpha) (also referred to as NR2A1) is a transcription factor of the nuclear receptor family that was initially identified as a regulator of liver-specific gene expression (10Costa R.H. Grayson D.R. Darnell Jr., J.E. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes.Mol. Cell. Biol. 1989; 9: 1415-1425Crossref PubMed Scopus (428) Google Scholar, 11Sladek F.M. Zhong W.M. Lai E. Darnell Jr., J.E. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (854) Google Scholar). Since its initial discovery in the liver, HNF4α has also been detected in the kidneys, pancreas, stomach, small intestine and colon (12Tanaka T. Jiang S. Hotta H. Takano K. Iwanari H. Sumi K. Daigo K. Ohashi R. Sugai M. Ikegame C. Umezu H. Hirayama Y. Midorikawa Y. Hippo Y. Watanabe A. Uchiyama Y. Hasegawa G. Reid P. Aburatani H. Hamakubo T. Sakai J. Naito M. Kodama T. Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.J. Pathol. 2006; 208: 662-672Crossref PubMed Scopus (130) Google Scholar). HNF4α is crucial for the development and maintenance of hepatocyte function, including lipid homeostasis, transport and metabolism, as well as the detoxification of xenobiotics (13Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis.Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (862) Google Scholar, 14Wortham M. Czerwinski M. He L. Parkinson A. Wan Y.J. Expression of constitutive androstane receptor, hepatic nuclear factor 4 alpha, and P450 oxidoreductase genes determines interindividual variability in basal expression and activity of a broad scope of xenobiotic metabolism genes in the human liver.Drug Metab. Dispos. 2007; 35: 1700-1710Crossref PubMed Scopus (116) Google Scholar, 15Yin L. Ma H. Ge X. Edwards P.A. Zhang Y. Hepatic hepatocyte nuclear factor 4alpha is essential for maintaining triglyceride and cholesterol homeostasis.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 328-336Crossref PubMed Scopus (109) Google Scholar). Additional functions for HNF4α in the gut and pancreas have also emerged (16Eeckhoute J. Moerman E. Bouckenooghe T. Lukoviak B. Pattou F. Formstecher P. Kerr-Conte J. Vandewalle B. Laine B. Hepatocyte nuclear factor 4 alpha isoforms originated from the P1 promoter are expressed in human pancreatic beta-cells and exhibit stronger transcriptional potentials than P2 promoter-driven isoforms.Endocrinology. 2003; 144: 1686-1694Crossref PubMed Scopus (66) Google Scholar, 17Wang H. Maechler P. Antinozzi P.A. Hagenfeldt K.A. Wollheim C.B. Hepatocyte nuclear factor 4alpha regulates the expression of pancreatic beta -cell genes implicated in glucose metabolism and nutrient-induced insulin secretion.J. Biol. Chem. 2000; 275: 35953-35959Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 18Drewes T. Senkel S. Holewa B. Ryffel G.U. Human hepatocyte nuclear factor 4 isoforms are encoded by distinct and differentially expressed genes.Mol. Cell. Biol. 1996; 16: 925-931Crossref PubMed Scopus (192) Google Scholar). In contrast to other types of NR, HNF4α is constitutively localized in the nucleus and does not require binding of a ligand to homodimerize and interact with the response elements of its target genes (19Yuan X. Ta T.C. Lin M. Evans J.R. Dong Y. Bolotin E. Sherman M.A. Forman B.M. Sladek F.M. Identification of an endogenous ligand bound to a native orphan nuclear receptor.PLoS ONE. 2009; 4: e5609Crossref PubMed Scopus (148) Google Scholar). HNF4α has long been considered an orphan nuclear receptor, although crystallography of the LBD initially revealed the presence of fatty acids of various compositions bound at the level of the ligand binding pocket of HNF4α (20Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. Crystal structure of the HNF4 alpha ligand binding domain in complex with endogenous fatty acid ligand.J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Subsequent studies have identified linoleic acid, a long chain polyunsaturated fatty acid (C18: 2ω6), as the molecule preferentially binding to its LBD (19Yuan X. Ta T.C. Lin M. Evans J.R. Dong Y. Bolotin E. Sherman M.A. Forman B.M. Sladek F.M. Identification of an endogenous ligand bound to a native orphan nuclear receptor.PLoS ONE. 2009; 4: e5609Crossref PubMed Scopus (148) Google Scholar); however, this binding is reversible and does not modulate the transcriptional activity of HNF4α. The nature of the HNF4α ligand therefore remains controversial, because linoleic acid is endogenously present and does not appear necessary for receptor activity, unlike the typical mode of action of NR requiring binding to their ligand. HNF4α recognizes DR1 sites (Direct Repeat 1), consisting of two repeated hexameric half-sites separated by a nucleotide, typically an adenosine (21Fang B. Mane-Padros D. Bolotin E. Jiang T. Sladek F.M. Identification of a binding motif specific to HNF4 by comparative analysis of multiple nuclear receptors.Nucleic Acids Res. 2012; 40: 5343-5356Crossref PubMed Scopus (75) Google Scholar). HNF4α also recognizes direct repeats separated by two nucleotides (DR2), but with lower specificity (22Jiang G. Sladek F.M. The DNA binding domain of hepatocyte nuclear factor 4 mediates cooperative, specific binding to DNA and heterodimerization with the retinoid X receptor alpha.J. Biol. Chem. 1997; 272: 1218-1225Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The consensus sequence of its half-sites, AGGTCA, is shared by most nonsteroidal NR (21Fang B. Mane-Padros D. Bolotin E. Jiang T. Sladek F.M. Identification of a binding motif specific to HNF4 by comparative analysis of multiple nuclear receptors.Nucleic Acids Res. 2012; 40: 5343-5356Crossref PubMed Scopus (75) Google Scholar). HNF4α is an exclusive homodimer, this form being stably found in solution and necessary to bind DNA (23Jiang G. Nepomuceno L. Hopkins K. Sladek F.M. Exclusive homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new subclass of nuclear receptors.Mol. Cell. Biol. 1995; 15: 5131-5143Crossref PubMed Scopus (173) Google Scholar). However, both RXRα/β/γ and RARα nuclear receptors, known for their ability to form heterodimers with several NR, do not assemble into heterodimers with HNF4α (23Jiang G. Nepomuceno L. Hopkins K. Sladek F.M. Exclusive homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new subclass of nuclear receptors.Mol. Cell. Biol. 1995; 15: 5131-5143Crossref PubMed Scopus (173) Google Scholar, 24Lee S. Privalsky M.L. Multiple mutations contribute to repression by the v-Erb A oncoprotein.Oncogene. 2005; 24: 6737-6752Crossref PubMed Scopus (11) Google Scholar). Alternative splicing is a major source of cellular protein diversity. The estimated percentage of human gene products undergoing alternative splicing has been proposed to be as high as 95% of multiexon genes, although it is still unclear how many of these splicing variants are expressed or functional (25Pan Q. Shai O. Lee L.J. Frey B.J. Blencowe B.J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.Nat. Genet. 2008; 40: 1413-1415Crossref PubMed Scopus (2555) Google Scholar). However, very few of these alternative protein isoforms have well-characterized cellular functions, given that studies on these proteins have either mostly concentrated on a single isoform, or do not specify which isoform was under study, hence leading to discrepancies or contradictions in protein function (26Kelemen O. Convertini P. Zhang Z. Wen Y. Shen M. Falaleeva M. Stamm S. Function of alternative splicing.Gene. 2013; 514: 1-30Crossref PubMed Scopus (491) Google Scholar). In addition, many alternatively spliced transcripts studied for protein-protein interaction by yeast two-hybrid assay were shown to display significant differences between reference and alternative isoforms, with many alternative isoforms interacting with different protein partners (27Yang X. Coulombe-Huntington J. Kang S. Sheynkman G.M. Hao T. Richardson A. Sun S. Yang F. Shen Y.A. Murray R.R. Spirohn K. Begg B.E. Duran-Frigola M. MacWilliams A. Pevzner S.J. Zhong Q. Trigg S.A. Tam S. Ghamsari L. Sahni N. Yi S. Rodriguez M.D. Balcha D. Tan G. Costanzo M. Andrews B. Boone C. Zhou X.J. Salehi-Ashtiani K. Charloteaux B. Chen A.A. Calderwood M.A. Aloy P. Roth F.P. Hill D.E. Iakoucheva L.M. Xia Y. Vidal M. Widespread expansion of protein interaction capabilities by alternative splicing.Cell. 2016; 164: 805-817Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). These differences in protein complexes underline the importance of considering each protein isoform to understand its unique role(s). In the present study, the transcriptional functions of the 12 annotated isoforms of HNF4α (28Babeu J.P. Boudreau F. Hepatocyte nuclear factor 4-alpha involvement in liver and intestinal inflammatory networks.World J Gastroenterol. 2014; 20: 22-30Crossref PubMed Scopus (120) Google Scholar, 29Sladek F.M. Seidel S.D. Hepatocyte nuclear factor 4α.in: Burris T.P. McCabe E.R.B. Nuclear Receptors and Genetic Disease. Academic Press, San Diego, CA2001: 309-361Google Scholar) were specifically characterized by generating stable lines expressing each HNF4α isoform in HCT 116 cells. The entire transcriptome associated with each isoform was analyzed by RNA sequencing, as well as their respective proteome by a BioID approach coupled to quantitative mass spectrometry. This analysis integrating the vast amount of transcriptomic and proteomic data enabled the identification of transcriptional regulatory mechanisms specific to each isoform, demonstrating the importance of considering all isoforms which can exhibit distinct functions. All cell lines were obtained from the ATCC. HCT 116 (human colorectal cancer (hCRC)), Caco-2/15 (hCRC), Capan-2 (human pancreatic adenoma), HepG2 (human hepatocellular carcinoma) and 293T (transformed human embryonic kidney) cell lines were cultured in DMEM. AsPC-1 (human pancreatic adenocarcinoma), COLO 205 (hCRC), and DLD-1 (hCRC) cell lines were cultured in RPMI. The T84 (hCRC) cell line was cultured in DMEM/F-12. The LoVo (hCRC) cell line was cultured in F-12K. The HT-29 (hCRC) cell line was cultured in McCoy's 5A. All cultured media were supplemented with 10% FBS and cell lines were grown in a humidified incubator at 37 °C with 5% CO2. The Human Digestive System MTC Panel cDNA library was purchased from Clontech Laboratories (Mountain View). The cDNA preparations found in this library are derived from the combination of cDNAs from several healthy Caucasian individuals between 18 and 61 years of age and were provided at a concentration of 1.0 ng/μl following first-strand cDNA preparation for each tissue. A total of 12 tissues of the digestive system were included in the bank: liver, stomach, esophagus, duodenum, jejunum, ileum, ileocecum, cecum, ascending, transverse and descending colon, as well as the rectum. Total RNA of HCT 116, Caco-2/15, T84, COLO 205, LoVo, DLD-1, HT-29, HepG2, AsPC-1, Capan-2, and HCT 116 HNF4α (1Zhang Z. Burch P.E. Cooney A.J. Lanz R.B. Pereira F.A. Wu J. Gibbs R.A. Weinstock G. Wheeler D.A. Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution from the rat genome.Genome Res. 2004; 14: 580-590Crossref PubMed Scopus (171) Google Scholar, 2Robinson-Rechavi M. Escriva Garcia H. Laudet V. The nuclear receptor superfamily.J. Cell Sci. 2003; 116: 585-586Crossref PubMed Scopus (367) Google Scholar, 3Lavery D.N. McEwan I.J. Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations.Biochem. J. 2005; 391: 449-464Crossref PubMed Scopus (153) Google Scholar, 4Khorasanizadeh S. Rastinejad F. Nuclear-receptor interactions on DNA-response elements.Trends Biochem. Sci. 2001; 26: 384-390Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 5Germain P. Staels B. Dacquet C. Spedding M. Laudet V. Overview of nomenclature of nuclear receptors.Pharmacol. Rev. 2006; 58: 685-704Crossref PubMed Scopus (460) Google Scholar, 6Pawlak M. Lefebvre P. Staels B. General molecular biology and architecture of nuclear receptors.Curr. Top Med. Chem. 2012; 12: 486-504Crossref PubMed Scopus (90) Google Scholar, 7Giguere V. Orphan nuclear receptors: from gene to function.Endocr. Rev. 1999; 20: 689-725Crossref PubMed Scopus (714) Google Scholar, 8Heery D.M. Kalkhoven E. Hoare S. Parker M.G. A signature motif in transcriptional co-activators mediates binding to nuclear receptors.Nature. 1997; 387: 733-736Crossref PubMed Scopus (1772) Google Scholar, 9Patel S.R. Skafar D.F. Modulation of nuclear receptor activity by the F domain.Mol. Cell. Endocrinol. 2015; 418: 298-305Crossref PubMed Scopus (23) Google Scholar, 10Costa R.H. Grayson D.R. Darnell Jr., J.E. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes.Mol. Cell. Biol. 1989; 9: 1415-1425Crossref PubMed Scopus (428) Google Scholar, 11Sladek F.M. Zhong W.M. Lai E. Darnell Jr., J.E. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (854) Google Scholar, 12Tanaka T. Jiang S. Hotta H. Takano K. Iwanari H. Sumi K. Daigo K. Ohashi R. Sugai M. Ikegame C. Umezu H. Hirayama Y. Midorikawa Y. Hippo Y. Watanabe A. Uchiyama Y. Hasegawa G. Reid P. Aburatani H. Hamakubo T. Sakai J. Naito M. Kodama T. Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.J. Pathol. 2006; 208: 662-672Crossref PubMed Scopus (130) Google Scholar) -GFP cell lines was isolated using RNeasy RNA isolation kit (Qiagen). The stable HCT 116 HNF4α (1Zhang Z. Burch P.E. Cooney A.J. Lanz R.B. Pereira F.A. Wu J. Gibbs R.A. Weinstock G. Wheeler D.A. Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution from the rat genome.Genome Res. 2004; 14: 580-590Crossref PubMed Scopus (171) Google Scholar, 2Robinson-Rechavi M. Escriva Garcia H. Laudet V. The nuclear receptor superfamily.J. Cell Sci. 2003; 116: 585-586Crossref PubMed Scopus (367) Google Scholar, 3Lavery D.N. McEwan I.J. Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations.Biochem. J. 2005; 391: 449-464Crossref PubMed Scopus (153) Google Scholar, 4Khorasanizadeh S. Rastinejad F. Nuclear-receptor interactions on DNA-response elements.Trends Biochem. Sci. 2001; 26: 384-390Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 5Germain P. Staels B. Dacquet C. Spedding M. Laudet V. Overview of nomenclature of nuclear receptors.Pharmacol. Rev. 2006; 58: 685-704Crossref PubMed Scopus (460) Google Scholar, 6Pawlak M. Lefebvre P. Staels B. General molecular biology and architecture of nuclear receptors.Curr. Top Med. Chem. 2012; 12: 486-504Crossref PubMed Scopus (90) Google Scholar, 7Giguere V. Orphan nuclear receptors: from gene to function.Endocr. Rev. 1999; 20: 689-725Crossref PubMed Scopus (714) Google Scholar, 8Heery D.M. Kalkhoven E. Hoare S. Parker M.G. A signature motif in transcriptional co-activators mediates binding to nuclear receptors.Nature. 1997; 387: 733-736Crossref PubMed Scopus (1772) Google Scholar, 9Patel S.R. Skafar D.F. Modulation of nuclear receptor activity by the F domain.Mol. Cell. Endocrinol. 2015; 418: 298-305Crossref PubMed Scopus (23) Google Scholar, 10Costa R.H. Grayson D.R. Darnell Jr., J.E. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes.Mol. Cell. Biol. 1989; 9: 1415-1425Crossref PubMed Scopus (428) Google Scholar, 11Sladek F.M. Zhong W.M. Lai E. Darnell Jr., J.E. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (854) Google Scholar, 12Tanaka T. Jiang S. Hotta H. Takano K. Iwanari H. Sumi K. Daigo K. Ohashi R. Sugai M. Ikegame C. Umezu H. Hirayama Y. Midorikawa Y. Hippo Y. Watanabe A. Uchiyama Y. Hasegawa G. Reid P. Aburatani H. Hamakubo T. Sakai J. Naito M. Kodama T. Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.J. Pathol. 2006; 208: 662-672Crossref PubMed Scopus (130) Google Scholar) -GFP cell lines were induced for 48 h with 2.5 μg/ml doxycycline (Clontech Laboratories, Mountain View) before extraction of total RNAs. cDNA synthesis was performed with the SuperScript IV-RT reverse transcriptase enzyme (Thermo Fisher Scientific, Waltham). Two micrograms of RNA were added in a total volume of 10 μl by supplementing with DEPC water. A mixture containing 2.4 μl of 0.5 μg/μl poly (dT) oligos (Amersham Biosciences, Little Chalfont, United Kingdom) and 0.8 μl of 25 mm dNTPs (Amersham Biosciences, Little Chalfont, United Kingdom) was added to the RNA, then heated for 5 min at 75 °C and placed on ice for an additional 5 min. A 10 μl volume of RT reaction mixture according to the manufacturer's conditions was added to the RNA. The reaction was incubated for 1 h at 50 °C, before inactivating the SuperScript IV-RT by heating for 5 min at 95 °C. The cDNA samples were subsequently stored at −20 °C. Oligonucleotides specific for each isoform and the reference genes were obtained from Integrated DNA Technologies (IDT, San Jose) (supplemental TableS6). The cDNA from HCT 116 HNF4α (1Zhang Z. Burch P.E. Cooney A.J. Lanz R.B. Pereira F.A. Wu J. Gibbs R.A. Weinstock G. Wheeler D.A. Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution from the rat genome.Genome Res. 2004; 14: 580-590Crossref PubMed Scopus (171) Google Scholar, 2Robinson-Rechavi M. Escriva Garcia H. Laudet V. The nuclear receptor superfamily.J. Cell Sci. 2003; 116: 585-586Crossref PubMed Scopus (367) Google Scholar, 3Lavery D.N. McEwan I.J. Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations.Biochem. J. 2005; 391: 449-464Crossref PubMed Scopus (153) Google Scholar, 4Khorasanizadeh S. Rastinejad F. Nuclear-receptor interactions on DNA-response elements.Trends Biochem. Sci. 2001; 26: 384-390Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 5Germain P. Staels B. Dacquet C. Spedding M. Laudet V. Overview of nomenclature of nuclear receptors.Pharmacol. Rev. 2006; 58: 685-704Crossref PubMed Scopus (460) Google Scholar, 6Pawlak M. Lefebvre P. Staels B. General molecular biology and architecture of nuclear receptors.Curr. Top Med. Chem. 2012; 12: 486-504Crossref PubMed Scopus (90) Google Scholar, 7Giguere V. Orphan nuclear receptors: from gene to function.Endocr. Rev. 1999; 20: 689-725Crossref PubMed Scopus (714) Google Scholar, 8Heery D.M. Kalkhoven E. Hoare S. Parker M.G. A signature motif in transcriptional co-activators mediates binding to nuclear receptors.Nature. 1997; 387: 733-736Crossref PubMed Scopus (1772) Google Scholar, 9Patel S.R. Skafar D.F. Modulation of nuclear receptor activity by the F domain.Mol. Cell. Endocrinol. 2015; 418: 298-305Crossref PubMed Scopus (23) Google Scholar, 10Costa R.H. Grayson D.R. Darnell Jr., J.E. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes.Mol. Cell. Biol. 1989; 9: 1415-1425Crossref PubMed Scopus (428) Google Scholar, 11Sladek F.M. Zhong W.M. Lai E. Darnell Jr., J.E. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (854) Google Scholar, 12Tanaka T. Jiang S. Hotta H. Takano K. Iwanari H. Sumi K. Daigo K. Ohashi R. Sugai M. Ikegame C. Umezu H. Hirayama Y. Midorikawa Y. Hippo Y. Watanabe A. Uchiyama Y. Hasegawa G. Reid P. Aburatani H. Hamakubo T. Sakai J. Naito M. Kodama T. Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.J. Pathol. 2006; 208: 662-672Crossref PubMed Scopus (130) Google Scholar) -GFP lines was used as a positive control for each isoform. Expression levels of the HPRT and PUM1 genes were used as references, with an amplification of 26 cycles at a hybridization temperature of 60 °C and an elongation time of 20 s. The expression of the 12 HNF4α isoforms in the different human gastrointestinal tract tissues was assessed by PCR. The primers specific to each HNF4α isoform (supplemental Table S6) were used to assess the expression of the isoforms by PCR in each of the previously listed tissues. The reagents used for the amplification were the same as described previously, the template DNA being in this instance a volume of 3.7 μl of cDNA. The PCR reactions were performed according to the PCR conditions detailed in the previous section, for a first round of 30 cycles. A 10 μl aliquot of the reaction was retrieved, and the remainder of the reaction was supplemented again at 20 μl at the initial reaction concentrations for a second round of PCR of 6 additional cycles. The expression levels of the POLR2A and PSMB2 genes were used as references, with an amplification of 26 cycles. A PCR product corresponding to each isoform was sequenced to ensure the specificity of the amplification. Plasmid pUC19 was digested with the SmaI restriction enzyme (New England Biolabs, Ipswich) in CutSmart buffer (New England Biolabs, Ipswich) for 1 h at 25 °C, and subsequently purified on gel agarose. Ligation between the PCR product and the digested pUC19 plasmid was carried out in a 20:1 ratio (insert: vector) with T4 DNA ligase (New England Biolabs, Ipswich) for 3 h at room temperature. Sequencing was performed via the Genome Sequencing and Genotyping Platform (Université Laval, Quebec, Canada). The 12 HNF4α isoforms were cloned into the donor vector pENTR11 (Thermo Fisher Scientific, Waltham) in three distinct steps consisting in initially cloning the sequence common to the 12 isoforms, inserting the different N-terminal termini (A/B domains) on each side, followed by the different C termini (F domain). Th" @default.
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