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• W3203802214 abstract "CCAAT enhancer binding protein (CEBP) transcription factors (TFs) are known to promote adipocyte differentiation; however, suppressors of CEBP TFs have not been reported thus far. Here, we find that homologous chromosome pairing protein 2 (Hop2) functions as an inhibitor for the TF CEBPα. We found that Hop2 mRNA is highly and specifically expressed in adipose tissue, and that ectopic Hop2 expression suppresses reporter activity induced by CEBP as revealed by DNA transfection. Recombinant and ectopically expressed Hop2 was shown to interact with CEBPα in pull-down and coimmunoprecipitation assays, and interaction between endogenous Hop2 and CEBPα was observed in the nuclei of 3T3 preadipocytes and adipocytes by immunofluorescence and coimmunoprecipitation of nuclear extracts. In addition, Hop2 stable overexpression in 3T3 preadipocytes inhibited adipocyte differentiation and adipocyte marker gene expression. These in vitro data suggest that Hop2 inhibits adipogenesis by suppressing CEBP-mediated transactivation. Consistent with a negative role for Hop2 in adipogenesis, ablation of Hop2 (Hop2−/−) in mice led to increased body weight, adipose volume, adipocyte size, and adipogenic marker gene expression. Adipogenic differentiation of isolated adipose-derived mesenchymal stem cells showed a greater number of lipid droplet–containing colonies formed in Hop2−/− adipose-derived mesenchymal stem cell cultures than in wt controls, which is associated with the increased expression of adipogenic marker genes. Finally, chromatin immunoprecipitation revealed a higher binding activity of endogenous CEBPα to peroxisome proliferator–activated receptor γ, a master adipogenic TF, and a known CEBPα target gene. Therefore, our study identifies for the first time that Hop2 is an intrinsic suppressor of CEBPα and thus adipogenesis in adipocytes. CCAAT enhancer binding protein (CEBP) transcription factors (TFs) are known to promote adipocyte differentiation; however, suppressors of CEBP TFs have not been reported thus far. Here, we find that homologous chromosome pairing protein 2 (Hop2) functions as an inhibitor for the TF CEBPα. We found that Hop2 mRNA is highly and specifically expressed in adipose tissue, and that ectopic Hop2 expression suppresses reporter activity induced by CEBP as revealed by DNA transfection. Recombinant and ectopically expressed Hop2 was shown to interact with CEBPα in pull-down and coimmunoprecipitation assays, and interaction between endogenous Hop2 and CEBPα was observed in the nuclei of 3T3 preadipocytes and adipocytes by immunofluorescence and coimmunoprecipitation of nuclear extracts. In addition, Hop2 stable overexpression in 3T3 preadipocytes inhibited adipocyte differentiation and adipocyte marker gene expression. These in vitro data suggest that Hop2 inhibits adipogenesis by suppressing CEBP-mediated transactivation. Consistent with a negative role for Hop2 in adipogenesis, ablation of Hop2 (Hop2−/−) in mice led to increased body weight, adipose volume, adipocyte size, and adipogenic marker gene expression. Adipogenic differentiation of isolated adipose-derived mesenchymal stem cells showed a greater number of lipid droplet–containing colonies formed in Hop2−/− adipose-derived mesenchymal stem cell cultures than in wt controls, which is associated with the increased expression of adipogenic marker genes. Finally, chromatin immunoprecipitation revealed a higher binding activity of endogenous CEBPα to peroxisome proliferator–activated receptor γ, a master adipogenic TF, and a known CEBPα target gene. Therefore, our study identifies for the first time that Hop2 is an intrinsic suppressor of CEBPα and thus adipogenesis in adipocytes. Adipose tissues play a significant role in the maintenance of metabolic homeostasis. It stores energy in the form of fat, offers insulation and protection to soft vital organs, and produces more than 600 secretory molecules, collectively named adipokines, to regulate food intake, energy expenditure, insulin sensitivity, fat distribution, inflammation, and blood pressure (1Lehr S. Hartwig S. Lamers D. Famulla S. Muller S. Hanisch F.G. Cuvelier C. Ruige J. Eckardt K. Ouwens D.M. Sell H. Eckel J. Identification and validation of novel adipokines released from primary human adipocytes.Mol. Cell. Proteomics. 2012; 11 (M111.010504)Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 2Fasshauer M. Bluher M. Adipokines in health and disease.Trends Pharmacol. Sci. 2015; 36: 461-470Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). However, excessive fat accumulation in obese individuals is a serious pandemic currently affecting 42% of adult Americans (3Hales C.M. Carroll M.D. Fryar C.D. Ogden C.L. Prevalence of obesity and severe obesity among adults: United States, 2017-2018.NCHS Data Brief. 2020; : 1-8Google Scholar). Obesity increases the risk for many diseases, including cardiovascular disease, stroke, type 2 diabetes, physical disability (4Haslam D.W. James W.P. Obesity.Lancet. 2005; 366: 1197-1209Abstract Full Text Full Text PDF PubMed Scopus (3337) Google Scholar, 5Van Gaal L.F. Mertens I.L. De Block C.E. Mechanisms linking obesity with cardiovascular disease.Nature. 2006; 444: 875-880Crossref PubMed Scopus (1897) Google Scholar), and cancer (6LeRoith D. Novosyadlyy R. Gallagher E.J. Lann D. Vijayakumar A. Yakar S. Obesity and type 2 diabetes are associated with an increased risk of developing cancer and a worse prognosis; epidemiological and mechanistic evidence.Exp. Clin. Endocrinol. Diabetes. 2008; 116 Suppl 1: S4-S6Crossref PubMed Scopus (95) Google Scholar). The cellular basis of obesity involves an increased number and size of adipocytes, resulting from proliferation and differentiation of adipocyte progenitor cells or preadipocytes (7Faust I.M. Johnson P.R. Stern J.S. Hirsch J. Diet-induced adipocyte number increase in adult rats: A new model of obesity.Am. J. Physiol. 1978; 235: E279-E286Crossref PubMed Google Scholar, 8Klyde B.J. Hirsch J. Isotopic labeling of DNA in rat adipose tissue: Evidence for proliferating cells associated with mature adipocytes.J. Lipid Res. 1979; 20: 691-704Abstract Full Text PDF PubMed Google Scholar). Adipocytes contribute more than 90% of the adipose tissue volume in the human body and other vertebral animals (9Eto H. Suga H. Matsumoto D. Inoue K. Aoi N. Kato H. Araki J. Yoshimura K. Characterization of structure and cellular components of aspirated and excised adipose tissue.Plast. Reconstr. Surg. 2009; 124: 1087-1097Crossref PubMed Scopus (175) Google Scholar). Understanding the molecular and cellular basis of adipocyte proliferation and differentiation is critical to improving the management of obesity and associated diseases. The molecular regulation of adipocyte differentiation has been extensively investigated. A transcriptional cascade, which was identified 3 decades ago by in vitro studies of the cultured preadipocyte 3T3-L1 cell line (10Cao Z. Umek R.M. McKnight S.L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells.Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1315) Google Scholar, 11Tontonoz P. Hu E. Spiegelman B.M. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor.Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3046) Google Scholar), plays a central role in the regulation of this process. Upon the stimulation of an adipogenic “cocktail” containing insulin and glucocorticoids, two basic (b) leucine zipper (Zip) containing transcription factors (TFs), the CCAAT enhancer binding protein (CEBP) β and CEBPδ, are expressed. The two CEBP proteins form a heterodimer to bind promoters of the peroxisome proliferator–activated receptor γ (PPARγ), a member of the nuclear receptor superfamily of ligand-activated TFs, and CEBPα. The PPARγ and CEBPα can activate themselves and each other to form a feed-forward loop that maintains a high level of PPARγ expression in terminally differentiated adipocytes (12Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. McKeon C. Darlington G.J. Spiegelman B.M. Cross-regulation of C/EBP alpha and PPAR gamma controls the transcriptional pathway of adipogenesis and insulin sensitivity.Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). The PPARγ and CEBPα have been shown by RNA sequencing and chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-Seq) to synergistically activate many key metabolic adipocyte marker genes, including adipocyte protein 2 (aP2), glucose transporter 4 (Glut4), phosphoenolpyruvate carboxykinase (Pepck), leptin (Lep), and adiponectin (An) involved in fatty acid transport, triglyceride synthesis, and insulin sensitivity (13Madsen M.S. Siersbaek R. Boergesen M. Nielsen R. Mandrup S. Peroxisome proliferator-activated receptor gamma and C/EBPalpha synergistically activate key metabolic adipocyte genes by assisted loading.Mol. Cell. Biol. 2014; 34: 939-954Crossref PubMed Scopus (112) Google Scholar). These in vitro findings are supported by genetic data that CEBPβ−/− mice display reduced body fat mass and resist to diet-induced obesity (14Millward C.A. Heaney J.D. Sinasac D.S. Chu E.C. Bederman I.R. Gilge D.A. Previs S.F. Croniger C.M. Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta are protected against diet-induced obesity.Diabetes. 2007; 56: 161-167Crossref PubMed Scopus (72) Google Scholar, 15Schroeder-Gloeckler J.M. Rahman S.M. Janssen R.C. Qiao L. Shao J. Roper M. Fischer S.J. Lowe E. Orlicky D.J. McManaman J.L. Palmer C. Gitomer W.L. Huang W. O'Doherty R.M. Becker T.C. et al.CCAAT/enhancer-binding protein beta deletion reduces adiposity, hepatic steatosis, and diabetes in Lepr(db/db) mice.J. Biol. Chem. 2007; 282: 15717-15729Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and CEBPβ/δ double mutant mice have a greater reduction in body adiposity, accompanied with decreased expression of CEBPα and PPARγ transcripts (16Tanaka T. Yoshida N. Kishimoto T. Akira S. Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene.EMBO J. 1997; 16: 7432-7443Crossref PubMed Scopus (618) Google Scholar, 17Wang N.D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Impaired energy homeostasis in C/EBP alpha knockout mice.Science. 1995; 269: 1108-1112Crossref PubMed Scopus (818) Google Scholar). Similarly, CEBPα−/− or PPARγ−/− mice have no white adipose, and their adipocytes fail to accumulate lipid and express adipocyte marker genes, whereas cell lineage commitment is not affected (12Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. McKeon C. Darlington G.J. Spiegelman B.M. Cross-regulation of C/EBP alpha and PPAR gamma controls the transcriptional pathway of adipogenesis and insulin sensitivity.Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar, 17Wang N.D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Impaired energy homeostasis in C/EBP alpha knockout mice.Science. 1995; 269: 1108-1112Crossref PubMed Scopus (818) Google Scholar, 18Darlington G.J. Wang N. Hanson R.W. C/EBP alpha: A critical regulator of genes governing integrative metabolic processes.Curr. Opin. Genet. Dev. 1995; 5: 565-570Crossref PubMed Scopus (110) Google Scholar, 19Barak Y. Nelson M.C. Ong E.S. Jones Y.Z. Ruiz-Lozano P. Chien K.R. Koder A. Evans R.M. PPAR gamma is required for placental, cardiac, and adipose tissue development.Mol. Cell. 1999; 4: 585-595Abstract Full Text Full Text PDF PubMed Scopus (1585) Google Scholar). Thus, both in vitro and in vivo studies demonstrated this cascade of TFs in which the CEBPβ and CEBPδ activate PPARγ and CEBPα to promote adipocytes to acquire and maintain terminally differentiated phenotypes. In addition to the CEBPs, activating transcription factor 4 (ATF4), a member of the ATF/CREB subfamily of the bZip superfamily subfamilies (20Vallejo M. Ron D. Miller C.P. Habener J.F. C/ATF, a member of the activating transcription factor family of DNA-binding proteins, dimerizes with CAAT/enhancer-binding proteins and directs their binding to cAMP response elements.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4679-4683Crossref PubMed Scopus (205) Google Scholar), has also been shown to promote adipogenesis by dimerizing with CEBPβ in both human mesenchymal stem cell and 3T3-L1 preadipocyte cultures (21Cohen D.M. Won K.J. Nguyen N. Lazar M.A. Chen C.S. Steger D.J. ATF4 licenses C/EBPbeta activity in human mesenchymal stem cells primed for adipogenesis.Elife. 2015; 4e06821Crossref PubMed Google Scholar, 22Yu K. Mo D. Wu M. Chen H. Chen L. Li M. Chen Y. Activating transcription factor 4 regulates adipocyte differentiation via altering the coordinate expression of CCATT/enhancer binding protein beta and peroxisome proliferator-activated receptor gamma.FEBS J. 2014; 281: 2399-2409Crossref PubMed Scopus (24) Google Scholar). Supporting these in vitro findings, Atf4−/− mice are lean and resist diet-induced obesity (23Yang X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li L. Brancorsini S. Sassone-Corsi P. Townes T.M. Hanauer A. Karsenty G. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin-Lowry syndrome.Cell. 2004; 117: 387-398Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 24Seo J. Fortuno 3rd, E.S. Suh J.M. Stenesen D. Tang W. Parks E.J. Adams C.M. Townes T. Graff J.M. Atf4 regulates obesity, glucose homeostasis, and energy expenditure.Diabetes. 2009; 58: 2565-2573Crossref PubMed Scopus (145) Google Scholar, 25Wang C. Huang Z. Du Y. Cheng Y. Chen S. Guo F. ATF4 regulates lipid metabolism and thermogenesis.Cell Res. 2010; 20: 174-184Crossref PubMed Scopus (79) Google Scholar, 26Xiao G. Zhang T. Yu S. Lee S. Calabuig-Navarro V. Yamauchi J. Ringquist S. Dong H.H. ATF4 protein deficiency protects against high fructose-induced hypertriglyceridemia in mice.J. Biol. Chem. 2013; 288: 25350-25361Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 27Yoshizawa T. Hinoi E. Jung D.Y. Kajimura D. Ferron M. Seo J. Graff J.M. Kim J.K. Karsenty G. The transcription factor ATF4 regulates glucose metabolism in mice through its expression in osteoblasts.J. Clin. Invest. 2009; 119: 2807-2817Crossref PubMed Scopus (169) Google Scholar). Using the bZip motif as a bait, we recently identified that homologous pairing protein 2 (Hop2) also binds to ATF4 (28Zhang Y. Lin T. Lian N. Tao H. Li C. Li L. Yang X. Hop2 interacts with ATF4 to promote osteoblast differentiation.J. Bone Miner. Res. 2019; 34: 2287-2300Crossref PubMed Scopus (7) Google Scholar). Hop2 was originally identified in yeast as a meiosis-specific protein required for interchromosomal interaction/paring during meiosis (29Leu J.Y. Chua P.R. Roeder G.S. The meiosis-specific Hop2 protein of S. cerevisiae ensures synapsis between homologous chromosomes.Cell. 1998; 94: 375-386Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). A human homolog of Hop2 has been identified by its suppression of the HIV replication suppressor TBP1 (30Ijichi H. Tanaka T. Nakamura T. Yagi H. Hakuba A. Sato M. Molecular cloning and characterization of a human homologue of TBPIP, a BRCA1 locus-related gene.Gene. 2000; 248: 99-107Crossref PubMed Scopus (15) Google Scholar, 31Tanaka T. Nakamura T. Takagi H. Sato M. Molecular cloning and characterization of a novel TBP-1 interacting protein (TBPIP):enhancement of TBP-1 action on Tat by TBPIP.Biochem. Biophys. Res. Commun. 1997; 239: 176-181Crossref PubMed Scopus (20) Google Scholar). Somatic mutations in Hop2 are prevalent in sporadic breast, ovarian, and fallopian tube cancer (32Peng M. Yang Z. Zhang H. Jaafar L. Wang G. Liu M. Flores-Rozas H. Xu J. Mivechi N.F. Ko L. GT198 splice variants display dominant-negative activities and are induced by inactivating mutations.Genes Cancer. 2013; 4: 26-38Crossref PubMed Scopus (13) Google Scholar, 33Peng M. Zhang H. Jaafar L. Risinger J.I. Huang S. Mivechi N.F. Ko L. Human ovarian cancer stroma contains luteinized theca cells harboring tumor suppressor gene GT198 mutations.J. Biol. Chem. 2013; 288: 33387-33397Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). In mammals, the mRNA of Hop2 is robustly expressed in the testis and genetic ablation of Hop2 in mice, in which the first three exons of the Hop2 gene were replaced with the neomycin-resistant gene (Neo), leads to infertility because of a failure in the formation of haploid gametes (34Petukhova G.V. Romanienko P.J. Camerini-Otero R.D. The Hop2 protein has a direct role in promoting interhomolog interactions during mouse meiosis.Dev. Cell. 2003; 5: 927-936Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Previous cell culture studies found a nonmeiotic role that Hop2 plays by acting as a tissue-specific coactivator of nuclear receptors, which is consistent with the observation that the expression of Hop2 mRNA was broader than originally reported (28Zhang Y. Lin T. Lian N. Tao H. Li C. Li L. Yang X. Hop2 interacts with ATF4 to promote osteoblast differentiation.J. Bone Miner. Res. 2019; 34: 2287-2300Crossref PubMed Scopus (7) Google Scholar). Our previous work demonstrated that in osteoblasts, the binding of Hop2 to ATF4 stimulates ATF4-mediated transactivation and promotes osteoblast differentiation in vitro and in vivo. Genetic interaction between ATF4 and Hop2 was best demonstrated by osteopenia (low bone mass) in the Atf4+/−:Hop2+/− double heterozygous (het) mice, which is identical to a low bone mass in Hop2−/− mice and milder than osteoporosis in Atf4−/− mice (23Yang X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li L. Brancorsini S. Sassone-Corsi P. Townes T.M. Hanauer A. Karsenty G. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin-Lowry syndrome.Cell. 2004; 117: 387-398Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 28Zhang Y. Lin T. Lian N. Tao H. Li C. Li L. Yang X. Hop2 interacts with ATF4 to promote osteoblast differentiation.J. Bone Miner. Res. 2019; 34: 2287-2300Crossref PubMed Scopus (7) Google Scholar). Interestingly, Hop2−/− mice display an increased body weight and adipose tissue mass under normal chow, and this abnormal adipose phenotype is absent in the Atf4+/−:Hop2+/− double het mice. This important observation led us to hypothesize that Hop2 in adipocytes interacts with one or more of the three adipocyte bZip TFs to regulate adipogenesis. This hypothesis is further supported by our previous finding that ATF4 is an extremely labile protein that is undetectable in adipose tissue (35Ducy P. Karsenty G. Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.Mol. Cell. Biol. 1995; 15: 1858-1869Crossref PubMed Scopus (510) Google Scholar, 36Schinke T. Karsenty G. Characterization of Osf1, an osteoblast-specific transcription factor binding to a critical cis-acting element in the mouse osteocalcin promoters.J. Biol. Chem. 1999; 274: 30182-30189Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 37Yang X. Karsenty G. ATF4, the osteoblast accumulation of which is determined post-translationally, can induce osteoblast-specific gene expression in non-osteoblastic cells.J. Biol. Chem. 2004; 279: 47109-47114Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In this study, we identified another Hop2-interacting protein, CEBPα, in adipocytes. We provide data demonstrating that the binding of Hop2 to CEBPα suppresses the DNA binding of CEBPα to its target genes. In vivo, ablation of Hop2 in mice leads to increased adiposity compared with wt or het (+/−) littermates, whereas, in cells, forced expression of Hop2 in preadipocytes suppresses adipocyte differentiation. Consistent with an inhibitory role, the Hop2 protein level is high in preadipocytes but low in differentiated adipocytes. Thus, our study for the first time reveals a novel function of Hop2 in adipose tissues. With the C-terminal bZip motif of ATF4 on a yeast hybrid screening of mouse complementary DNA (cDNA) library, we previously identified an atypical Zip-containing protein Hop2 that binds and stimulates ATF4-dependent transcription and osteoblast differentiation (28Zhang Y. Lin T. Lian N. Tao H. Li C. Li L. Yang X. Hop2 interacts with ATF4 to promote osteoblast differentiation.J. Bone Miner. Res. 2019; 34: 2287-2300Crossref PubMed Scopus (7) Google Scholar). Intriguingly, our data revealed that this well-characterized meiotic-specific protein is also highly and specifically expressed in adipose tissue among 13 somatic tissues examined (28Zhang Y. Lin T. Lian N. Tao H. Li C. Li L. Yang X. Hop2 interacts with ATF4 to promote osteoblast differentiation.J. Bone Miner. Res. 2019; 34: 2287-2300Crossref PubMed Scopus (7) Google Scholar). Quantitative RT–PCR (qRT–PCR) of total RNA isolated from 2-month-old male mouse tissues revealed that Hop2 mRNA level is about 10 and 12 times higher in brown and white fat adiposes than in skin, bone, lung, and kidney, but approximately ten times lower than in testis (Fig. 1A). The endogenous expression of Hop2 in preadipocytes and adipocytes was then validated by qRT–PCR and Western blot with validated antibody (Fig. S1) analyses of differentiating primary adipocyte-derived mesenchymal stem cells (aMSCs) and 3T3-L1 preadipocytes, and interestingly, Hop2 mRNA and protein levels are higher in undifferentiated 3T3-L1 preadipocytes than in fully differentiated adipocytes (Fig. 1, B–G). These results suggest an intrinsic role that Hop2 plays in the regulation of adipocyte differentiation and function. To begin to address the functional relevance of the expression of Hop2 in adipocyte differentiation, we tested whether Hop2 influences the CEBP-dependent transactivation activity in vitro. The three adipocyte CEBP isoforms, CEBPα, CEBPβ, and CEBPδ, share over 90% homology in their DNA-binding domains and recognize and activate the same consensus sequences (38Osada S. Yamamoto H. Nishihara T. Imagawa M. DNA binding specificity of the CCAAT/enhancer-binding protein transcription factor family.J. Biol. Chem. 1996; 271: 3891-3896Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Two reporter constructs, C3-Luc and Leptin-Luc, were used in our DNA cotransfection assays. The complement 3 (C3) contains a promoter fragment covering −1 to −199 bp of the human C3 with two direct repeats of a CEBP consensus binding sequence at −110 (TTGAGAAAT) and −123 (TTAGGAAAT) (39Wilson D.R. Juan T.S. Wilde M.D. Fey G.H. Darlington G.J. A 58-base-pair region of the human C3 gene confers synergistic inducibility by interleukin-1 and interleukin-6.Mol. Cell. Biol. 1990; 10: 6181-6191Crossref PubMed Scopus (66) Google Scholar, 40Juan T.S. Wilson D.R. Wilde M.D. Darlington G.J. Participation of the transcription factor C/EBP delta in the acute-phase regulation of the human gene for complement component C3.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2584-2588Crossref PubMed Scopus (121) Google Scholar) (Fig. 1H), respectively, whereas the Leptin-Luc reporter construct contains a 456-bp mouse Leptin proximal promoter (Fig. 1I) with a perfect CEBPα binding palindrome (TTGCGCAA) (41He Y. Chen H. Quon M.J. Reitman M. The mouse obese gene. Genomic organization, promoter activity, and activation by CCAAT/enhancer-binding protein alpha.J. Biol. Chem. 1995; 270: 28887-28891Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Reporter assays showed that cotransfection of CEBPα, CEBPβ, and CEBPδ expression plasmids, individually, with the C3-Luc increases the reporter's activity by 6.1-, 4.5-, and 5.7-fold, respectively. Hop2 cotransfection brought down the CEBP-induced reporter's activity to 1.6-, 3.1-, and 2.2-fold, corresponding to a 74%, 31%, and 61% suppression on CEBPα-, CEBPβ-, and CEBPδ-dependent activation, respectively (Fig. 1, J–L). This inhibitory effect is specific for Hop2 because cotransfection of ATF4, a bZip TF reported dimerizing with CEBPβ (21Cohen D.M. Won K.J. Nguyen N. Lazar M.A. Chen C.S. Steger D.J. ATF4 licenses C/EBPbeta activity in human mesenchymal stem cells primed for adipogenesis.Elife. 2015; 4e06821Crossref PubMed Google Scholar, 22Yu K. Mo D. Wu M. Chen H. Chen L. Li M. Chen Y. Activating transcription factor 4 regulates adipocyte differentiation via altering the coordinate expression of CCATT/enhancer binding protein beta and peroxisome proliferator-activated receptor gamma.FEBS J. 2014; 281: 2399-2409Crossref PubMed Scopus (24) Google Scholar) and Hop2 (28Zhang Y. Lin T. Lian N. Tao H. Li C. Li L. Yang X. Hop2 interacts with ATF4 to promote osteoblast differentiation.J. Bone Miner. Res. 2019; 34: 2287-2300Crossref PubMed Scopus (7) Google Scholar), had no significant suppression on the CEBPα- and CEBPβ-dependent reporter's activity, except for a 30% inhibition on CEBPδ-induced transactivation. The Hop2-specific suppression on the CEBP-mediated transactivation was also observed on the Leptin-Luc reporter, in which Hop2 reduces CEBP-induced reporter's activity from 35.7- to 12.2-fold, 21.0- to 1.7-fold, and 31.9- to 1.4-fold representing a 66%, 92%, and 97% inhibition for CEBPα, CEBPβ, and CEBPδ, respectively (Fig. 1, M–O). Again, ATF4 cotransfection did not cause a significant change in the CEBP-dependent activation. These data indicated that Hop2 conveys a strong and specific inhibition of the transactivation potential of all three CEBP isoforms. Leucine zipper domain or Zip is a common structural motif mediating protein–protein interactions (42Landschulz W.H. Johnson P.F. McKnight S.L. The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins.Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2495) Google Scholar), and our previous studies established that the Zip domain of Hop2 is necessary and sufficient to mediate its interaction with the bZip domains of ATF4 (28Zhang Y. Lin T. Lian N. Tao H. Li C. Li L. Yang X. Hop2 interacts with ATF4 to promote osteoblast differentiation.J. Bone Miner. Res. 2019; 34: 2287-2300Crossref PubMed Scopus (7) Google Scholar). These findings led us to hypothesize that Hop2 suppresses the CEBP-dependent transactivation by interacting with these bZip TFs in adipocytes. Given the fact that genetic deletion of CEBPα alone was sufficient to block adipocyte differentiation in vivo (17Wang N.D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Impaired energy homeostasis in C/EBP alpha knockout mice.Science. 1995; 269: 1108-1112Crossref PubMed Scopus (818) Google Scholar), we focused our next set of experiments on this founding member of the CEBP subfamily (43Landschulz W.H. Johnson P.F. McKnight S.L. The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite.Science. 1989; 243: 1681-1688Crossref PubMed Scopus (417) Google Scholar) (Fig. 2A). To detect a direct binding between Hop2 and CEBPα, we purified recombinant glutathione-S-transferase (GST)-Hop2 and His-CEBPα fusion proteins expressed in bacteria. Pull-down assays demonstrated that GST-Hop2, but not GST, was able to pull down His-CEBPα protein, and conversely, a Ni-charged affinity chromatography retained GST-Hop2 only in the presence of His-CEBPα (Fig. 2, B and C). These results thus show that Hop2 and CEBPα interact directly in vitro. To determine whether Hop2 interacts with CEBPα in cells, we transfected COS1 cells transiently with expressing plasmids encoding hemagglutinin (HA)-tagged Hop2 (HA-Hop2) and Flag-CEBPα singly or together. Nuclear extracts (NEs) of COS1 cells ectopically expressing HA-Hop2 and Flag-CEBPα were subjected to coimmunoprecipitation (co-IP). As expected, an anti-HA antibody (Ab) precipitated HA-Hop2 and an anti-Flag Ab precipitated Flag-CEBPα from NEs of COS1 cells expressing either HA-Hop2 or Flag-CEBPα alone (positive control; Fig. 2D). Moreover, the anti-HA cross precipitated Flag-CEBPα, whereas the anti-Flag Ab brought down HA-Hop2 from the NEs of COS1 cells coexpressing both HA-Hop2 and Flag-CEBPα, but not expressing each of these proteins singly (negative controls). Furthermore, immunocytochemistry of COS1 cells transiently overexpressing both HA-Hop2 and Flag-CEBPα and of 3T3-L1 preadipocytes transiently overexpressing HA-Hop2 detected double-positive signals (orange) with anti-HA (green) and anti-Flag (red) or anti-CEBPα Abs in the nuclei of these cells (Fig. S2, A and B), confirming a colocalization of Hop2 and CEBPα when overexpressed. To further examine whether endogenous Hop2 interacts with adipocyte CEBP bZip TFs under physiological conditions, we isolated NEs from visceral adipose tissues (VATs) and performed co-IP with Abs against Hop2 and CEBP bZips. Anti-Hop2 Abs, but not anti-IgG Abs (negative control), precipitated all three isoforms of the CEBPα, CEBP-β, and CEBP-δ; and reciprocally, anti-CEBPα, anti-CEBPβ, and anti-CEBPδ Abs, but not anti-IgG Abs, precipitated Hop2 (Fig. 2, E–G). In addition, immunofluorescence of mouse VAT verified that endogenous Hop2 (red) and CEBPα (green) are colocalized in the nuclei (blue) of adipocytes (Fig. 2H), supporting that Hop2 plays an intrinsic physiological role in adipocytes. To further explore at which stage(s) of adipocyte differentiation that the interaction between Hop2 and CEBPs takes place, we analyzed the temporal and dynamic expression pattern of the three CEBP bZip TFs. Western blot found that the protein expression levels of CEBPβ and CEBP-δ in 3T3-L1 cells are low in undifferentiated cultures (day 0), increased in 2-day cultures, and then reduced to undetectable in 4-day cultures upon adipocyte induction (Fig. S2C). On the" @default.
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• W3203802214 date "2021-11-01" @default.
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• W3203802214 title "Hop2 interacts with the transcription factor CEBPα and suppresses adipocyte differentiation" @default.
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