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• W2018945542 abstract "We used a yeast two-hybrid screening approach to identify novel interactors of CCAAT/enhancer-binding protein α (C/EBPα) that may offer insight into its mechanism of action and regulation. One clone obtained was that for CA150, a nuclear protein previously characterized as a transcriptional elongation factor. In this report, we show that CA150 is a widely expressed co-repressor of C/EBP proteins. Two-hybrid and co-immunoprecipitation analyses indicated that CA150 interacts with C/EBPα. Overexpression of CA150 inhibited the transactivation produced by C/EBPα and was also able to reverse the enhancing effect of the co-activator p300 on C/EBPβ-mediated transactivation. Analysis of C/EBPα mutants indicated that CA150 interacts with C/EBPα primarily through a domain spanning amino acids 135–150. Chromatin immunoprecipitation assays showed that CA150 was present on a promoter that is repressed by C/EBPα but not present on a promoter that is activated by C/EBPα. Finally, we showed that in cells in which growth arrest had been induced by ectopic expression of C/EBPα, CA150 was able to release them from growth arrest. Interestingly, CA150 could not reverse the growth arrest produced by the minimal growth-arrest domain of C/EBPα (amino acids 175–217), suggesting that the effect of CA150 was directed at a region of C/EBPα outside of this minimal domain, consistent with our two-hybrid analysis. Taken together, these data indicate that CA150 is a co-repressor of C/EBP proteins and provides a possible mechanism for how C/EBPα can repress transcription of specific genes. We used a yeast two-hybrid screening approach to identify novel interactors of CCAAT/enhancer-binding protein α (C/EBPα) that may offer insight into its mechanism of action and regulation. One clone obtained was that for CA150, a nuclear protein previously characterized as a transcriptional elongation factor. In this report, we show that CA150 is a widely expressed co-repressor of C/EBP proteins. Two-hybrid and co-immunoprecipitation analyses indicated that CA150 interacts with C/EBPα. Overexpression of CA150 inhibited the transactivation produced by C/EBPα and was also able to reverse the enhancing effect of the co-activator p300 on C/EBPβ-mediated transactivation. Analysis of C/EBPα mutants indicated that CA150 interacts with C/EBPα primarily through a domain spanning amino acids 135–150. Chromatin immunoprecipitation assays showed that CA150 was present on a promoter that is repressed by C/EBPα but not present on a promoter that is activated by C/EBPα. Finally, we showed that in cells in which growth arrest had been induced by ectopic expression of C/EBPα, CA150 was able to release them from growth arrest. Interestingly, CA150 could not reverse the growth arrest produced by the minimal growth-arrest domain of C/EBPα (amino acids 175–217), suggesting that the effect of CA150 was directed at a region of C/EBPα outside of this minimal domain, consistent with our two-hybrid analysis. Taken together, these data indicate that CA150 is a co-repressor of C/EBP proteins and provides a possible mechanism for how C/EBPα can repress transcription of specific genes. CCAAT/enhancer binding proteins (C/EBPs) 2The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; CAT, chloramphenicol acetyltransferase; ChIP, chromatin immunoprecipitation; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; HNF6, hepatic nuclear factor 6; PEPCK, phosphoenolpyruvate carboxykinase; RSV, Rous sarcoma virus; Cdk, cyclin-dependent kinase. are eukaryotic transcription factors that regulate a large number of genes. There are eight members in the C/EBP protein family, and the first two members that were identified, C/EBPα and C/EBPβ, are the most extensively examined and characterized (1Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). Although they are expressed in a variety of cell types, both family members are enriched in liver and have been demonstrated to regulate the expression of a number of genes that are associated with energy metabolism (2Roesler W.J. Annu. Rev. Nutr. 2001; 21: 141-165Crossref PubMed Scopus (82) Google Scholar). C/EBPα and -β have been generally observed to be activators of transcription. The transactivation domains of these proteins reside in the N terminus of each protein, and can function independently of the basic region-leucine zipper DNA-binding domain if linked to a heterologous DNA-binding domain (1Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). There appear to be several sub-domains within the transactivation domains that mediate transactivation (3Nerlov C. Ziff E. EMBO J. 1995; 14: 4318-4328Crossref PubMed Scopus (134) Google Scholar, 4Nerlov C. Ziff E. Genes Dev. 1994; 8: 350-362Crossref PubMed Scopus (118) Google Scholar, 5Roesler W.J. Park E.A. McFie P.J. J. Biol. Chem. 1998; 273: 14950-14957Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 6Wilson H.L. McFie P.J. Roesler W.J. Mol. Cell. Endocrinol. 2001; 181: 27-34Crossref PubMed Scopus (15) Google Scholar, 7Jurado L.A. Song S. Roesler W.J. Park E.A. J. Biol. Chem. 2002; 277: 27606-27612Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 8Trautwein C. Walker D.L. Plumpe J. Manns M.P. J. Biol. Chem. 1995; 270: 15130-15136Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), although the precise boundaries of these regions are difficult to determine from the available studies because they are assigned based on the arbitrary design of the mutants used. For C/EBPα, studies indicate that the majority of the transactivation potential lies within amino acid residues 1–150 (5Roesler W.J. Park E.A. McFie P.J. J. Biol. Chem. 1998; 273: 14950-14957Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), whereas for C/EBPβ, the transactivation domain appears to lie within amino acid residues 1–108 (9Park E.A. Song S. Vinson C. Roesler W.J. J. Biol. Chem. 1999; 274: 211-217Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In addition to its transcriptional role, C/EBPα is also a strong inhibitor of cell proliferation. It inhibits proliferation of cultured cells when overexpressed, and it inhibits proliferation in newborn liver (10Timchenko N.A. Harris T.E. Wilde M. Bilyeu T.A. Burgess-Beusse B.L. Finegold M.J. Darlington G.J. Mol. Cell. Biol. 1997; 17: 7353-7361Crossref PubMed Google Scholar) and in liver of adult animals (11Tan E.H. Hooi S.C. Laban M. Wong E. Ponniah S. Wee A. Wang N.-d. Cancer Res. 2005; 65: 10330-10337Crossref PubMed Scopus (49) Google Scholar). C/EBPα exerts its growth-arrest activity by several different mechanisms, depending on the tissue. In liver, C/EBPα binds Cdk 2 and prevents its interaction with cyclins (12Wang H. Iakova P. Wilde M. Welm A. Goode T. Roesler W.J. Timchenko N.A. Mol. Cell. 2001; 8: 817-828Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). In adipose, C/EBPα represses E2F-dependent transcription of several genes, including S phase and mitosis-specific genes (13Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1981) Google Scholar). Precisely how the transactivation domains of C/EBPs confer their effects onto the preinitiation complex is poorly understood. For C/EBPα, part of the basis for the constitutive activity of this domain may be explained by its ability to physically interact with the TATA-binding protein and with TFIIB (3Nerlov C. Ziff E. EMBO J. 1995; 14: 4318-4328Crossref PubMed Scopus (134) Google Scholar). Moreover, the transactivation potential of this C/EBP isoform can be enhanced by CBP/p300 and Retinoblastoma protein (14Erickson R.L. Hemati N. Ross S.E. MacDougald O.A. J. Biol. Chem. 2001; 276: 16348-16355Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 15Mink S. Haenig B. Klempnauer K. Mol. Cell. Biol. 1997; 17: 6609-6617Crossref PubMed Google Scholar, 16Chen P. Riley D. Chen Y. Lee W. Genes Dev. 1996; 10: 2794-2804Crossref PubMed Scopus (394) Google Scholar). C/EBPβ, upon binding to a promoter, can recruit CBP/p300, which leads to enhanced transactivation (15Mink S. Haenig B. Klempnauer K. Mol. Cell. Biol. 1997; 17: 6609-6617Crossref PubMed Google Scholar, 17Oelgeschlager M. Janknecht R. Krieg J. Schreek S. Luscher B. EMBO J. 1996; 15: 2771-2780Crossref PubMed Scopus (194) Google Scholar). In addition, both C/EBPα and -β can recruit SWI/SNF, a chromatin remodeling complex, to gene promoters (18Kowenz-Leutz E. Leutz A. Mol Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). C/EBPα specifically has been shown to enhance histone H3 acetylation by recruiting a co-regulator containing histone acetylase activity (19Zhang W.-H. Srihari R. Day R.N. Schaufele F. J. Biol. Chem. 2001; 276: 40373-40376Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Thus, the evidence to date suggests that there are several mechanisms for C/EBP-dependent transactivation of gene promoters. Herein, we report the identification and characterization of a novel co-repressor for C/EBPs that was uncovered during a yeast two-hybrid screen. Not only is CA150 a transcriptional co-repressor, but it also inhibits the growth-arrest activity of C/EBPα. Materials—DNA-modifying enzymes were purchased from New England Biolabs (Mississauga, Ontario, Canada) and Promega (Nepean, Ontario, Canada). Acetyl-[3H]CoA (10 Ci/mmol) was purchased from PerkinElmer Life Sciences. Tissue culture supplies were from Invitrogen. HepG2 cells were acquired from American Type Culture Collection. Yeast media were from Difco (Detroit, MI), whereas the amino acids and 3-amino-1,2,4-triazole were from Sigma-Aldrich. Biotinylated oligonucleotides were purchased from Invitrogen. Yeast Two-hybrid Assay—A human liver cDNA MATCHMAKER library, containing 3 × 106 independent clones, and screening kit were purchased from Clontech-BD Biosciences. The transactivation domain of C/EBPα with three residues mutated (Y67A, F77A, and L78A) (5Roesler W.J. Park E.A. McFie P.J. J. Biol. Chem. 1998; 273: 14950-14957Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) was cloned in-frame into the bait vector pGBT9, which resulted in the expression of a GAL4-C/EBPα bait protein. Approximately 12 × 106 clones were screened in yeast strain CG-1945 as per the supplier's instructions and were doubly selected based on their ability to grow in the absence of histidine/presence of 3-amino-1,2,4-triazole (through activation of the His3 reporter gene) and intensity of blue colony formation (through activation of the lacZ reporter gene). Selected clones were verified for the requirement for both bait and prey plasmids for reporter gene activation. The plasmids containing the prey cDNAs were isolated and the insert sequenced. Quantitative β-Galactosidase Assay—A quantitative β-galactosidase assay was performed as described in the manual provided with the Clontech MATCHMAKER kit, which can be used as an indirect assessment of the strength of interaction (20Felinski E.A. Quinn P.G. J. Biol. Chem. 1999; 274: 11672-11678Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Briefly, yeast were transformed with either bait plasmid, prey plasmid, or both, and plated out onto the appropriate media. Pooled or individual colonies were inoculated into 5 ml of appropriate liquid media, and cultured overnight at 30 °C with shaking. 2 ml of this overnight culture were then used to inoculate 8 ml of YPD liquid media (1% yeast extract, 2% bactopeptone, 2% dextrose), and further shaken at 30 °C for 4 h. Aliquots were removed, and cell extracts were prepared and assayed for β-galactosidase activity. Units are expressed in terms of the ΔA420 per minute per equivalent number of yeast cells. The data shown were obtained from screening of 6–10 colonies from at least two independent transformations. The C/EBPα bait protein used in this assay was the same as that used in the two-hybrid library screen described above. The various GAL4-C/EBPα mutants used to identify the interaction domain have been reported previously (5Roesler W.J. Park E.A. McFie P.J. J. Biol. Chem. 1998; 273: 14950-14957Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). All of the deletion mutants that contained amino acid residues 67, 77, and 78 had these three residues mutated to alanines to reduce the background activity of the bait proteins, as described above. The C/EBPβ bait protein consisted of the transactivation domain of C/EBPβ (amino acids 1–108) linked to the GAL4 DNA-binding domain, whereas the CREB bait contained the transactivation domain (amino acids 3–203) linked to the GAL4 DNA binding domain. The CA150 prey protein consisted of amino acid residues 89–480 of CA150 (representing the region contained in the clone pulled out in the yeast two-hybrid screen) fused to the GAL4 transactivation domain. mutCA150 contained amino acid residues 643–1098 of CA150, linked to the GAL4 transactivation domain. Mammalian Reporter Gene Experiments—Transfections were performed in HepG2 cells by the calcium phosphate precipitation method as described previously (21Roesler W. Simard J. Graham J. McFie P. J. Biol. Chem. 1994; 269: 14276-15890Abstract Full Text PDF PubMed Google Scholar). RSV-βgal was included in all transfections as a control for transfection efficiency. The amount of each plasmid used is indicated in the appropriate figure legend. Reporter genes –68FX4 (22Roesler W. McFie P. Puttick D. J. Biol. Chem. 1993; 268: 3791-3796Abstract Full Text PDF PubMed Google Scholar) and –68GX4 (5Roesler W.J. Park E.A. McFie P.J. J. Biol. Chem. 1998; 273: 14950-14957Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), expression plasmids for C/EBPα (23Park E.A. Roesler W.J. Liu J. Klemm D.J. Gurney A.L. Thatcher J.D. Shuman J.D. Friedman A.D. Hanson R.W. Mol. Cell. Biol. 1990; 10: 6264-6272Crossref PubMed Scopus (174) Google Scholar), C/EBPβ (5Roesler W.J. Park E.A. McFie P.J. J. Biol. Chem. 1998; 273: 14950-14957Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), CA150 (24Sune C. Garcia-Blanco M.A. Mol. Cell. Biol. 1999; 19: 4719-4728Crossref PubMed Scopus (49) Google Scholar), p300 (14Erickson R.L. Hemati N. Ross S.E. MacDougald O.A. J. Biol. Chem. 2001; 276: 16348-16355Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and the catalytic subunit of protein kinase A (25Mellon P. Clegg C. Correll L. McKnight G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4887-4891Crossref PubMed Scopus (201) Google Scholar), as well as expression plasmids for GAL4 fusions of C/EBPα (Gα) and related mutants (5Roesler W.J. Park E.A. McFie P.J. J. Biol. Chem. 1998; 273: 14950-14957Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), C/EBPβ (Gβ) (9Park E.A. Song S. Vinson C. Roesler W.J. J. Biol. Chem. 1999; 274: 211-217Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), GAL4-Sp1 (26Nakano K. Mizuno T. Sowa Y. Orita T. Yoshino T. Okuyama Y. Fujita T. Ohtani-Fujita N. Matsukawa Y. Tokino T. Yamagishi H. Oka T. Nomura H. Sakai T. J. Biol. Chem. 1997; 272: 22199-22206Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), and CREB (G-CREB) (27Roesler W.J. Graham J.G. Kolen R. Klemm D.J. McFie P.J. J. Biol. Chem. 1995; 270: 8225-8232Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) have been described previously. (The GAL4-C/EBPα mutant N150 was previously termed N135; however, resequencing of this mutant indicated that its coding region contained residues 6–150.) Assays for chloramphenicol acetyltransferase activity (CAT), β-galactosidase activity, and protein determination were performed as previously described (22Roesler W. McFie P. Puttick D. J. Biol. Chem. 1993; 268: 3791-3796Abstract Full Text PDF PubMed Google Scholar). Co-immunoprecipitation Assay—Co-immunoprecipitation of C/EBPα and CA150 was performed using mouse liver nuclear extract prepared as described previously (28Wang G.-L. Timchenko N.A. Mol. Cell. Biol. 2005; 25: 1325-1338Crossref PubMed Scopus (50) Google Scholar). 400 μg of nuclear extract was precleared with 50 μl of a 50% slurry of Protein G-agarose for 1 h at 4 °Cina total volume of 400 μl of co-immunoprecipitation buffer (20 mm Tris, pH 7.2, 1 mm EDTA, 0.1% Triton X-100, 150 mm NaCl) plus 1 mg/ml bovine serum albumin. The pre-cleared extract was then incubated overnight at 4 °C with the specific antibody that had been pre-attached to Protein G-agarose beads. The beads were pelleted and washed three times with 1 ml of co-immunoprecipitation buffer. The pellet was resuspended in SDS-PAGE loading buffer, boiled, and subjected to SDS-PAGE and Western blot analysis. Western Blot Analysis—Western blot analysis was performed as previously described (29Davies G. Khandelwal R. Roesler W. Mol. Cell. Biol. Res. Commun. 1999; 2: 202-208Crossref PubMed Scopus (33) Google Scholar), using Western Lightning™ chemiluminescent reagent (PerkinElmer Life Sciences) to detect the antigen-antibody complexes. Anti-C/EBPα (14AA) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-CREB antibody (9192) was purchased from Cell Signaling, and anti-CA150 antibodies (BL456) were purchased from Bethyl Laboratories (Montgomery, TX). Preparation of Nuclear Extracts—Nuclear extracts were prepared from several mouse tissues as described previously (30Wang G.-L. Iakova P. Wilde M. Awad S. Timchenko N.A. Genes Dev. 2004; 18: 912-925Crossref PubMed Scopus (117) Google Scholar). Briefly, tissues were homogenized in 5 volumes of buffer with a Dounce homogenizer, and the crude nuclear pellet was isolated by centrifugation at 12,000 × g for 5 min. The pellet was resuspended in buffer containing a final concentration of 400 mm NaCl and placed on ice for 15 min. This mixture was centrifuged at 12,000 × g for 20 min. The supernatant contained the soluble nuclear protein fraction. Growth Arrest Assay—Growth-arrest assays were performed in COS7 and HEK293 cells as previously described (30Wang G.-L. Iakova P. Wilde M. Awad S. Timchenko N.A. Genes Dev. 2004; 18: 912-925Crossref PubMed Scopus (117) Google Scholar). Briefly, cells were transfected with pAdTrack-C/EBPα, which expresses both green fluorescent protein and C/EBPα from distinct mRNAs. As indicated in the figure and corresponding legend, other experiments involved co-transfection with full-length CA150 or mutCA150, both expressed as hemagglutinin-tagged proteins using a vector described by King et al. (31King T.R. Fang Y. Mahon E.S. Anderson D.H. J. Biol. Chem. 2000; 275: 36450-36456Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). After 3 days, the number of green cells per colony (1, 2, or >2) as a percent of total green cells was assessed. Preliminary co-transfection experiments were performed to determine an optimal ratio of C/EBPα and CA150 under which the inhibitory effect of CA150 is maximal. The data shown were obtained by using a 1:4 ratio of C/EBPα to CA150 expression plasmids. Chromatin Immunoprecipitation Assay—The chromatin immunoprecipitation assay was performed with mouse liver tissues using the ChIP-IT kit (Active Motif). Briefly, the chromatin solutions were sheared by enzymatic digestion according to the instruction manual. The size of DNA fragments produced averaged between 500 and 1000 bp in length. Antibodies against C/EBPα (14AA), CA150 (N19), and cdk2 (all purchased from Santa Cruz Biotechnology) were added to each aliquot of precleared chromatin and incubated overnight. Protein G-agarose beads were added, and the mixture was incubated for 1.5 h at 4 °C. After reversing the cross-links, DNA was isolated and used for PCR reactions with primers specific for PEPCK and HNF6 promoter regions that contain the C/EBPα binding sites (–204/–3 and –175/+140, respectively). The sequences of the primers for these promoters are as follows. PEPCK-C/EBP-F: 5′-GGCCTCCCAACATTCATTAAC-3′; PEPCK-C/EBP-R: 5′-GTAGCCCGCCCTCCTTGCTTTAA-3′. HNF6-C/EBP-F: 5′-GCTCGAGCTGGCGGGCGGCACAGGC-3′, HNF6-C/EBP-R: 5′-AGGAGTCCAGTCTTCACATCGGCTG-3′. As a control for the appropriate shearing of DNA, primers were designed for a region ∼3.4 kb upstream of the C/EBP site in the PEPCK promoter (–3429/–3081) and ∼1.5 kb upstream in the HNF6 promoter (–1579/–1210). The sequences of these primers are as shown below. PEPCK-Control-F: 5′-AACCAACTGGCCCTAACTCACAGA-3′, PEPCK-Control-R: 5′-GCTGCAGTCCAGCTAATGCAACAA-3′. HNF-Control-F: 5′-AGGCAGGCACTTGGGTTAAGAGAT-3′, HNF-Control-R: 5′-AGAGCCTTGTCTGCTTAGGTGCTT-3′. PCR mixtures were amplified for 1 cycle of 95 °C for 5 min, annealing temperature for primers (62 °C) for 5 min, and 72 °C for 2 min. Then PCR mixtures were amplified for 34 cycles of 95 °C for 1 min, annealing temperature for 2 min, and 72 °C for 1.5 min. PCR products were separated by 1.5% agarose gel electrophoresis or by 4% PAGE. To search for co-regulators of C/EBPα, we employed a yeast two-hybrid approach to screen a human liver cDNA library. The “bait” used to screen the library was a fusion protein consisting of the GAL4 DNA-binding domain fused to a mutated transactivation domain of C/EBPα. This transactivation domain contained amino acids 6–217 of C/EBPα, with three amino acids (Tyr-67, Phe-77, and Leu-78) mutated to alanines. The three point mutations have been shown previously to abrogate the constitutive activity of the transactivation domain (3Nerlov C. Ziff E. EMBO J. 1995; 14: 4318-4328Crossref PubMed Scopus (134) Google Scholar), yet not significantly alter the integrity of the domain based on the observation that the protein kinase A-inducible activity of this mutant remains functional (5Roesler W.J. Park E.A. McFie P.J. J. Biol. Chem. 1998; 273: 14950-14957Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The use of this mutant as the bait was necessary, because the wild-type C/EBPα transactivation domain displayed significant activity in yeast (data not shown) and thus gave a high background of reporter gene expression. Several potential interactors were obtained by this screen and sequenced, and one of these was a previously identified gene product called CA150 (32Sune C. Hayashi T. Liu Y. Lane W. Young R. Garcia-Blanco M. Mol. Cell. Biol. 1997; 17: 6029-6039Crossref PubMed Scopus (83) Google Scholar). CA150 was first characterized as a nuclear protein from HeLa cells that was associated with RNA polymerase II and involved in Tat-activated transcription of the human immunodeficiency viral promoter. It contains glutamine- and alanine-rich repeats that are characteristic of transcriptional regulators. CA150 has been shown to repress RNA polymerase II transcription, although in a specific fashion, because it doesn't repress transcription of all genes (24Sune C. Garcia-Blanco M.A. Mol. Cell. Biol. 1999; 19: 4719-4728Crossref PubMed Scopus (49) Google Scholar). CA150-mediated repression of elongation requires a TATA box, and overexpression of the TATA-binding protein alleviates the repression, although there appears to be no direct physical interaction between the TATA-binding protein and CA150 (24Sune C. Garcia-Blanco M.A. Mol. Cell. Biol. 1999; 19: 4719-4728Crossref PubMed Scopus (49) Google Scholar). CA150 has been shown to bind directly to the phosphorylated carboxyl-terminal domain of RNA polymerase II. The strength of the genetic interaction observed in the yeast two-hybrid assay between the transactivation domain of C/EBPα and CA150 was quantified and compared with that of two, well described interactors using a liquid β-galactosidase assay. This assay quantifies the amount of lacZ reporter gene activity in yeast that results from the bait and prey proteins interacting on a GAL4-driven promoter. As expected, the empty bait vector alone, expressing only the GAL4 DNA-binding domain, resulted in only a small amount of β-galactosidase expression (Fig. 1). Transformation of yeast with the CA150 prey vector (expressing the GAL4 transactivation domain fused to CA150), alone or in combination with the empty bait vector, also resulted in little activation of the lacZ reporter gene. When yeast were transformed with the mutant C/EBPα bait vector, some stimulation of β-galactosidase activity was observed, but this was not increased further with co-transformation with the empty prey vector. However, when the C/EBPα bait and CA150 prey vectors were co-transformed, a significant induction of lacZ reporter gene activity was observed, suggesting that an interaction between the C/EBPα and CA150 occurred (Fig. 1). Comparison of this interaction with that of the positive control TD1 and VA3 vectors, which express a p53 bait protein and large T-antigen prey protein, respectively, indicated that the degree of genetic interaction between C/EBPα and CA150 is relatively strong. One study examining the expression of CA150 suggested that the levels of mRNA were very low in liver (33Bohne J. Cole S. Sune C. Lindman B. Ko V. Vogt T. Garcia-Blanco M. Mamm. Genome. 2000; 11: 930-933Crossref PubMed Scopus (7) Google Scholar), raising the question of whether it is expressed at significant levels in this tissue. To address this issue, nuclear extracts from several mouse tissues were prepared, and Western blots were performed to assess protein levels of CA150 and to compare them with the relative levels of CREB, a ubiquitously expressed transcription factor. As shown in Fig. 2, CA150 protein levels in liver were easily detectable and were similar to those present in kidney, brain, and lung. Heart, skeletal muscle, and spleen showed low levels of CA150, however, an abundant protein of molecular mass ∼200 kDa was detected in muscle extracts. No CA150 was detected in cytosolic fractions, confirming that it localizes to nuclei (data not shown, see Fig. 9A). CREB showed a similar pattern of expression to CA150 except that spleen expression was abundant. Thus, CA150 appears to be widely expressed albeit at varying levels.FIGURE 9CA150 associates with a promoter that is repressed by C/EBPα. ChIP analysis was performed on mouse liver DNA. A, a Western blot was performed to identify the presence of CA150 in cytosolic (C) or nuclear (N) fractions (100 μg) of the mouse liver homogenates. The corresponding positions of the molecular weight markers are shown on the left. B, the results of ChIP analysis from two mouse livers (#1 and #2) are shown. The cross-linked DNA fragments were immunoprecipitated with either no antibody (No ab), or with anti-C/EBPα, -CA150, or -cdk2. The immunoprecipitated fragments were then subjected to PCR with primers directed against the C/EBP binding region of the PEPCK or HNF6 promoters, or with control primers directed at a region ∼1.5 kb upstream of each corresponding C/EBP site. The PCR signal from each input set of DNA samples is shown in the first lane.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next performed co-immunoprecipitation assays to see if we could detect complexes between C/EBPα and CA150. Mouse liver nuclear extracts were incubated with antibodies for C/EBPα, CREB, or with no antibodies, and assessed for the presence of CA150 in the immunoprecipitate. The efficacy of C/EBPα depletion of the nuclear extract was first assessed. As shown in Fig. 3A, addition of Protein G-agarose beads alone (Pre-cleared input), or co-immunoprecipitation with no antibody or CREB antibody, did not deplete the extract of C/EBPα. However, immunoprecipitation with anti-C/EBPα effectively depleted the extract. The immunoprecipitates were then analyzed for the presence of CA150 (Fig. 3B). CA150 was detected only in the immunoprecipitates obtained using anti-C/EBPα. C/EBPβ shares some amino acid sequence similarity with C/EBPα, including sub-regions within the transactivation domain (3Nerlov C. Ziff E. EMBO J. 1995; 14: 4318-4328Crossref PubMed Scopus (134) Google Scholar, 6Wilson H.L. McFie P.J. Roesler W.J. Mol. Cell. Endocrinol. 2001; 181: 27-34Crossref PubMed Scopus (15) Google Scholar). This suggested that CA150 might also interact with this isoform. This hypothesis was tested by quantitative yeast two-hybrid analysis (Fig. 4). Transformation of yeast with the CA150 prey vector alone produced no stimulation of β-galactosidase activity. As expected, the C/EBPβ bait vector, expressing a fusion protein consisting of the GAL4 DNA-binding domain fused to the transactivation domain (amino acids 1–108) of C/EBPβ, stimulated the β-galactosidase reporter gene, indicating that this transactivation domain is active in yeast. When the CA150 prey and C/EBPβ bait vectors were co-transformed, a significant induction of reporter gene activity was observed, suggesting that the GAL4 transactivation domain on the CA150 prey protein was recruited to the reporter gene. The specificity of this interaction was demonstrated by the lack of activation of the reporter gene observed when a CREB bait vector and CA150 prey vector were co-transformed into yeast (Fig. 4). We next examined what effect CA150 had on the transactivation potential of C/EBPs, using transient transfection of reporter genes in HepG2 cells. Initially, we assessed the activity of CA150 on –68FX4 (22Roesler W. McFie P. Puttick D. J. Biol. Chem. 1993; 268: 3791-3796Abstract Full Text PDF PubMed Google Scholar), which contains a chimeric promoter consisting of four copies of a region of the PEPCK promoter (–355/–200) that possesses three C/EBP binding sites, linked to a minimal promoter. Thus, it is a promoter that is highly sensitive to C/EBP-dependent transactivation. Overexpression of C/EBPα or C/EB" @default.
• W2018945542 created "2016-06-24" @default.
• W2018945542 creator A5002970194 @default.
• W2018945542 creator A5003288313 @default.
• W2018945542 creator A5015867733 @default.
• W2018945542 creator A5019057172 @default.
• W2018945542 creator A5027354549 @default.
• W2018945542 creator A5047085626 @default.
• W2018945542 date "2006-06-01" @default.
• W2018945542 modified "2023-10-11" @default.
• W2018945542 title "Identification of a Co-repressor That Inhibits the Transcriptional and Growth-Arrest Activities of CCAAT/Enhancer-binding Protein α" @default.
• W2018945542 cites W131975224 @default.
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