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• W1969345252 abstract "FRA-2/FOSL2 is a basic region-leucine zipper motif transcription factor that is widely expressed in mammalian tissues. The functional repertoire of this factor is unclear, partly due to a lack of knowledge of genomic sequences that are targeted. Here, we identified novel, functional FRA-2 targets across the genome through expression profile analysis in a knockdown transgenic rat. In this model, a nocturnal rhythm of pineal gland FRA-2 is suppressed by a genetically encoded, dominant negative mutant protein. Bioinformatic analysis of validated sets of FRA-2-regulated and -nonregulated genes revealed that the FRA-2 regulon is limited by genomic target selection rules that, in general, transcend core cis-sequence identity. However, one variant AP-1-related (AP-1R) sequence was common to a subset of regulated genes. The functional activity and protein binding partners of a candidate AP-1R sequence were determined for a novel FRA-2-repressed gene, Rgs4. FRA-2 protein preferentially associated with a proximal Rgs4 AP-1R sequence as demonstrated by ex vivo ChIP and in vitro EMSA analysis; moreover, transcriptional repression was blocked by mutation of the AP-1R sequence, whereas mutation of an upstream consensus AP-1 family sequence did not affect Rgs4 expression. Nocturnal changes in protein complexes at the Rgs4 AP-1R sequence are associated with FRA-2-dependent dismissal of the co-activator, CBP; this provides a mechanistic basis for Rgs4 gene repression. These studies have also provided functional insight into selective genomic targeting by FRA-2, highlighting discordance between predicted and actual targets. Future studies should address FRA-2-Rgs4 interactions in other systems, including the brain, where FRA-2 function is poorly understood. FRA-2/FOSL2 is a basic region-leucine zipper motif transcription factor that is widely expressed in mammalian tissues. The functional repertoire of this factor is unclear, partly due to a lack of knowledge of genomic sequences that are targeted. Here, we identified novel, functional FRA-2 targets across the genome through expression profile analysis in a knockdown transgenic rat. In this model, a nocturnal rhythm of pineal gland FRA-2 is suppressed by a genetically encoded, dominant negative mutant protein. Bioinformatic analysis of validated sets of FRA-2-regulated and -nonregulated genes revealed that the FRA-2 regulon is limited by genomic target selection rules that, in general, transcend core cis-sequence identity. However, one variant AP-1-related (AP-1R) sequence was common to a subset of regulated genes. The functional activity and protein binding partners of a candidate AP-1R sequence were determined for a novel FRA-2-repressed gene, Rgs4. FRA-2 protein preferentially associated with a proximal Rgs4 AP-1R sequence as demonstrated by ex vivo ChIP and in vitro EMSA analysis; moreover, transcriptional repression was blocked by mutation of the AP-1R sequence, whereas mutation of an upstream consensus AP-1 family sequence did not affect Rgs4 expression. Nocturnal changes in protein complexes at the Rgs4 AP-1R sequence are associated with FRA-2-dependent dismissal of the co-activator, CBP; this provides a mechanistic basis for Rgs4 gene repression. These studies have also provided functional insight into selective genomic targeting by FRA-2, highlighting discordance between predicted and actual targets. Future studies should address FRA-2-Rgs4 interactions in other systems, including the brain, where FRA-2 function is poorly understood. IntroductionFos-related antigen 2 (FRA-2/FOSL2) is a member of the FOS/JUN subgroup of bZIP 3The abbreviations used are: bZIPbasic region-leucine zipperAP-1RAP-1-related sequenceAP-1FAP-1 family sequenceCBPcAMP-response element-binding protein-binding proteinCREcAMP response elementDN-FRA-2dominant negative FRA-2TFtranscription factorANOVAanalysis of variancePWMposition weight matrixTGtransgenicQPCRquantitative PCRTSStranscriptional start sitedfdegrees of freedom. transcription factors (TFs) that function in concert with regulatory DNA sequences in target genes (1Vinson C. Myakishev M. Acharya A. Mir A.A. Moll J.R. Bonovich M. Mol. Cell. Biol. 2002; 22: 6321-6335Crossref PubMed Scopus (339) Google Scholar). The various FOS proteins appear to serve distinct developmental, physiological, and pathological roles (2Jochum W. Passegué E. Wagner E.F. Oncogene. 2001; 20: 2401-2412Crossref PubMed Scopus (590) Google Scholar, 3Wagner E.F. Ann. Rheum. Dis. 2010; 69: i86-i88Crossref PubMed Scopus (100) Google Scholar), and current studies are seeking to define these individual roles and the mechanisms involved. FRA-2 exerts a specific action in bone development (4Bozec A. Bakiri L. Hoebertz A. Eferl R. Schilling A.F. Komnenovic V. Scheuch H. Priemel M. Stewart C.L. Amling M. Wagner E.F. Nature. 2008; 454: 221-225Crossref PubMed Scopus (146) Google Scholar) and appears to have selective physiological and pathological roles in diverse processes, including photoperiodic regulation (5Engel L. Gupta B.B. Lorenzkowski V. Heinrich B. Schwerdtle I. Gerhold S. Holthues H. Vollrath L. Spessert R. Neuroscience. 2005; 132: 511-518Crossref PubMed Scopus (14) Google Scholar), some cancers (6Carro M.S. Lim W.K. Alvarez M.J. Bollo R.J. Zhao X. Snyder E.Y. Sulman E.P. Anne S.L. Doetsch F. Colman H. Lasorella A. Aldape K. Califano A. Iavarone A. Nature. 2010; 463: 318-325Crossref PubMed Scopus (896) Google Scholar), and pulmonary fibrosis (7Eferl R. Hasselblatt P. Rath M. Popper H. Zenz R. Komnenovic V. Idarraga M.H. Kenner L. Wagner E.F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 10525-10530Crossref PubMed Scopus (135) Google Scholar). This accumulated evidence of functional specialization indicates that mechanisms confer specificity to the actions of the FRA-2 TF relative to other members of the FOS/JUN group.One aspect of this selectivity that remains to be fully characterized is selective genomic targeting by FRA-2. Selection at the level of cis-sequence specificity is clearly one aspect of this targeting; numerous studies established that different combinations of FOS and JUN proteins (28 dimeric combinations in total) have different binding affinities for activator protein-1 (AP-1) and cAMP-response element (CRE) sequences (8Hai T. Curran T. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 3720-3724Crossref PubMed Scopus (1098) Google Scholar, 9Ryseck R.P. Bravo R. Oncogene. 1991; 6: 533-542PubMed Google Scholar). A study using tethered protein dimers (10Bakiri L. Matsuo K. Wisniewska M. Wagner E.F. Yaniv M. Mol. Cell. Biol. 2002; 22: 4952-4964Crossref PubMed Scopus (156) Google Scholar) showed that whereas the c-FOS/c-JUN dimer interacts exclusively with AP-1-like sequences (core consensus, tga(g/c)tca), other dimers, including FRA-2/c-JUN, are less selective and also bind CRE elements (core consensus, tgacgtca). However, it is also apparent that there are other levels of selection because about one-third of vertebrate promoters contain consensus AP-1 family sites (AP-1F; MatInspector, Genomatix), but not all these genes are regulated by AP-1 (11Zhou H. Zarubin T. Ji Z. Min Z. Zhu W. Downey J.S. Lin S. Han J. DNA Res. 2005; 12: 139-150Crossref PubMed Scopus (26) Google Scholar).FRA-2, like FRA-1, differs from FOS and FOSB in lacking a potent C-terminal transactivation domain (12Suzuki T. Okuno H. Yoshida T. Endo T. Nishina H. Iba H. Nucleic Acids Res. 1991; 19: 5537-5542Crossref PubMed Scopus (193) Google Scholar). As may be predicted from this structural difference, FRA-2 can act to suppress transcription, for example at promoters induced by c-JUN homodimers (12Suzuki T. Okuno H. Yoshida T. Endo T. Nishina H. Iba H. Nucleic Acids Res. 1991; 19: 5537-5542Crossref PubMed Scopus (193) Google Scholar). However, in heterodimeric combination with JUND, FRA-2 can enhance transcription relative to JUND homodimers (12Suzuki T. Okuno H. Yoshida T. Endo T. Nishina H. Iba H. Nucleic Acids Res. 1991; 19: 5537-5542Crossref PubMed Scopus (193) Google Scholar). These findings exemplify the varied actions of FRA-2 and underline the critical role that AP-1 protein composition plays in transactivation potential (13Kerppola T.K. Curran T. Mol. Cell. Biol. 1993; 13: 5479-5489Crossref PubMed Scopus (140) Google Scholar).The role of FRA-2 has been studied using a unique transgenic animal model in which FRA-2 function is perturbed in a tissue-selective manner using a dominant negative (C-terminally truncated) genetic construct (DN-FRA-2 (14Smith M. Burke Z. Humphries A. Wells T. Klein D. Carter D. Baler R. Mol. Cell. Biol. 2001; 21: 3704-3713Crossref PubMed Scopus (51) Google Scholar)). In this model, DN-FRA-2 is stably expressed in transgenic rats under the control of a conditional promoter (Aanat (15Baler R. Covington S. Klein D.C. Biol. Cell. 1999; 91: 699-705Crossref PubMed Google Scholar, 16Burke Z. Wells T. Carter D. Klein D. Baler R. J. Neurochem. 1999; 73: 1343-1349Crossref PubMed Scopus (39) Google Scholar)) that confers cellularly (pinealocyte) and temporally (nocturnal) specific expression to the pineal gland. DN-FRA-2 binds DNA normally, but because it is unable to transactivate target genes, it competes with endogenous FRA-2 for target sites and thereby inhibits endogenous FRA-2 activity. In addition, it has been shown that this construct also knocks down expression of endogenous Fra-2 via competition of autoregulatory feedback (14Smith M. Burke Z. Humphries A. Wells T. Klein D. Carter D. Baler R. Mol. Cell. Biol. 2001; 21: 3704-3713Crossref PubMed Scopus (51) Google Scholar).The conditional nature of this transgenic model is of notable importance because it allows examination of the role of FRA-2 throughout life under physiological conditions. In contrast, the study of FRA-2 function in conventional knock-out animal models is limited by neonatal lethality of Fra-2 null alleles (17Zenz R. Wagner E.F. Int. J. Biochem. Cell Biol. 2006; 38: 1043-1049Crossref PubMed Scopus (130) Google Scholar). The model is also able to reveal true physiological roles of FRA-2 because the pineal gland has an endogenous rhythm of gene expression (linked to rhythms of hormone production) that is regulated by FRA-2 (14Smith M. Burke Z. Humphries A. Wells T. Klein D. Carter D. Baler R. Mol. Cell. Biol. 2001; 21: 3704-3713Crossref PubMed Scopus (51) Google Scholar, 18Humphries A. Weller J. Klein D. Baler R. Carter D.A. J. Neurochem. 2004; 91: 946-955Crossref PubMed Scopus (37) Google Scholar); when induced in this animal model, DN-FRA-2 therefore intervenes in an autonomous physiological response. Accordingly, this model is superior to experimental paradigms where gene expression is artificially induced.In previous studies with the DN-FRA-2 model, we have shown that FRA-2 mediates both positive and negative transcriptional regulation of genes (14Smith M. Burke Z. Humphries A. Wells T. Klein D. Carter D. Baler R. Mol. Cell. Biol. 2001; 21: 3704-3713Crossref PubMed Scopus (51) Google Scholar). These opposite modes of regulation by FRA-2 are intriguing because studies have shown that the nocturnal AP-1 binding complex in the rat pineal gland is largely composed of FRA-2/JUND that is inconsistent, prima facie, with multiple modes of regulation (14Smith M. Burke Z. Humphries A. Wells T. Klein D. Carter D. Baler R. Mol. Cell. Biol. 2001; 21: 3704-3713Crossref PubMed Scopus (51) Google Scholar). FRA-2 phosphorylation in the nocturnal pineal gland is also progressive and homogeneous (14Smith M. Burke Z. Humphries A. Wells T. Klein D. Carter D. Baler R. Mol. Cell. Biol. 2001; 21: 3704-3713Crossref PubMed Scopus (51) Google Scholar), arguing against a role for different protein isoforms underlying differential regulation. We therefore hypothesize that differential regulation of target gene expression is mediated at the level of a cis-acting sequence. To pursue this, we have now sought to explain the complexity of gene regulation by FRA-2 through further analysis using transcriptome-scale expression profiling. This strategy was adopted to obtain a dataset of FRA-2-regulated genes for bioinformatic analysis of cis-regulatory sequences. The results of these studies provide insight into selective genomic targeting of FRA-2 and reveal discordance between predicted and real targets.RESULTSExpression profiling with the Affymetrix 230A rat genome microarray (15866 probe sets) demonstrated that a large number of gene transcripts are dysregulated in the DN-FRA-2 rat model. The full set of (predicted) FRA-2 targets are listed in supplemental Table S2, and a subset of Northern blot-validated targets are listed in Table 1. Representative Northern blots are shown in Fig. 1 and supplemental Fig. S1. Our (statistically verified) validation procedure also involved Northern blot analysis of five nonregulated transcripts (Table 1) and confirmed the microarray/GeneSpring-based prediction of FRA-2 targets. These targets (supplemental Table S2) include proteins of diverse molecular function and biological process, with cellular locations from plasma membrane to nucleus. ModuleMiner analysis confirmed that no Gene Ontology terms are over-represented in the FRA-2-regulated gene set; therefore FRA-2 does not appear to be associated with one particular aspect of cellular function. To gain an understanding of the general molecular mechanisms used by FRA-2, we next conducted bioinformatic analysis of validated target genes.In Silico Analysis of Candidate Gene PromotersFRA-2 actions may be either direct or indirect, i.e. involve intermediating TFs that themselves directly target gene promoters. One potential intermediary factor (CREM/ICER (31Stehle J.H. Foulkes N.S. Molina C.A. Simonneaux V. Pévet P. Sassone-Corsi P. Nature. 1993; 365: 314-320Crossref PubMed Scopus (355) Google Scholar)) is not regulated in the DN-FRA-2 model (Table 1); however, by using in silico analysis, we sought to obtain positive evidence of conserved sequences that could mediate direct genomic actions of FRA-2.Large scale (−10,000 bp from TSS) regulatory module homology analysis (ModuleMiner (27Van Loo P. Aerts S. Thienpont B. De Moor B. Moreau Y. Marynen P. Genome Biol. 2008; 9: R66Crossref PubMed Scopus (31) Google Scholar)) showed that AP-1 elements were over-represented in the regulated group of genes versus validated nonregulated genes (Table 2). Further division of the regulated genes into positively and negatively regulated subgroups generated an additional interesting finding, namely that only repressed genes exhibited an enrichment of AP-1 sites. This finding might indicate that some (positively regulated) genes may be indirectly regulated.TABLE 2Analysis of cis-regulatory module conservation in FRA-2-regulated gene setsGene setIndividual PWM (top 5)PWM weightTop PWM groupaTOP PWM GROUP is a ModuleMiner output that provides a summed score for the most represented PWM group.Top PWM group weightAll FRA-2 regulated genesV$AP1_Q2_011.08V$AP1_01VĊX_Q50.869V$AP1_Q2_01V$NFY_Q6_010.659V$AP1_Q4_011.76V$ALPHACP1_010.591V$BACH2_01V$AP1_010.382FRA-2 repressed genesV$AP1_010.955V$ALPHACP1_010.855V$AP1_01V$LEF1_Q20.765V$AP1_Q6_011.87V$AP1_Q6_010.735V$AP1_Q6V$PAX4_030.55FRA-2 enhanced genesVĀEF2_Q60.97RUSH1-a0.776V$AP4_010.699VĀEF2_Q60.97V$NFY_Q6_010.563V$ALPHACP1_010.514FRA-2-nonregulated genesV$ER_Q6_020.955V$NFY_Q6_01Arnt-Ahr0.605NF-YV$KROX_Q60.594V$NFY_010.988V$AP2_Q60.409V$NFY_Q6V$NFY_010.295a TOP PWM GROUP is a ModuleMiner output that provides a summed score for the most represented PWM group. Open table in a new tab However, analysis of the validated genes employing a more proximal (−2000 to +100) sequence and MatInspector, which distinguishes different classes of AP-1 sites, delivered a less clear outcome (Table 3). We found the following: (i) the presence of AP-1 family (AP-1F, Fig. 1C) and CRE sequences in the majority (but not all) of both FRA-2-regulated and nonregulated genes; (ii) no obvious distinction in the distribution of these sites between FRA-2-regulated and nonregulated genes; (iii) no clear distinction in the distribution of these sites between FRA-2-positively and -negatively regulated genes. At the same time, MatInspector analysis revealed the presence of some interesting alternative sites in the regulated genes set; the most common (8 of 9 genes) AP-1 sites were of the AP-1-related (AP-1R, Fig. 1C) matrix family. This was of interest because this family of elements includes the sequence tgcgtca (the highly conserved AP-1F “A” at position 3 is substituted by a “C”, Fig. 1C) that is present in the FRA-2-regulated Nr4a1 gene and binds FRA-2 (18Humphries A. Weller J. Klein D. Baler R. Carter D.A. J. Neurochem. 2004; 91: 946-955Crossref PubMed Scopus (37) Google Scholar). The tgcgtca sequence (present in Nr4a1, FRA-2, and Rgs4) includes, on the reverse DNA strand, a CRE half-site which, in some sequence contexts, can bind CREB with high affinity (32Flammer J.R. Popova K.N. Pflum M.K. Biochemistry. 2006; 45: 9615-9623Crossref PubMed Scopus (18) Google Scholar). In Rgs4, this CRE site has been predicted to be a functional CRE (CRE-TATA module) as defined by whole genome bioinformatics and ChIP-Chip analysis (33Conkright M.D. Guzmán E. Flechner L. Su A.I. Hogenesch J.B. Montminy M. Mol. Cell. 2003; 11: 1101-1108Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 34Zhang X. Odom D.T. Koo S.H. Conkright M.D. Canettieri G. Best J. Chen H. Jenner R. Herbolsheimer E. Jacobsen E. Kadam S. Ecker J.R. Emerson B. Hogenesch J.B. Unterman T. Young R.A. Montminy M. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4459-4464Crossref PubMed Scopus (756) Google Scholar). The potential role of this AP-1R site in mediating the effects of FRA-2 was therefore addressed in further extensive studies of Rgs4, which is robustly expressed in the pineal gland as a single transcript of ∼3 kb (Fig. 1A).TABLE 3Search for AP1/CRE elements in FRA-2-regulated and nonregulated genesGenesAP1F elements relative to CREBach2.01 elements relative to CREtgcgtca sequenceValidated FRA-2 regulated genes (repressed)Atf-4Four up and down of eight CREtwo up and down of eight CRE0Cox6a2Two up of one CRENone0Dio2One up and down of five CRETwo up of one CRE0Nr4a1No AP1F, six CREFour down and over six CRE4Rgs4Three up and down of four CRETwo down and over two CRE1Validated FRA-2 regulated genes (enhanced)Cd24Two up of three CREOne up and down of three CRE0Fra2(Fosl2)NoneTwo down and over six CRE2Mt1aOne up and down of five CRETwo up of one CRE0Opn1swNoneOne upstream of three CRE0Validated FRA-2 nonregulated genesAanatOne up and down of five CRENone0DbpNoneOne down of two CRE0E4bp4 (Nfil3)One up and down of two CRENone0Crem (ICER)Two up and down of four CREOne down of four CRE0Id1Three up and down of six CREFour up and down of six CRE0Per2Three up & down of 5 CREOne up & down of 5 CRE0Syt4NoneThree up and down of eight CRE0 Open table in a new tab Rgs4 as a Model FRA-2-regulated GeneThe Rgs4 AP-1R site, which starts at position −155 (relative to TSS in rat, Fig. 1D), is 100% conserved across rat, mouse, and human genomic sequences and is generally highly conserved in mammalian species and many higher vertebrate species (supplemental Fig. S2). Furthermore, this is the only AP-1R element in the rat Rgs4 genomic locus between −5 kb of the 5′-flanking sequence to +5kb of the 3′-flanking sequence (chromosome 13, 85,533,882–85,540,173; Ensembl rat genome build Feb. 2006). No other AP-1 or CRE elements were found to be positionally conserved (in rat, mouse, and human) across this region. A consensus AP-1F (tgactca) sequence in the rat 5′-flanking sequence at position −415 is conserved neither in mouse, human (no AP-1F elements in −1500 to +100 region, MatInspector), nor other mammals. The −155AP-1R site of the rat Rgs4 promoter is therefore a promising candidate sequence for mediating the regulatory action of FRA-2.Ex Vivo ChIP Analysis of Rgs4 Promoter OccupancyTo confirm that FRA-2 regulation of Rgs4 is associated with a direct interaction of FRA-2 with the rat Rgs4 genomic locus, we next conducted ChIP analysis on rat pineal gland chromatin, targeting a region of the Rgs4 promoter that contains the AP-1R element discussed above. Using this approach, we found a specific enrichment of proximal Rgs4 promoter sequence following chromatin immunoprecipitation with a FRA-2 antiserum as compared with a control (FRA-2) preimmune serum (Fig. 2). Control ChIP assays also showed relative enrichment of both this Rgs4 sequence and a β-actin sequence when using a RNA polymerase II antiserum (Fig. 2).FIGURE 2Association of FRA-2 with Rgs4 promoter sequence in vivo. ChIP assays were conducted using chromatin extracted from rat pineal glands; gene promoter sequences were amplified by PCR and visualized by ethidium bromide staining of agarose gels. A, ChIP analysis reveals enrichment of pineal chromatin when precipitated with FRA-2 and RNA polymerase II (Pol II) antisera compared with a FRA-2 preimmune serum and IgG. IN, input chromatin; Con, water PCR control. Note that input chromatin is diluted relative to ChIP chromatin. A parallel assay conducted with primers specific for the control β-actin gene revealed enrichment only with polymerase II antisera. B, summated data from multiple Rgs4 ChIP assay. Values are expressed as % of input DNA (mean ± S.E. n = 3 assays from three individual groups of rats). Group means that are significantly different (p < 0.05) are indicated (paired Student's t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Functional Analysis of the Rgs4 PromoterFollowing our demonstration that the Rgs4 promoter is directly associated with FRA-2, we next conducted a functional investigation of Rgs4 promoter sequence via transfection assays in cultured cells. These experiments were designed to independently test the function of both the −155 AP-1R and the −415 AP-1F sequences. PC12 cells were selected for this analysis because it is a rat cell line that expresses Rgs4 (30Pepperl D.J. Shah-Basu S. VanLeeuwen D. Granneman J.G. MacKenzie R.G. Biochem. Biophys. Res. Commun. 1998; 243: 52-55Crossref PubMed Scopus (72) Google Scholar), FRA-2 (35Eriksson M. Taskinen M. Leppä S. J. Cell. Physiol. 2007; 210: 538-548Crossref PubMed Scopus (52) Google Scholar), and p-CREB (36Iwamoto T. Mamiya N. Masushige S. Kida S. Cytotechnology. 2005; 47: 107-116Crossref PubMed Scopus (9) Google Scholar). The nuclear environment in these cells therefore has characteristics of the nocturnal rat pineal gland in which both FRA-2 and p-CREB activity are strongly induced (14Smith M. Burke Z. Humphries A. Wells T. Klein D. Carter D. Baler R. Mol. Cell. Biol. 2001; 21: 3704-3713Crossref PubMed Scopus (51) Google Scholar, 37Baler R. Klein D.C. J. Biol. Chem. 1995; 270: 27319-27325Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 38Maronde E. Pfeffer M. Olcese J. Molina C.A. Schlotter F. Dehghani F. Korf H.W. Stehle J.H. J. Neurosci. 1999; 19: 3326-3336Crossref PubMed Google Scholar).Analysis of Rgs4 promoter construct expression in PC12 cells revealed that the Rgs4 sequences generated between 20- and 40-fold higher expression levels than the pGL4.11 plasmid alone (Fig. 3A). Mutation of the −155 tgcgtca sequence significantly enhanced expression compared with the wild type sequence (Fig. 3A; p < 0.05, ANOVA and Duncan's post hoc test; F = 31.294, df = 31). The longer (−426) construct was not associated with an altered expression level compared with the −167 wild-type construct, and mutation of the −415 tgactca sequence did not affect expression (Fig. 3A). Our finding that the AP-1R sequence appeared to mediate transcriptional suppression in the basal state was initially surprising, but Western blot analysis (Fig. 3B) revealed high levels of FRA-2 in the PC12 cell cultures used for transfection studies.FIGURE 3Functional analysis of Rgs4 promoter activity in transfected cells. Rgs4 constructs cloned into pGL4.11 were transfected into PC12 cells. Levels of expression were determined by luciferase (Luc) assays and corrected against a co-transfected Renilla luciferase construct. A, expression levels (luciferase activity) of the different constructs relative to empty pGL4.11 (mean ± S.E., n = 8; *, p < 0.05 versus all other groups, ANOVA and post hoc test). The position of the −415 AP-1F, −155 AP-1R, and −42 TATA elements (see text) are indicated by filled symbols and are shown as open symbols where mutated (Rgs4-167m and Rgs4-426m constructs, see text). B, Western blot of whole cell extracts (pineal 24.00 h, 50 μg; PC12 cells, 10 μg) probed sequentially with antisera to FRA-2 and GAPDH. Note the relatively high level of FRA-2 in unstimulated PC12 cells. Arrows indicate two (differentially phosphorylated (14Smith M. Burke Z. Humphries A. Wells T. Klein D. Carter D. Baler R. Mol. Cell. Biol. 2001; 21: 3704-3713Crossref PubMed Scopus (51) Google Scholar)) FRA-2 bands. Horizontal bars indicate molecular weight markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In Vitro Analysis of Rgs4 Promoter Protein BindingWe next examined the composition of protein complexes that bound the Rgs4 promoter. EMSA of interactions between an oligonucleotide probe containing the rat Rgs4 tgcgtca sequence and pineal gland nuclear protein revealed a sequence-specific interaction (Fig. 4). We discovered that two major shifted bands were observed in the presence of pineal protein extracts, and only one of these bands was found with extracts of brain cortex (Fig. 4A). Comparison of multiple pineal extracts obtained at either 12.00 or 24.00 h revealed a 3–4-fold increase in protein binding to the probe at night (Fig. 4B).FIGURE 4Protein composition and specificity of DNA binding activity at the Rgs4 AP-1R sequence in vitro. Representative chemiluminescent images of EMSAs showing bands of biotin-labeled oligonucleotide probe shifted in the presence of nuclear protein extracts (1.2 μg) from either pineal gland (P) or brain cortex (Cx). A, note that two shifted bands (upper and lower arrows) are observed in the pineal gland, whereas only the equivalent upper band is observed in the cortex together with two additional slower migrating bands (arrowheads). Note that both EMSA bands are more abundant in pineal samples extracted at 12.00 versus 24.00 h. Unbound (free) probe is indicated at the gel base. B, summated results of multiple EMSAs comparing the abundance of the upper and lower shifted bands in pineal glands sampled at either 12.00 and 24.00 h. Values are fold-difference compared with the level at 12.00 h (mean ± S.E., n = 6, *, p < 0.05 versus equivalent 12.00-h group, paired Student's t test). C, expanded image of shifted bands (only) showing the effect of different antisera on the abundance of pineal EMSA bands. Two different FRA-2 and two different CREB-1 antisera were compared, and a rabbit IgG was used in the Control lane. Note that the lower EMSA band is completely abrogated by both FRA-2 antibodies, whereas both EMSA bands are diminished by the two CREB-1 antibodies; the CREB-9197 (9197, Cell Signaling Technology) antibody is somewhat more effective in abrogating the bands compared with CREB-1–186 (sc186, Santa Cruz Biotechnology Inc.) antibody. Unbound probe is not shown. D and E, comparison of the effects of CREB-1 and phospho-CREB antisera on the abundance of EMSA bands observed in pineal glands sampled at either 12.00 or 24.00 h. Note that 24.00-h protein extracts were diluted 2-fold to equalize the intensity of binding activity relative to the 12.00-h samples. Expanded images of shifted bands (only) are shown in D, and summated results of multiple EMSAs are shown in E. Note that the CREB-1 antibody (sc186) diminishes the pineal EMSA bands to a similar extent in both groups, whereas the p-CREB antibody (9197, Cell Signaling Technology) significantly diminishes only the 24.00-h group. Histogram values are fold-difference compared with the control (IgG) level (mean ± S.E., n = 4; *, p < 0.05 versus equivalent control group (IgG), paired Student's t test). F and G, comparison of the effects of either unlabeled wild-type Rgs4 AP-1R probe (Rgs4; 4-, 8-, or 16-fold molar excess) with unlabeled mutant Rgs4 AP-1R probe (mutRgs4, similar molar excess) on the abundance of pineal EMSA bands. Expanded images of shifted bands (only) are shown in F, and summated results of multiple EMSAs are shown in G. Note that the unlabeled wild-type probe significantly competes with the labeled probe at all molar concentrations whereas the mutant probe competes significantly only at 16-fold molar excess. Histogram values are fold-difference compared with the control (No competitor, 1st bar in each group; mean ± S.E., n = 3; *, p < 0.05 versus no competitor, ANOVA, and post hoc test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Pineal proteins interacting with the EMSA probe were shown to include both FRA-2 and CREB based on abrogation in the presence of antisera (Fig. 4C). Complete abrogation of the lower band with FRA-2 antibodies indicated that this band was wholly composed of FRA-2-containing complexes. In contrast, the diminution of both bands in the presence of CREB antisera indicated that CREB was a constituent of both shifted bands. Because FRA-2 is apparently restricted to the lower band, this" @default.
• W1969345252 created "2016-06-24" @default.
• W1969345252 creator A5015461261 @default.
• W1969345252 creator A5021220986 @default.
• W1969345252 creator A5070313847 @default.
• W1969345252 date "2011-04-01" @default.
• W1969345252 modified "2023-09-26" @default.
• W1969345252 title "Selective Genomic Targeting by FRA-2/FOSL2 Transcription Factor" @default.
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