Báo cáo khoa học: The )148 to )124 region of c-jun interacts with a positive regulatory factor in rat liver and enhances transcription Dipali Sharma*, Sujata Ohri and Aparna Dixit ppt

The )148 to )124 region of c- jun interacts with a positive regulatory factor in rat liver and enhances transcription Dipali Sharma*, Sujata Ohri and Aparna Dixit Gene Regulation Laboratory, Center for Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India The c-jun gene encodes the protein Jun, a component of the essential transcription factor, AP1. Jun/AP-1 occupies a central position in signal transduction pathways as it is responsible for the induction of a number of genes in response to growth promoters. However, the exact mecha- nisms leading to an enhanced expression of the c-jun gene itself during proliferation, differentiation, cell growth and development are not fully understood. Cell culture studies have given some insight in the mechanisms involved in the up-regulation of c-jun expression by UV irradiation and phorbol esters. However, it is well known that transformed cells do not accurately reflect the biology of a normal cell. We now report the identification of a positive regulatory factor from normal rat liver that activates transcription from the c-jun promoter by binding to the )148 to )124 region of c-jun. Preincubation of fractionated rat liver nuclear extract with an oligonucleotide encompassing this region of the gene significantly reduced transcription from cloned c-jun pro- moter. In vitro transfection studies using green fluorescent protein as a reporter gene under the control of the c-jun promoter with ()148 to +53) and without ()123 to +53) this region further confirmed its role in transcription. A DNA-binding protein factor, interacting with this region of c-jun was identified from rat liver by using electrophoretic mobility shift assays. This factor binds to its recognition sequence only in the phosphorylated form and exhibits high affinity and specificity. UV cross-linking studies, South- Western analysis and affinity purification collectively indi- cated the factor to be 40 kDa and to bind to its recognition sequence as a dimer. Keywords:c-jun; DNA–protein interaction; in vitro tran- scription; rat liver positive regulatory factor; transcriptional regulation. Elucidation of the molecular mechanisms regulating eu- karyotic gene expression is essential for an understanding of the complex processes that occur during normal cellular development, differentiation and oncogenic transformation. Proto-oncogene c-jun encodes a protein Jun, a major component of transcription factor AP-1 [1–3]. Jun/AP-1 plays a role in the flow of information from cell surface receptors to the nucleus [4,5]. Jun has been reported to be involved in different aspects of cell growth, differentiation and development [6–8]. Expression of the c-jun gene is induced as an early response by serum active phorbol esters, ionizing radiation and tumour necrosis factor-alpha [9–11]. An increase in the expression of c-jun precedes DNA synthesis in proliferating cells. Jun/AP-1 is responsible for the induction of a number of genes in response to phorbol ester and tumour promoters and thus holds a central place in the signal transduction pathway. However, the exact mechanism(s) regulating c-jun expression during cell prolif- eration, differentiation, growth and development are not clearly understood except for its autoregulation by AP-1. AP-1 is known to autoregulate c-jun expression by binding to the AP-1 site present within the c-jun promoter [4,5]. Further, AP-1 transcription factors of different composition have been reported to control c-jun transcription in resting or stimulated cells [12]. c-jun expression and activity are partly regulated by Jun N-terminal kinases (JNKs) and mitogen activated protein kinases. JNKs phosphorylate the N terminus of the trans- acting domain of Jun, thereby increasing its transactiva- tion potency [13–16]. Inhibition of the stress-dependent signal cascade (JNK/SAPK pathway) by culture confluency inhibits c-jun N-terminal phosphorylation in response to platelet-derived growth factor, epidermal growth factor or UV irradiation [14]. Hence, Jun/AP-1 activity is regulated at two different levels. Immediately after stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA), a post-trans- lational event leads to an increased activity of pre-existing Jun/AP-1 molecules. The second step involves increased synthesis of Jun mediated by the interaction of activated Jun/AP-1 with the jun promoter, resulting in transcrip- tional activation [4,5]. The positive autoregulation of c-jun can therefore function as a major genetic switch respon- sible for the conversion of transient early events in signal transduction into long lasting effects on cellular gene expression. Correspondence to A. Dixit, Gene Regulation Laboratory, Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India. Fax: +91 11 6198234, Tel.: +91 11 6102164, E-mail: adix2100@mail.jnu.ac.in; adixit7@yahoo.com Abbreviations: RNE-d, rat liver nuclear extract-fraction D; EMSA, electrophoretic mobility shift assay; TPA, 12-O-tetradecanoyl phrobol 13-acetate. *Present address: The Johns Hopkins Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA. (Received 10 September 2002, revised 4 November 2002, accepted 6 November 2002) Eur. J. Biochem. 270, 181–189 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03369.x Regulation of c-jun is likely to involve many more cis- acting elements and a number of factors differentially interacting with these elements under different physiological conditions and may vary between cell types. All of the studies to understand c-jun transcriptional regulation have been conducted in cultured cells which do not mimic in vivo conditions. The present investigation was therefore under- taken to develop an understanding of regulation of c-jun expression in quiescent rat liver. We have identified a positive regulatory factor from normal rat liver that binds to the region )148 to )124 of c-jun and stimulates transcription. Materials and methods Reagents and animals All chemicals were of reagent grade and were from Sigma Chemical Co. unless stated otherwise. Healthy female inbred rats of Wistar strain weighing 150–170 g were procured from the Animal Facility, Jawaharlal Nehru University, New Delhi, India. Animals were fed water and standard rat chow ad libitum. Plasmid DNA isolation Escherichia coli cells, HB101 transformed with plasmid )1100/+170 jun-CAT were grown in liquid culture and plasmid DNA was isolated by the alkaline lysis method [17]. Plasmid )1100/+170 jun-CAT consists of the indicated region of the c-jun gene upstream of the promoterless CAT gene [4]. Fractionation of nuclear extract Animals were killed by cervical dislocation, livers were removed immediately, washed in chilled saline and pro- cessed further for the preparation of nuclear extract as described [18,19]. The fraction designated RNE-d contain- ing maximum RNA polymerase II activity and essential transcription factors was used in in vitro transcription assay and electrophoretic mobility shift assay (EMSA). In vitro run-off transcription assay In vitro transcription reactions were carried out using conditions described earlier [19,20]. The transcription reac- tion was carried out using 12 lgÆmL )1 EcoRI linearized plasmid )1100/+170 jun-CAT and 1.6 mgÆmL )1 nuclear protein (RNE-d) at 30 °C for 30 min. Transcripts extracted with phenol/chloroform/isoamylalcohol (25 : 24 : 1) were precipitated with ethanol and separated on a 6% acryl- amide, 8 M urea gel in 1 · Tris/borate/EDTA buffer [17]. The transcripts were visualized by autoradiography. EcoRI linearized plasmid )1100/+170 jun-CAT should yield a 370-nucleotides long run-off transcript. Transient transfection and reporter gene assay Promoter constructs. Green fluorescent protein (GFP) does not require any exogenous substrate and cofactors for its fluorescence and its expression can be used to monitor gene expression [21]. Also, GFP is a highly stable protein and fluorescence from GFP can be used as a quantitative measure of GFP content per cell [22]. Therefore, to assay jun promoter activity, two promoter constructs ) p123jun-eGFP and p148jun-eGFP ) were made by cloning PCR amplified )123 to +53 region and )148 to +53 region of c-jun, respectively. For both the amplifications, AseIandEcoRI restriction sites were included in the forward and reverse primers, respectively. PCR amplified fragments digested with AseIandEcoRI were cloned into AseI–EcoRI digested plasmid pEGFP-N1 (GenBank Accession # U55762, Invitrogen), thus placing the GFP coding region under the control of the )123 to +53 and )148 to +53 regions of c-jun in p123jun-eGFP and p148jun-eGFP, respectively. Recombinant clones were confirmed for insertion of the promoter regions of c-jun by sequencing. Cells and cell culture. Chinese hamster ovary (CHO) cells were maintained in Eagle’s modified essential medium (Biological Industries, Israel) supplemented with 10% heat- inactivated foetal bovine serum, 100 UÆmL )1 penicillin and 100 lgÆmL )1 streptomycin at 37 °C in a humidified atmos- phere containing 5% CO 2 . Transfection assay. CHO cells were plated at a density of 2 · 10 5 cells per well (35 mm diameter) in 2 mL Eagle’s modified essential medium containing foetal bovine serum, penicillin and streptomycin in six-well tissue culture plates (Falcon, Becton Dickinson) to achieve 50–80% confluency in 24 h. The cells were transfected with 2.5 lgeither p123jun-eGFP or p148jun-eGFP DNA and 5 lL Lipofec- tin reagent (Gibco-BRL) according to the manufacturer’s protocol. One lg pSV-bgal (Promega) was included as a control plasmid to monitor transfection efficiency. Twenty- four h after transfection, the DNA-containing medium was replaced with 2 mL normal growth medium and incubated at 37 °Cina5%CO 2 incubator for an additional 48 h. Medium was again removed and the cells were rinsed with NaCl/P i followed by an incubation in 500 lL lysis buffer (100 m M Tris/HCl pH 7.4, 0.15 M NaCl, 1.5 m M magne- sium acetate, 0.5% NP-40) at 37 °C for 5 min. The lysates were assayed for both GFP and b-galactosidase activity. GFP activity was assessed by measuring the fluorescence at 480 nm (excitation maximum) and 507 nm (emission maximum) in a Varian fluorescence spectrofluorometer (Varian Ltd, Germany). The b-galactosidase activity was measured using O-nitrophenol b- D -galactoside in phosphate buffer as per the manufacurer’s protocol. The results are reported as the ratio of the observed fluorescence to b-galactosidase activity in the respective sample to account for differences in transfection efficiency. EMSA EMSA using fraction RNE-d and a- 32 P-labelled oligonu- cleotide encompassing the )148 to )124 region of c-jun (designated Jun)25) was performed essentially as described by Garg et al. [23]. Two complementary synthetic oligonu- cleotides [(a) 5¢-CTAGGGTGGAGTCTCCATGGT GAC-3¢ ()148 to )124 of c-jun)and(b)5¢-GTCACCATG GAGACTCCA-3¢ (designed in such a way as to leave a seven base 5¢ overhang upon annealing with oligonucleotide 182 D. Sharma et al. (Eur. J. Biochem. 270) Ó FEBS 2003 ÔaÕ)] were obtained from Rama Biotechnologies (Hyderabad, India). Annealed oligonucleotide (Jun)25) was labelled by end filling using Klenow fragment and [a- 32 P]dCTP and purified on 15% polyacrylamide gel prior to its use in EMSA [23]. Various concentrations of RNE-d (prein- cubated with 500 ng fragmented calf thymus DNA for 20 min) were incubated with 1 ng (0.06 pmol) labelled Jun)25 ( 10 4 c.p.m.), in a reaction mixture containing 1 · binding buffer (10 m M Tris/HCl pH 7.5, 50 m M NaCl, 2.5 m M MgCl 2 ,1m M dithiothreitol, 1 m M EDTA, 0.1% Triton-X-100, 5% glycerol) in a final reaction volume of 40 lLat30°C for 30 min (unless otherwise stated). The complex was immediately loaded on a pre-electrophoresed 6% nondenaturing polyacrylamide gel and electrophoresed in 1 · Tris/glycine buffer (0.192 M glycine, 25 m M Tris/ HClpH8.3)at11VÆcm )1 for 3 h. The products were analysed by autoradiography. For competition experi- ments, unlabeled Jun)25 oligonucleotide or nonspecific DNA (pBR322 and fragmented calf thymus DNA) were added to the reaction mixture prior to the addition of labelled Jun)25. Alkaline phosphatase treatment Fraction RNE-d (100 lg nuclear protein) was treated with 2–20 U calf intestine alkaline phosphatase (Boehringer Manheim, Germany) for 30 min at 37 °C [24] in the presence of 1 · binding buffer. RNE-d treated with heat-inactivated phosphatase was used as a control. Phos- phatase-treated nuclear extracts were assayed for their DNA-binding capacity in standard EMSA. UV crosslinking of DNA–protein adduct The EMSA reaction was carried out using 1 ng labelled Jun)25 and 100 lg nuclear protein as described earlier. After 15 min, the reaction mixture was placed on ice and UV irradiated (254 nm) for 15 min [25]. Following irradi- ation, the mixture was separated by SDS/PAGE (15% acrylamide) and analysed by autoradiography. South-Western blot analysis South-Western analysis of RNE-d with labelled probe (tetramer of Jun)25) was performed essentially as described by Philippe [26]. Fraction RNE-d of rat liver nuclear extract was separated by SDS/PAGE on a 12% acrylamide gel and transferred electrophoretically to a nitrocellulose mem- brane. All of the following steps were performed at 4 °C. The membrane strip containing the sample was cut and incubated in denaturing solution (6 M guanidine/HCl in 1 · binding buffer) for 10 min. To this, an equal volume of 1 · binding buffer was sequentially added to dilute guanidine/ HCl in the denaturing buffer to 3 M ,1.5 M ,0.75 M ,0.38 M and 0.185 M with a 5-min incubation after each addition. The membrane was then blocked for 1 h in blocking buffer (5% BSA in 1 · binding buffer) and washed four times with 1 · binding buffer for 10 min each. Finally, 1 · binding buffer consisting of labelled tetramer of Jun)25 (10 6 c.p.m.ÆmL )1 ), fragmented calf thymus DNA (10 lgÆmL )1 ) and 0.25% BSA was added and allowed to incubate overnight. The strip was washed with three changes of 1 · binding buffer over a period of 30 min and autoradiographed. Affinity purification of the factor(s) interacting with the )148 to )124 region of c- jun This was carried out essentially as described by Kadonaga and Tjian [27]. First, 220 lg annealed oligonucleotides encompassing the )148 to )124 region of c-jun were 5¢end labelled using polynucleotide kinase and [c- 32 P]ATP. The radiolabelled oligonucleotides were ligated and analysed for the presence of oligomers ranging from 3 · to 75 · of Jun)25 on nondenaturing PAGE. The concatemers were coupled to commercially available CNBr-activated seph- arose CL-4B resin in the presence of 10 m M potassium phosphate pH 8.0. The oligonucleotide-affinity resin thus prepared was collected on a sintered glass funnel, washed with 200 mL H 2 O and 100 mL 1 M ethanolamine/HCl pH 8.0. The oligonucleotide-affinity resin was finally sus- pended in 14 mL 1 M ethanolamine/HCl. All procedures were carried out at 4 °C. DNA-affinity resin was poured in a syringe column plugged with glass wool and equilibrated with 1 · binding buffer excluding Triton-X-100. The salt concentration of the protein sample (RNE-d) was adjusted to 0.1 M NaCl. Fraction RNE-d (10 mg) was then incuba- ted for 10 min on ice with fragmented calf thymus DNA at 100 ngÆlg )1 protein to block nonspecific binding followed by incubation with the resin in a 15-mL tube with end-over- end mixing for 30 min at 4 °C. Resin incubated with RNE-d and fragmented calf thymus DNA was packed in a 3-mL syringe column followed by washing with binding buffer and was eluted with binding buffer containing increasing concentrations of NaCl at a flow rate of 15 mLÆh )1 .The fractions collected were frozen rapidly in liquid nitrogen and stored at )70 °C. Aliquots from the various fractions were analysed by EMSA. The fractions were also analysed by SDS/PAGE and silver staining [28]. Results and discussion Role of the )148 to )124 region of c- jun in transcription Angel et al. [4] have reported that binding of AP-1 to its consensus sequence within the c-jun promoter positively autoregulates c-jun expression. It was also reported that sites further upstream of the AP-1 site may be involved in the transcriptional regulation of c-jun [29]. In order to investi- gate the functional properties of upstream regions of c-jun, several oligonucleotides encompassing various upstream regions were synthesized and analysed for their role in transcription, if any. Fractionated nuclear extract prepared from normal rat liver could accurately transcribe EcoRI- linearized plasmid )1100/+170 jun-CAT (Fig. 1A). Prein- cubation of RNE-d with the )148 to )124 region of c-jun resulted in a significant decrease in intensity of the transcripts obtained (Fig. 1B, lanes 5–7) while no decrease in the transcription was obtained when RNE-d was preincubated with equimolar concentrations of pBR322 (lanes 2–4). These results suggest that this region specifically binds to some positive regulatory factors present in normal rat liver and preincubation with this oligonucleotide Ó FEBS 2003 Regulation of c-jun expression in rat liver (Eur. J. Biochem. 270) 183 titrates out these factors thus resulting in a decreased transcription. To establish the direct role of this region in c-jun transcription, CHO cells were transfected with p123jun- eGFP and p148jun-eGFP plasmids containing GFP as a reporter gene as shown in Fig. 2A. It is clear from Fig. 2B that the presence of the )148 to )124 region significantly increased GFP expression when compared to the control promoter present in pjun123-eGFP, substantiating the positive role of this region in c-jun transcription in normal rat liver. The )148 to )124 region of c- jun binds to factors present in fractionated rat liver nuclear extract As preincubation of nuclear extract with the oligonucleotide ()148 to )124) had resulted in a decrease in transcription, suggesting its interaction with positive factors present therein, binding reactions were carried out using different amount of RNE-d. As shown in Fig. 3A, optimum complex formation was obtained with 100, 150 and 200 lg nuclear protein in RNE-d (lanes 2–4) while at higher concentrations of RNE-d (250 and 300 lg, lanes 5 and 6), a decrease in the complex formation was observed. The factor(s) involved in the complex formation are designated as RLjunRP [rat liver jun regulatory protein(s)]. Binding of factors, present in normal liver, with this region of c-jun intrigued us as earlier studies [30,31] had shown that the )139 to )129 region of c-jun is recognized by NF-jun or NF-jun-like transcription factors present in cellular extracts from TPA-induced leukaemic cells. This activity was reported to be absent from nonproliferating diploid cells. Sequence-specific binding of RLjunRP Specificity of the complex formation between the factors and the )148 to )124 region of c-jun was examined (Fig. 3B) by preincubating 100 lg of the fraction RNE-d with a 100-fold excess of unlabelled nonspecific DNA [fragmented calf thymus DNA (lane 7), pBR322 (lane 8) and unlabelled oligonucleotide (5–20 ng, lanes 3–6)] prior to the addition of labelled oligonucleotide Jun)25 (1 ng). As is evident, the complex formation was completely abolished when RNE-d was preincubated with unlabelled Jun)25 whereas no effect on the complex formation was observed when a 100- to 200-fold excess of nonspecific DNA was used for competition, indicating the specificity of complex formation. The complex formation did not take place in the Fig. 2. Effect of )148 to )124regiononc-jun promoter activity. (A) Schematic diagram of plasmids p123jun-eGFP and p148jun-eGFP used in reporter gene assay. Plasmid p123jun-eGFP consists of the )123 to +53 region of c-jun cloned upstream of the GFP coding region and p148jun-eGFP consists of the )148 to +53 region of c-jun cloned upstream of the GFP coding region. (B) Transfection assay and GFP expression under the control of the c-jun promoter. CHO cells (2 · 10 5 cellsÆmL )1 , in triplicate) were transfected with 2.5 lg p123jun- eGFP or p148jun-eGFP along with 1 lgofpSV-bgal plasmid. Cells transfected with 2.5 lgpEGFP-N1and1lgpSV-bgal served as a positive control. Relative fluorescence shown here represent mean + SEM of three independent transfections performed in tripli- cate for the respective plasmids. Fig. 1. (A) In vitro transcription of EcoRI-linearized )1100/+ + 170 jun- CAT plasmid with fractionated rat liver nuclear extract (RNE-d) and (B) effectofthe)148 to )124 region of c-jun on in vitro transcription of linearized )1100/+ + 170 jun-CAT plasmid. (A) Linearized template (12 lgÆmL )1 ) was transcribed with rat liver fraction RNE-d (0.4 and 0.8 lgÆmL )1 , lanes 1 and 2, respectively). The arrow points to the 370-nucleotide-long run-off transcript and M indicates end-labelled molecularmassmarkers/X174 DNA digested with HaeIII. (B) In vitro transcription reactions were carried out using 10 lgÆmL )1 EcoRI linearized plasmid )1100/+170 jun-CAT as template and 1.6 mgÆmL )1 RNE-d (lane 1). Lanes 5–7 represent the transcripts obtained from in vitro transcription reactions carried out with fract- ionated nuclear extract preincubated with 10, 20 and 40 ng oligonu- cleotide Jun)25, encomapassing the )148 to )124 region of c-jun for 20 min prior to the addition of template. Lanes 2–4 represent tran- scription reaction carried out with RNE-d preincubated with equi- molar concentrations of pBR322 to the amount of oligonucleotide used in lanes 5–7, respectively. 184 D. Sharma et al. (Eur. J. Biochem. 270) Ó FEBS 2003 presence of 7.5% formamide further confirming the speci- ficity of protein–oligo interaction (lane 2) as formamide is known to dissociate the protein factors from the recognition sequence. The presence of high affinity of RLjunRP for its cognate sequence was established by performing binding reactions in the absence of fragmented calf thymus DNA (Fig. 3B, lane 2) which is used to titrate out nonspecific DNA binding protein. RLJunRP present in crude nuclear extract could bind even in the absence of nonspecific DNA showing that it has a high binding affinity enabling it to compete with the nonspecific DNA-binding proteins present in the extract. Regulatory proteins are known to bind to their specific recognition sites with higher affinity than unrelated DNA sequence [32]. Specific DNA-binding proteins can bind nonspecifically to nontarget DNA, albeit with low affinity. Therefore, if excessive nonspecific DNA is added, it will compete for the specific factor of interest and the level of the specific complex will decrease. Binding reactions were performed using 100 lg RNE-d and 1 ng labelled )148 to )124 region of c-jun in the presence of much higher excess of fragmented calf thymus DNA to inspect the specificity of the interac- tions between RLjunRP and the )148 to )124 region of c-jun. When RNE-d was incubated with labelled Jun)25 oligonucleotide in the presence of a 1000-, 10 000-, 20 000- and 40 000-fold excess of nonspecific fragmented calf thymus DNA (Fig. 3C; lanes 1–4), specific DNA–protein adducts were observed confirming the remarkable specificity of RLjunRP. The optimum concentration of monovalent cations was determined by carrying out EMSA using 100 lg nuclear proteins and 1 ng labelled )148 to )124 region of c-jun in the presence of different concentrations of NaCl. Complex formation was observed over a range of concentration of monovalent cations, i.e. 25–250 m M (Fig. 4A, lanes 1–5). At 500 m M (lane6),therewasadecreaseinthecomplex formation. The fact that RLjunRP retained its binding activity even in the presence of 0.5 M NaCl indicated that the factor has a higher than usual affinity to the recognition sequence. Most of the DNA-binding proteins exhibit binding activity with a rather limited range of monovalent cations with optimal binding at either low or high salt concentrations. The RNA polymerase II transcription factor TFIIB (which is considered to be unusual in terms of high salt resistance) can be stripped off its cognate DNA sequence by high salt concentrations [33]. It was observed that TFIIB could bind to its specific sequence only at low salt concentration, following which it can withstand increa- ses in NaCl concentration. However, TFIIB cannot bind at high salt concentration. RLjunRP, in contrast, can actually bind to its recognition sequence at a relatively higher salt concentration. The fact that the complex formation between RLjunRP and the )148 to )124 region of c-jun was not highly affected by the fluctuation in NaCl concentration indicates that the protein–DNA association is probably through interactions that are nonionic. The involvement of divalent cations that are required for certain protein–cognate sequence interaction was investi- gated by carrying out EMSA in the presence of EDTA (Fig. 4B). Inclusion of 100 m M EDTA in the binding reaction resulted in a slight decrease in complex formation (lane 3) and no complex was observed in the presence of 150 m M EDTA (lane 4). It is likely that in the presence of 50 or 100 m M EDTA (Fig. 4B, lanes 2 and 3, respectively), most of the divalent cations are chelated but there might still be small amounts of free divalent cations (unchelated), which are sufficient for complex formation. When the EDTA concentration is raised to 150 m M (Fig. 4B, lane 4), all of these ions are chelated and no complex formation is observed. These data suggest that very small amounts of divalent cations are necessary for the formation of complex between RLjunRP and the )148 to )124 region of c-jun, and so the optimum amount of MgCl 2 required for complex formation was then titrated (Fig. 4C). Complex formation couldbeseeninthepresenceof1m M MgCl 2 (lane 1). Binding was found to be maximal in the presence of 2.5 m M MgCl 2 (lane 2). Studies on the effect of temperature (Fig. 4D) on complex formation revealed that the factors present in RNE-d formed the complex even at temperature as low as 0 °C Fig. 3. Specificity of complex formation between )148 to )124 region of c-jun and factors present in RNE-d. (A) Titration of optimum concen- tration of nuclear extract for binding. EMSA reactions were carried out in the presence of 1 ng )148 to )124 region of c-jun and various con- centrations of nuclear proteins as indicated. (B) jun-RP forms specific complex with the )148 to )124 region of c-jun. Lane 1 represents the interaction of factor(s) present in fraction RNE-d with 1 ng )148 to )124 region of c-jun. EMSA reactions were carried out using 100 lgof RNE-d preincubated with a 100-fold excess of unlabelled nonspecific DNA [fragmented calf thymus DNA (lane 7), pBR322 (lane 8)], and in the presence of various concentrations of unlabeled Jun)25 oligo- nucleotide encompassing the )148 to )124 region of c-jun (lanes 3–6) prior to the addition of labelled Jun)25. Lane 2 depicts the binding reaction carried out in the presence of 7.5% of formamide. (C) RLjunRP can form complexes even in the presence of a 40 000-fold excess of fragmented calf thymus DNA. The binding reactions were carried out with 1 ng labelled )148 to )124 region of c-jun and 100 lg fractionated nuclear extracts in the presence of 1 lg(lane1),10lg (lane 2), 20 lg(lane3)and40 lg (lane 4)fragmented calf thymus DNA. Ó FEBS 2003 Regulation of c-jun expression in rat liver (Eur. J. Biochem. 270) 185 (lane 1). No significant change in complex formation was observed untill 30 °C (lanes 2–6). However, very little complex formation occurred when EMSA was carried out at 45 °C (lane 7) and no complex was formed at 55 °C onwards. Unlike TATA binding protein that becomes totally inactivated within 15 min of heat treatment at 47 °C [34], junRP retains its DNA-binding activity, although at a relatively low level, even when the binding reaction was carried out at 45 °C for 30 min. Phosphorylation of RLjunRP is imperative for its DNA-binding activity Inducible phosphorylation or dephosphorylation of tran- scription factors is an important mechanism of signal dependent gene regulation in eukaryotic cells [35,36]. It is generally assumed that protein phosphorylation stabilizes different conformational states of the regulated and regulatory molecule to enhance or inhibit biological activity [36–40]. To check whether RLjunRP interacts with the )148 to )124 region of c-jun in the phospho- rylated or dephosphorylated form, nuclear extract from normal liver was treated with various concentrations of calf intestinal alkaline phosphatase prior to its addition to the EMSA reaction (Fig. 4E). A decrease in complex formation was observed with increasing concentrations of alkaline phosphatase from 4 U upwards and the treat- ment of RNE-d with 20 U of enzyme completely abolished DNA binding (lane 2) suggesting that RLjunRP interacts with the cis-element only in phos- phorylated form. The inhibitory effect of phosphorylation on DNA binding is depicted by a number of trans-acting factors whereas phosphorylation is necessary for DNA binding in very few cases [35], making RLjunRP unique in this respect. It is possible that phosphorylation of RLjunRP is imperative to maintain its DNA-binding domain in an active conformation. RLjunRP is an  40 kDa protein that forms an 80-kDa protein–DNA adduct To assess approximate molecular mass of the factors interacting with the )148 to )124 region of c-jun, RLjunRP complexed with this region was UV irradiated (254 nm) for 15 min. After separation by SDS/PAGE on a 15% acrylamide gel, the complex was visualized by autoradio- graphy (Fig. 5A). The molecular mass of the cross-linked junRP was  80 kDa as evident from lane 1. The protein– DNA complex shows a retarded electrophoretic mobility as compared with the free DNA fragment. The parameter for the degree of retardation of a linear DNA fragment bound in a complex with its specific factors reflects the molecular mass of the bound protein(s), as the molecular mass of DNA is negligible [41–43] in terms of the charge : mass ratio required to alter the mobility of the complex. South- Western analysis of rat liver nuclear extract using the )148 to )124 region as a probe, revealed a hybridized band of  40 kDa (Fig. 5B). These data suggest that RLjunRP binds to its recognition sequence as a dimer. Affinity purified RLjunRP is a protein of  40 kDa To confirm that RLjunRP is indeed a protein of  40 kDa, it was affinity purified from rat liver nuclear extract (Fig. 6). Major peak fractions eluted between 0.1 M and 0.2 M NaCl (Fig. 6A) contained nonspecific DNA binding proteins, as these fractions did not show any complex formation in EMSA (Fig. 6B). The factor(s) interacting with the )148 to Fig. 4. Binding characteristics of RLjunRP. (A) Titration of optimum monovalent cation concentration. Binding reactions between junRP and the )148 to )124 region of c-jun were carried out in the presence of 25, 50, 75, 100, 250 and 500 m M NaCl (lanes 1–6, respectively) using 100 lg nuclear extract and 1 ng labelled )148 to )124 region of c-jun. (B) Divalent cations are absolutely essential for the binding activity of junRP(s). EMSA were carried out with 100 lgfractionatednuclear extract RNE-d and 1 ng labelled )148 to )124 region of c-jun in the presence of 25, 50, 100 and 150 m M EDTA (lanes 1–4, respectively). (C) Determination of optimum divalent cation concentration for complex formation. EMSA were carried out using 1 ng labelled )148 to )124 region of c-jun and 100 lg fractionated nuclear extract from normal rat liver in the presence of 1 m M (lane 1), 2.5 m M (lane 2), 5 m M (lane 3), 10 m M (lane 4), 15 m M (lane 5) and 20 m M (lane 6) MgCl 2 and analysed by nondenaturing PAGE on 6%acrylamide gels.(D) Complex between Jun)25 and RLjunRP forms over a wide temperature range. The binding reactions between 100 lg fraction RNE-d from normal liver and 1 ng labelled )148 to )124 region of c-jun were carried out at temperatures ranging from 0 to 65 °C (lanes 1–9). (E) Phosphorylation of RLjunRP is necessary for its DNA-binding activity. One-hundred micrograms fractionated nuclear extract from normal rat liver was treated with different concentrations of calf intestine alkaline phos- phatase (shown at the top) prior to its addition to EMSA. Lane 1 shows the complex formed between RNE-d treated with heat inactivated alkaline phosphatase (10 U) and labelled Jun)25. 186 D. Sharma et al. (Eur. J. Biochem. 270) Ó FEBS 2003 )124 region of c-jun eluted in the 2.0 M NaCl fraction (fractions 38–42) as evident from the formation of retarded complex with labelled )148 to )124 region of c-jun.Allof the proteins that do not interact with the )148 to )124 region of c-jun, may nonspecifically bind to the affinity matrix and be eluted at a lower salt concentration. SDS/ PAGE of different peaks obtained from affinity chroma- tography showed a band of  40 kDa (Fig. 6C, lane P). Presence of a purified factor of  40 kDa is consistent with our South-Western data. These data further confirm that RLjunRP is indeed a protein of  40 kDa and binds to its recognition sequence as a dimer. Dimerization of several transcription factors has been found to be necessary for their interaction with recognition sequence [44,45]. It is likely that dephosphorylation (which results in complete loss of complex formation) results in the dissociation of the dimers and the monomers are not able to bind to the )148 to )124 region of c-jun. This study thus provides an insight into the molecular mechanisms regulating the c-jun expression in quiescent cells. The data indicate that the )148 to )124 region of c-jun is a functional motif present upstream of the gene promoter region, interacting with positive regulatory trans-acting factors present in rat liver. Although previous studies have reported the presence of an inducible factor, NF-jun, in human myeloid leukaemia cells that protected the )139 to )129 region of c-jun [30], NF-jun binding activity was found to be absent from nonproliferating diploid cells and appeared to be restricted to dividing cells [30,31] as growth arrested human embryonic lung fibroblasts, granulocytes and resting human T cells did not express NFjun constitu- tively. Further in Hela cells, it has been shown that NF-jun is already bound to its recognition sequence (before transcriptional activation of c-jun by TPA and UV irradi- ation). Thus, NF-jun behaves differently in different cell types, being translocated from the cytosol to the nucleus upon induction by an external stimulus in human myeloid leukaemia cells but found already bound to c-jun gene in uninduced Hela cells. Thus, RLjunRP differs from the factor NF-jun reported by Brach et al. [30] (that interacts with the )139 to )132 Fig. 6. Affinity Purification of factors interacting with the )148 to )124 region of c-jun. (A) Spectrophotometric elution Profile: RNE-d was subjected to sequence-specific affinity column chromatography and all fractions obtained were analysed spectrophotometrically. Absorbance at 280 nm was measured and plotted. (B) Assessment of complex formation ability of eluted fractions from DNA affinity column. Presence of RLjunRP in different fractions obtained by affinity chro- matography was checked using EMSA with labelled )148 to )124 oligonucleotide fragment of c-jun. L represents EMSA reaction with the loaded fraction and the numbers on top represent the fraction numbers. The numbers at the bottom represent the salt concentration in the respective fraction. (C) SDS/PAGE of RLjunRP-positive frac- tion. The fractionated nuclear extract, RNE-d fraction (L), flow- throughfraction(F)andpeakfractionnumber38(P)showingDNA binding ability in EMSA, were subjected to SDS/PAGE and silver stained. M represents the mid-range molecular mass markers. Fig. 5. UV cross-linking and South-Western blot analysis. (A) Deter- mination of the molecular mass of complex between junRP and the )148 to )124 region of c-jun by UV cross-linking. Complex between RLjunRP (lane 1) with its cognate sequence was formed under standard conditions using 100 lgRNE-dand1 ng)48 to )124 region of c-jun followed by UV irradiation (254 nm) for 15 min. DNA–pro- tein complex was separated from free DNA by SDS/PAGE. Autora- diography revealed the presence of complex (shown by arrowhead). Numbers represent protein molecular mass markers. (B) South-West- ern blot analysis of fraction RNE-d with Jun)25. Fifty and 75 lg nuclear extract fraction RNE-d were fractionated by SDS/PAGE (lanes 1 and 2), transferred onto a nitrocellulose sheet and probed with radiolabelled tetramer of Jun)25 oligonucleotide. The molecular mass of the markers is shown on the left. Ó FEBS 2003 Regulation of c-jun expression in rat liver (Eur. J. Biochem. 270) 187 region) with respect to it being present in resting liver cells whereas NF-jun is found to be restricted to rapidly dividing cells such as myeloid leukaemia cells and is not detectable in nonproliferating diploid lung fibroblasts, blood monocytes, granulocytes or resting T cells. Thus, in vivo occupancy of the )148 to )124 region in the c-jun promoter with RLjunRP cannot generally be associated with the prolifer- ative state of the cells. Further, NF-jun forms DNA–protein adducts of 55 and 125 kDa as established by UV cross- linking studies suggesting that it can bind to the sequence both as a monomer and dimer [20]. Unlike NF-jun, RLjunRP shows only a single complex at  80 kDa in UV cross-linking studies whereas the purified protein is only  40 kDa, suggesting that it binds only as a dimer. Absence of an  40 kDa DNA-protein adduct in UV cross-linking studies indicates that RLjunRP is not able to bind as a monomer. Thus, we have clearly demonstrated a direct involvement of the )148 to )124 region of c-jun in its transcription and its interaction with positive regulatory factor (RLjunRP) in normal rat liver. The positive regulatory factor interacting with this region was purified to homogeneity and the cDNA cloning of the gene encoding this factor is in progress to help in understanding its structural and functional aspects. Acknowledgements P. Angel, Institute for Genetik, Kernforschungszentrum Karlruhe, GmBH Postfach 3640 D-76021, Karlsruhe, Germany is gratefully acknowledged for providing the )1100/+170 jun-CAT plasmid. This work was supported by a research grant (#37(834)/94-EMR-II) from the Council of Scientific and Industrial Research (CSIR), India to A.D. CSIR, India is duly acknowledged for the Senior Research Fellowships to D.S. and S.O. The technical assistance of S. Singh is sincerely appreciated. 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The )148 to )124 region of c- jun interacts with a positive regulatory factor in rat liver and enhances transcription Dipali Sharma*, Sujata Ohri and Aparna. quiescent rat liver. We have identified a positive regulatory factor from normal rat liver that binds to the region )148 to )124 of c-jun and stimulates transcription. Materials