Interaction of an  40 kDa protein from regenerating rat liver with the )148 to )124 region of c-jun complexed with RLjunRP coincides with enhanced c-jun expression in proliferating rat liver Sujata Ohri*, Dipali Sharma† and Aparna Dixit Gene Regulation Laboratory, Center for Biotechnology, Jawaharlal Nehru University, New Delhi, India The c-jun belongs to the family of proto-oncogenes and encodes for the protein Jun, a component of transcription factor AP-1 involved in regulation of the expression of genes indispensable for cell proliferation and differentiation. While the r ole o f c-jun in the r egulation o f s uch g enes has been well examined, the regulation of c-jun in proliferating cells is not fully understood. We have earlier reported that the )148 to )124 region of c-jun is involved in the positive regulation of c-jun transcription, and interacts with a pos- itive regulatory factor (rat liver jun regulatory protein; RLjunRP) present in rat liver. In this investigation, we report t hat t his region is d ifferentially recognized in prolif- erating liver as evidenced by the formation of a complex, different from that observed with normal liver extract. The new c omplex appears as early as 2 h after partial hepatec- tomy and i ts appearance coincides w ith the rise in c-jun mRNA levels after partial hepa tectomy. In regenerating rat liver nuclear extract, an additional protein of  40 kDa (rRLjunRP) interacts with a pre-existing dimer o f RLjunRP complexed with the )148 to )124 region of c-jun to f orm a slow-migrating complex. rRLjunRP a ppears to pre-exist in the cytosol and translocate to the nucleus as indicated by the co ntinued p resence of t he retarded complex in nuclear extract prepared from partially hepatectomized rats treated with cycloheximide. UV crosslinking studies, South-West- ern blot a nalysis, SDS/PAGE of affinity-purified factor(s), and 2D-PAGE analys is clearly d emonstrate t hat t he addi- tional factor in duced in response t o g rowth stimulus i s an  40 kDa, th at binds with the dimer of RLjunRP and enhances the c-jun transcriptio n. Keywords: c-jun; DNA–protein interac tion; positive regula- tory factor; r egenerating liver; t ranscriptional regulation. Precise and coordinated control of gene expression is a primary r equirement for normal growth, development and function of an organism. Major control of gene expression is exerted by regulating m RNA p roduction and involves complex i nteractions between an array of t ranscriptionally active proteins and s pecific regulatory DNA sequences. We have been interested in explicating t he underlying m olecular mechanisms of transcriptional regulation of c-jun in resting and proliferating rat liver. The protein Jun, a major component of the dimeric transcription factor complex activating protein-1 (A P-1), i s e ncoded by c-jun [1–5]. Be ing a component of AP-1, Jun is known to be involved in the regulation of a variety of cellular processes including cellular proliferation, differentiation, apoptosis and oncogenesis ([6–9], r eviewed i n [10]). As a n immediate early response gene, expression of c-jun is affected by a variety of extracellular stimuli including growth factors, cytokines, serum phorbol esters, tumour promoters, UV radiation a nd hormones [11–16]. Jun is known to regulate the expression of a myriad of genes i n a variety of tissues and c ell types [17]; however, transcriptional regulation o f c-jun itself still re mains elusive. Among known i mportant r egulatory elements previously identifiedinthec-jun promoter are the two AP-1 sites ()71 to )64 and )190 to )183) [18]. Pre-existing cJun homo- dimers and cJun/ATF-2 heterodimers can bind to these two AP-1 sites a nd activate transcription [13,18]. I nvolvement of the )148 to )124 region of c-jun in the positive regulation o f transcription from t he c- jun promoter through its interaction with a pos itive r egulatory f actor (rat liver jun regulatory protein; RLjunRP), which is present in quiescent rat liver nuclear extract, has been reported [19]. Brach and coworkers have earlier reported the presen ce of a factor, Nuclear factor-jun (NF-jun), in human myeloid l eukaemia cells that protected the )139 to )129 region of c-jun [20]. However, its Correspondence to A. Dixit, Centre for Biotechnology, Jawaharlal Nehru University, N ew Delhi - 110067, India. Fax: +91 11 26198234, Tel.: +91 11 26102164 or 26704085, E-mail: adix2100@mail.jnu.ac.in or adixit7@yahoo.com Abbreviations: AP-1, activating protein-1; C HX, cyclohexamide; EMSA, electrophoretic mobility shift assay(s); IL-6 DBP, interleukin- 6 dependent DNA binding protein; NF-jun, nuclear factor-jun; NF-jB, nuclear factor-jB; RLjunRP, rat liver jun regulatory protein; rRLjunRP, r egenerating rat liver jun regulatory protein; RNE-d, rat liver nuclear extract-fraction D; nRNE-d, normal rat liver nuclear extract-fraction D; rRNE-d, r egenerating rat liver nuclear extract- fraction D ; TFIIIA, 5S R NA gene-specific transcription factor IIIA; TFIIIC, 5S R NA gene-specific tr anscription factor IIIC. *Present address: Department of Pathology and Laboratory Medicine, University of Louisville, 511 S F loyd Street, Louisville, K Y-40202, USA. Present address: Department o f Hematology-Oncology, W inship Cancer Institute, E mory University Schoo l of Med icine, Clinic C, 1701 Uppergate D rive Rm # 4060, Atlanta, G A-30322, USA. (Received 9 Septem ber 2 004, rev ised 10 O ctober 2004, accepted 25 Octobe r 2004) Eur. J. Biochem. 271, 4892–4902 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04458.x activity was found to be absent from nonproliferating diploid cells and appeared to be restricted to dividing cells [20,21]. RLjunRP that binds to the )148 to )124 region of c-jun, identified in our laboratory previously, differs from NF-jun with respect to it being p resent in resting liver cells [19]. These findings suggest that expression of c-jun is like ly to differ from one cell type to another. Because d ifferential interaction of factors with the cis acting elements under different physiological conditions and in response to growth stimuli, in part, is known to regulate differential gene expression, it is likely that some o ther inducible factor interacts with R LjunRP bound to the )148 to )124 region, and further enhances c-jun transcription in proliferating liver. Most of the studies to gain an insight into the transcrip- tional r egulation of c-jun in response to g rowth s timuli have been conducted in cultured cells which do no t mimic in vivo conditions. We have therefore chosen regenerating rat l iver following partial h epatectomy, a s a source of proliferating tissue to mimic in v ivo conditions. Surgical removal of two- thirds of the liver results in regeneration of the remaining liver lobes until t he original liv er mass is regained [22–24]. Partially hepatectomized liver has been proposed as a model for hepatic neoplasia and is a well-suited system to study normal regulated growth [24–26]. I t serves as a s ource of relatively abundant quantities of homogeneous growing tissue. P artial h epatectomy l eads to an orchestrated regen- erative response, activating a cascade of cell signalling events necessary for cell c ycle pro gression and proliferation of hepatocytes. Because progression of liver proliferation can be followed u sing this system, regenerating liver allows us to follow c hanges in the specific factor(s) t hat may be involved i n the initiation o f regeneration, liver growth and development. The Jun protein has been reported to b e a major constituent of the AP-1 com plex both i n quiescent and e arly regenerating liver [27,28]. Activation of AP-1, in turn, influences the expression of several genes essential for the proliferation of hepatocytes [14,29]. It has also been shown that the liver specific deletion of c-jun leads to decreased hepatocyte p roliferation. Investigating regulation of c-jun in regenerating liver is thus of significance to study normal regulated growth in regenerating liver. The present investigatio n was ther efore undertaken with an attempt to e lucidate whether the )148 t o )124 r egion o f c-jun is differentially recognized by factors present in resting and proliferating liver, and its implication on e nhanced c-jun expression in re generating rat liver. Materials and methods Animals and partial hepatectomy Healthy female inbred rats of Wistar strain (150–170 g) were procured from the Animal Facility, Jawaharlal Nehru University, New Delhi, India. The rats were treated humanly using approved procedures in accordance with the guidelines of the Institutional Animal Ethics C ommittee at the Jawaharlal Nehru University, New Delhi. Animals were fed water and standard rat chow ( Hindustan Lever Ltd, Mumbai, India) ad libitum and maintained o n a 12 h light/dark cycle. P artial hepatectomy ( 70%) was performed on animals as described earlier [ 30]. For the i mmediate and early t ime points of 0 and 15 min postsurgery, the incision was covered with sterile gauze, saturated with sterile saline, before harvesting the liver remnant. For longer time points, incisions were sutur ed closed until the liver was harvested. Fractionation of nuclear extract Experimental animals were killed at different postoperation- al intervals. Livers were removed immediately, snap frozen in liquid n itrogen and s tored at )80 °C until processed further for the preparation of nuclear extract as described earlier [31,32]. The fraction designated rat liver nuclear extract- fraction D (RNE-d), containing maximum RNA poly- merase II activity and essential t ranscription factors, was used in electrophoretic mobility shift assays (EMSA). nRNE-d refers to nuclear extract prepared from normal rat liver and rRNE-d r efers to nuclear extract prepared from regenerating rat liver sacrificed at 8 h after surgery (time after partial hepatectomy at which c-jun transcription peaks in rat liver) [ 33]. CHX-rRNE-d refers to nuclear extracts prepared from partially hepatectomized rat livers from animals injected with cyclohexamide (CHX in saline; 6 mg per 100 g body weight, interperitoneally) immediately after surgery. Liver was excised at 3 h p ost surgery. Protein estimation was carried out by the method of Bradford [34]. EMSA EMSA using nuclear extracts prepared at different postsur- gery intervals a nd a- 32 P-labelled oligonucleotide encompas- sing the )148 t o )124 region of t he c-jun promoter (Jun-25) was performed as described by Sharma et al .[19].The binding reaction consisted of 10 lg RNE-d (preincubated with 500 ng fragmented calf thumus DNA for 20 min), 1 n g (0.06 p M ) labelled J un-25 (  166.5 B q), 1 0 m M Tris/HCl pH 7.5, 50 m M NaCl, 2.5 m M MgCl 2 ,1m M dithiothreitol, 1m M EDTA, 0.1% (v/v) Triton X-100 and 5% (v/v) glycerol in a final reaction volume of 40 lL unless otherwise stated. T he complex formation was allowed to take place at 30 °C for 30 min followed by electrophoresis on a pre- electrophoresed 6% nondenaturing polyacrylamide gel in 1· Tris/glycine buffer ( 0.0192 M glycine, 25 m M Tris/HCl pH 8.3) at 11 V Æcm )1 for 3 h. The products were analysed by autoradiography. UV crosslinking of DNA–protein adducts UV crosslinking was performed to determine t he approxi- mate molecular mass o f DNA–protein adducts [19]. EMSA was carried out as described above except that 100 lgof nuclear protein and 5 ng of labelled Jun-25 were used. Following the binding reaction, the reaction mixture on ice was exposed to UV radiations in a UV Stratalinker (Stratagene, La J olla, CA, USA) and auto-crosslinked twice (2 · 60 mJ). Following UV irradiation, the mixture was separated on a 12% SDS/PAGE as described by Laemmli [ 35]. South-Western blot analysis South-Western b lot analysis using nRNE-d and rRNE-d with labelled probe (Jun-25 tetramer) was carried out as Ó FEBS 2004 Expression of c-jun in regenerating rat liver (Eur. J. Biochem. 271) 4893 described earlier [ 19]. Fraction nRNE-d and rRNE-d were separated on 12% SDS/PAGE and electrophoretically transferred o nto a nitrocellulose membrane. The membrane was t hen incubated i n denaturing solution (6 M guanidine/ HCl i n 1 · binding buffer) for 10 m in. T o this, an equal volume of 1· binding buffer was sequentially added to dilute the guanidine/HCl in the denaturing buffer to 3 M , 1.5 M ,0.75 M ,0.38 M and 0.185 M with 5 min incubation after each addition. The m embrane w as then incubat ed in blocking buffer [ 5% (w/v) BSA in 1 · binding buffer] for 1 h followed by four washes (10 min each) with 1· binding buffer. Finally, 1· binding buffer containing labelled tetramer of Jun-25 (16650 Bq m L )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. In vitro DNase I footprinting analysis DNase I f ootprinting analysis to identify protected region s was p erformed as described [36]. The binding reaction was carried out as described in EMS A with a 5¢ end-labelled 266 bp fragment of c-jun ()284 t o )18;  333 Bq) and increasing concentration of nuclear proteins in a final reaction volume of 50 lL containing 10 m M Tris/HCl, pH 7.5, 50 m M NaCl, 2.5 m M MgCl 2 ,1m M dithiothreitol, 1m M EDTA, 0.1% (v/v) Triton X-100 and 5% (v/v) glycerol. The reaction mix w as incubated at 3 0 °Cfor 20 min. The reaction mix was then supplemented w ith 1m M CaCl 2 ,5m M MgCl 2 and 50 lgoffragmentedcalf thymus DNA followed by digestion with 0.20 UÆmL )1 of DNase I (Promega, Madison, WI, USA) for 90 s at 37 °C. The reaction was terminated by addition of EDTA (30 m M ) and SDS (1%). The products were purified by phenol/ chloroform extraction and ethanol precipitation. The products were dissolved i n f ormamide dye, denatur ed a t 100 °C for 2 min and separated on a pre-electrophoresed 6% urea/acrylamide sequencing gel. The gel was dried and autoradiographed at )70 °C. A s tandard M13mp18 sequencing r eaction with a n  40mer universa l primer w as used as a reference. Recognition sequence DNA-affinity chromatography Affinity purification was performed as described e arlier [19]. Radiolabelled Jun-25 concatamers were covalently bound to CNBr-activated sepharose CL-4B. Nuclear proteins (nRNE-d and rRNE-d) were i ncubated with the affinity matrix (pre-equilibrated w ith 1· binding buffer excluding Triton X-100) in the presence o f nonspecific DNA in 1 · binding buffer excluding Triton X-100. The proteins bound specifically to Jun-25 were eluted with binding buffer containing increasing concentrations of NaCl. Aliquots from different fractions were analysed by EMSA. The fractions showing the complex formation were analysed by SDS/PAGE and s ilver staining [37]. Isoelectric focusing and second dimension SDS/PAGE Affinity purified nuclear proteins were subjected to 2D-PAGE as described by Pollard [38] using a Mini- Protean II 2-D gel apparatus (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. The isoelectric focussing (IEF) gel c omposition was similar to that described by O’Farrell [39]. The concentrations of the a mpholytes used to establish the pH gradient were 1.6% (w/v) Bio-Lyte (pH 5–7) and 0.4% (w/v) Bio-Lyte (pH 3–10). IEF gels were allowed to polymerize for 1 h and prefocussed at 200 V for 10 min, 300 V and 400 V for 15 min each. Protein samples (50–100 lg) were loaded onto the IEF gel, o verlaid w ith 20–40 lL of sample overlay buffer [9 M urea, 1% (v/v) ampholyte (0.8%, pH range 5–7; 0 .2%, pH range 3 –10) and 0.0025% (w/v) b romophenol blue] a nd electrofocussed a t 500 V for 1 0 min followed b y 750 V for 3.5 h . After electrofocussing, the IEF gels were extruded from the capillary with a gel ejector and a llowed t o e quilibrate in the SDS e quilibration buffer [0.0625 M Tris/HCl, p H 6 .8, 2.3% SDS, 5% 2-mercaptoethanol (v/v), 10% glycerol ( v/v) and 0.0025% bromophenol blue] for 10 min prior to second dimension electrophoresis. The IEF gels were then placed on a 5% stacking gel and o verlayered with a tracking dy e. Electrophoresis was achieved at 1 00 V through t he stacking gel a nd at 200 V t hrough the 12% separating g el. Proteins were visualized by silver staining [37]. Results Establishment of differential complex formation between factor(s) present in normal and regenerating rat liver with the )148 to )124 region of c-jun In order to establish w hether the factors p resent in rRNE-d bind to the )148 to )124 region of c-jun and form a complex different than that observed w ith normal e xtract (nRNE-d), EMSA was carried out using labelled Jun-25 and different concentrations of the e xtracts prepared from normal a nd partially hepatectomized rat livers excised at 8 and 24 h after surgery (Fig. 1A). These t ime points were chosen based o n our earlier studies, which showed that the c-jun mRNA level in partially hepatectomized rat liver increased i mmediately after p artial hepatectomy a nd attained its maximum level a t 8h.At24h,c-jun mRNA levels declined a little but still remain significantly higher than that observed i n c ontrol liver [33]. T he appearance of a prominent slow-migrating complex C 2 with r RNE-d (lanes 3 and 4) can be d istinctly seen when compared to the complex C1 formed with nRNE-d (lanes1 and 2). A similar pattern was observed in nuclear ext ract p repared at 24 h after surgery (lanes 5 and 6). An a lmost complete d isappearance of complex C 1 (lanes 3 and 4) suggests that an additional factor, designated as regenerating rat liver Jun regulatory protein (rRLjunRP) induced by partial hepatectomy may interact with RLjunRP dimer, involved in complex C1 formation. Specificity of the complex formation was established (Fig. 1B,C) by incubating the rRNE-d (100 lg) with different amounts of unlabelled Jun-25 (lanes 3–6), 100- fold excess of n onspecific DNA, fragmented c alf thymus DNA (lane 7) a nd pBR322 (lane 8) prior to the a ddition of labelled oligonucleotide Jun-25. The complex formation was found to be highly specific as established b y the complete disappearance of the complex, when the extract was preincubated w ith unlabelled Jun-25, w hereas, no e ffect 4894 S. Ohri et al.(Eur. J. Biochem. 271) Ó FEBS 2004 was observed when a 100- to 200-fold excess of nonspecific DNA was used for c ompetition. Li ke the other re gulatory proteins that bind to their specific recognition site with higher affi nity [40], these factors c ould form a complex even in the presence of several thousan d-fold excess o f nonspe- cific DNA (Fig. 1C). The optimum concentration o f m onovalent cations was determined by carrying out EMSA in the presence of different concentrations of NaCl. Complex formation was observed over a range of NaCl c oncentrations, i.e. 25–100 m M (Fig. 1D, lanes 1–4), with maximal formation at 5 0 m M NaCl(lane2).Therewasadecreaseinthe complex formation from 100 m M onwards. At 250 and 500 m M NaCl, very little complex could b e seen (lanes 5 and 6, respectively). The optimum concentration of MgCl 2 required for the complex formation was titrated by c arrying out EMSA in the presence of different concentration s of MgCl 2 (Fig. 1 E). Complex formation could be seen in the presence o f 1 m M MgCl 2 (lane 1). Binding was found to be maximal in the presence of 2.5 m M MgCl 2 (lane 2). 1 C2 C1 + + + 23 4 56 C2 C1 C2 C1 C2 C1 MgCl 2 (mM) NaCl (mM) C2 C1 frag. CT DNA (µg) frag. CT DNA (100ng) Jun-25 (ng) 5101520 7.5% formamide pBR 322 (200ng) 1 110 25 50 75 100 250 500 1 2.5 5 10 15 20 20 40 234 1234 56 123456 123456 78 A B C D E Fig. 1. Spe cificity of complex formation between )148 to )124 region of c-jun (Jun-25) and fac tors present in rRNE-d, and determination of optimum c oncentrations of monovalent a nd divalent cations. (A) Differ- ential complex formation of nRNE -d and rRNE-d with Jun-25. EMSA reactions w ere c arried outin the presence of 1 ng of rad iolab elled Jun-25 and 100 lg(lanes1,3and5)and150lg (lanes 2, 4 and 6) o f nuclear extracts. Lanes 1 and 2 r e present EM SA performed with nRNE-d, lanes 3 and 4 represen t EMSA performed with nuclear extracts prepared 8 h after partial he patectomy an d lan es 5 and 6 represent EMSA performed with nuclear extracts prepared at 24 h after surgery. ( B) Factors in regenerating rat liver form sp ecific complex with the )148 to )124 re- gion of c-jun. L ane 1 represents the i nteraction o f factor(s) present in fraction rRNE-d with 1 n g o f rad iolabelled J un-25. E MSA r eactions were carried out using 100 lgofrRNE-dpreincubatedwith100-fold excess of unlabelled nonspecific DNA, namely , fragmented calf thymus (CT) DNA (lane 7), p BR322 (lane 8 ), and i n the pr esence of various concentrations of unlabelled Jun-25 (lanes 3–6) prior to t he addition of labelled Jun-25. L ane 2 d e picts the b in ding reaction carried out i n 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 o ut with 1 ng of labelled J un-25 and 100 lg of fractionated nuclear extracts, in the p resen ce of 1 lg(lane1), 10 lg(lane2),20lg(lane3)and40lg (lane 4) of fragmented calf thymus DNA. (D) Titration of optimum monovalent cation concen- trations. Binding reactions were carried out in the presence of 25, 50, 75, 100, 250 and 500 m M NaCl (lanes 1– 6, respectively) using 100 lgof rRNE-d and 1 ng of labelled Jun-25. (E ) D etermination of optimum divalent cation concentration for complex formation. EMSA were carried ou t using 1 ng l abelled Jun-25 a nd 100 lgofrRNE-dinthe 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 . C1 and C2 indicate the two DNA–protein complexes. Time (h) C2 C1 C2 C1 1 0 0.25 0.5 1 2 2.5 3 4 6 83.5 23 123 4 5 6 7 8 9 10 11 Fig. 2. Appe arance of additional factor i nteracting with )148 to )124 region of c-jun after partial hepatectomy. (A)Timeofappearanceofcomplex C2 after p artial hepatectomy. EMSA were carried out using 1 ng of labelled Jun-25 and nuclear extracts prepared from partially hepatectomized rat livers har vested at d ifferent time intervals (ind icated on top) after surge ry. The appearance of n ew complex C2 in lanes 5–11 can b e observed. (B) Complex f ormation in nuclear e xtract p r epared from partially hepatectomize d rat livers treated w ith CHX. EMSA was perform ed with 1 ng of labelled J un-25 and 10 lgeachofnRNE-d,rRNE-dandCHX-rRNE-d(lanes1–3,respectively). C1 and C2 indicate the t wo complexes. Ó FEBS 2004 Expression of c-jun in regenerating rat liver (Eur. J. Biochem. 271) 4895 Complex C2 appears as early as 2 h after partial hepatectomy It has been reported p reviously that c-jun m RNA levels in partial hepatectomized rat liver start to r ise a pproximately 2 h after surgery, attaining its maximum level at 8 h post partial hepatectomy [33]. Therefore, it was important to establish whether the increase in c-jun mRNA levels could b e correlated w ith the appearance of the factor, rRLjunRP, involved in the f ormation of complex C2, observed with rRNE-d, and if the time of appearance of this factor coincides with the increase in c-jun expression induced by partial hepatectomy i n a time dependent manner. For this purpose, EMSA was performed with nuclear extracts prepared from liver excised at various time intervals after surgery. As sh own in F ig. 2 A, only c omple x C1 is seen until 1 h after partial hepatectomy (lanes 1–4). Complex C2 could only be observed in nuclear extracts prepar ed at and after 2 h of s urgery (lanes 5–11). It h as already b een established that RLjunRP, involved in complex C1 formation, is a positive regulator of c-jun transcription. An increase in complex C1 can also be observed after 2 h of surgery, correlating with the enhanced c-jun mRNA levels reported earlier. However, when c-jun mRNA levels peak at 8 h , t he intensity of complex C2 remains higher than that of C1. Factor(s) involved in the C2 complex formation pre-exist in the cytosol In order t o establish w hether the synthesis of the rRLjunRP in rRNE-d, responsible for complex C2 for mation (conver- sion of C1 observed with nRNE-d to c omplex C2 observed with rRNE-d) is induced by partial h epatectomy or if it pre- exists in the cytosol, EMSA was performed with CHX- rRNE-d and labelled Jun-25 (Fig. 2B). Cycloheximide (CHX) i s a known inhibitor of protein synthesis. Therefore, if this factor is newly synthesized in response to a growth stimulus, no complex at the C2 position is expected to be present in EMSA carried out with CHX-rRNE-d. However, no difference in the pattern of complex formation was observed between rRNE-d (lane 2) and CHX-rRNE-d (lane 3). Interaction o f rRLjunRP a t the 3¢ end o f the )148 t o )124 region of c-jun is not absolutely essential for its interaction with R LjunRP complexed with the )148 to )124 region of c- jun. EMSA data (Fig. 1) u sing nuclear extracts from normal and regenerating liver indicated that the factor rRLjunRP induced by partial hepatectomy interacts with complex C1, resulting in t he formation of c omplex C2. I f rRLjunRP i s interacting with RLjunRP complexed with DNA, no significant difference in the footprinting pattern s hould be observed with nRNE-d a nd rRNE-d. DNase I foo tprinting analysis using t he 5 ¢ end-labelled fragment ( )284 to )18) of c-jun and nuclear extracts prepared from normal and regenerating liver, w as carried out to study whether a ny difference in the protection pattern exists betwee n t he two extracts. Figure 3 shows that while only the central portion of the )148 to )124 region i s protected with nRNE-d (protected region: )140 to )131) (lanes 3–5), the protection extends more towards the 3¢ end of this region (protected region: )140 to )125) with rRNE-d ( lanes 6–8). This indicates that w hile rRL junRP interacts with RLjunRP, it must also be interacting with the 3¢ region of the )148 to )124 region of c- jun. C2 C1 Jun-25 12 34 5 6 Jun-25A Jun-25B -64 -71 -87 -92 -107 -119 -124 -148 -183 -190 0 A B 1 GAC Protein (µg) T 23 456 78 01020 nRNE-d rRNE-d 20 10 20 20 Fig. 3. DNase I p rotection pattern of )284 to )18 fragment of c-jun by factors present in nRNE-d and nRNE-d, and EMSA with Jun-25 dele- tions. (A) DNase I footprinting analysis. The 5¢ end-labelled Cfr 91- AvaI f ragment of c-jun (encompassing )284 to )18 of c-jun)was incubated with the indicated amounts of nRNE-d (lanes 3–5) and rRNE-d (lanes 6– 8) foll owed by DNase I digestion. Lanes 1 and 2 represent DNase I-digested 5¢ end-labelled Cfr91-AvaI fragment of c-jun in the absence of n uclear e xtract. GACT are the seque ncing lanes (M13mp18 as template and  40 universal primer) electrophoresed simultaneously as a reference. T he marked regions represent protein binding sites identified earlier: AP-1 ()64 to )71 a nd )183 to )190), CTF ( )87 to )92), SP-1 ()107 to )119). The )148 to )124 r eg ion of c-jun is also shown. (B) E M SA with 5¢ an d 3¢ deletion mutants of Jun- 25. S tan dard binding reactions we re performed usin g 10 lgeachof nRNE-d (lanes 1, 3 and 5) and rRNE-d (lanes 2, 4 and 6) and 1 ng each of labelled Jun-25 (encompassing the )148 to )124 region of c-jun, lanes 1 and 2), J un-25A (encompassing the )139 to )124 region of c-jun; l an es 3 an d 4) and Jun-25B (encompassing the )148 to )134 region of c-jun). C1 and C2 indicate t he two complexes. 4896 S. Ohri et al.(Eur. J. Biochem. 271) Ó FEBS 2004 The 5¢ and 3¢ deletion mutants of Jun-25 (Jun-25A a nd Jun-25B, respectively) were used i n EMSA to establish whether the interaction of rRLjunRP at the 3¢ end of the )148 to )124 region of c-jun is necessary for its interaction with the RLjunRP dimer (Fig. 3B). It i s evident from Fig. 3 that w hen EMSA was p erformed w ith Jun-25A (encom- passing the )139 to )124 region of c-jun), a significant reduction in the complex formation w as observed ( lanes 3 and 4). H owever, only a slight decrease was observed o n the complex formation in EMSA reactions carried out with Jun-25B ( spanning the )148 to )134 reg ion of c-jun;lanes5 and 6 ), when compared to the complex formation observed in EMSA reactions performed with control J un-25. Complexes C1 and C2 are formed by the factor(s) binding on the minor groove of the )148 to )124 region of c-jun To establish the nature of interaction b etween trans-acting factor(s) and t he )148 to )124 r egion of c-jun,drugsthat specifically interact with the major or minor groove of DNA were evaluated for their ability to c ompete with trans-acting factor(s) in EMSA. EMSA were performed with both nRNE-d and r RNE-d i n t he presence of increasing concen- trations of methyl green, a majo r groove b inding drug [41] and distamycin A, a minor groove binding drug [42]. Increasing concentrations of distamycin A resulted in a decrease in the c omplex formation both w ith nRNE-d and rRNE-d (Fig. 4A,B, respectively) and virtually no complex formation was seen at a c oncentration of 1 0 l M . Another minor groove binding drug, actinomycin D also inhibited complex formation both in normal and regenerating liver extracts (Fig. 4 C,D, lanes 2 and 3 in both panels). On the other hand, me thyl green did not affect the f ormation of the complexes (lanes 4 and 5 ). The insensitivity of nRLjunRP and rRLjunRP to the major groove binding drug, m ethyl green, coupled with its sensitivity to minor groove binding drugs, actinomycin D and distamycin A, confirms that these are m inor gr oove binding proteins. rRLjunRP is an  40 kDa protein that interacts with the RLjunRP–Jun-25 adduct of  80 kDa To assess the approximate molecular mass o f the DNA– protein a dduct f ormed between factors present in rRNE-d and Jun-25 and to see if there exists any difference in the DNA–protein adduct formed between factors present in nRNE-d and Jun-25, UV crosslinked EMSA products were analysed on SDS/PAGE (Fig. 5A). The molecular mass of the crosslinked complex of nRNE-d was  80 kDa corresponding to complex C1 (lane 1). UV crosslinking of complexes formed between rRNE-d and Jun-25 displayed three addu cts of  40,  80, and  120 kDa (lane 2). The  80 and  120 k Da DNA– protein adducts correspond to the complexes C1 and C2, respectively. A diffused c omplex at  40 kDa (lane 2) seems to be formed between rRLjunRP and Jun-25. This is in line with our footprinting data where an a dditional protection at the 3¢ end of the )148 to )124 region of c-jun was observed (Fig. 3, lanes 6–8). These data suggest that rRLjunRP is also able to interact with Jun-25 independently. However, this interaction seems to be very weak as a smaller c omplex was not observed in E MSA. Thus, partial hepatectomy results in the a ppearance of an additional factor of  40 kDa that complexes with RLjunRP dimer bound to Jun-25. This was further confirmed by South-Western blot analysis of nRNE-d and rRNE-d using radiolabelled Jun-25 (Fig. 5 B). A single hybridized band of  40 kDa was observed w ith both nRNE-d (lane 1) and rRNE-d (lane 2). We h ave reported p reviously that the trans-acting f actor RLjunRP, present in nRNE-d, is a p rotein of  40 kDa [19] that binds to its recognition sequence as a dimer. UV crosslinking and South-Western blot analysis using rRNE-d and Jun-25 collectively suggest that an additional factor of  40 kDa is present only in rRNE-d, and binds to the  80 kDa DNA–protein adduct corresponding to complex C1 to give rise to the DNA–protein adduct of  120 kDa corresponding to the slow-migrating complex C2. Affinity purification of trans -acting factor(s) from rRNE-d The trans-acting factors present in r RNE-d interacting with Jun-25 were purified by recognition s equence affinity chromatography for further characterization. Major p e ak fractions eluted between 0.1 M and 0.5 M NaCl (Fig. 6A) did not show any complex formation with Jun-25 in EMSA. T he factors interacting with the )148 to )124 region of c-jun eluted in 1 M NaCl as evidenced from the Dist. A (mM) - 0.1 1.0 2.5 - - 0.5 1.5 1.50.5 123 45 1 2 345 - - 0.5 1.5 1.50.5 - 0.1 12341234567 1.0 2.510 25 50 Act. D (mM) Methyl Green (mM) A C B D Fig. 4. Determination o f the bind ing site of of factors int eracting with the )148 to )124 region of c-jun. (A,B) Effect of distamycin A (Dist. A) on complex f ormation. Standard EMSA reactions were carried o ut us ing 1 ng of labelled Jun-25 and 10 lg of nRNE-d (A) and rRNE-d ( B) in the presence of varying con- centrations of dist amyc in A (indicated on top). (C,D) E ffect of actinomycin D (Act. D) and methyl g reen on c omplex formation. EMSA we re p erformed using 1 ng of labelled Jun-25 an d of 10 lgrRNE-d(C)andrRNE-d (D) in t he pr esence of 0.5 and 1.5 m M of actinomycin D (lanes 2 and 3) and methyl green (lanes 4 and 5). Ó FEBS 2004 Expression of c-jun in regenerating rat liver (Eur. J. Biochem. 271) 4897 formation of complexes with Jun-25 (Fig. 6B). Residual complex formation could also be s een in t he fraction eluted with 2 M NaCl. A nalysis o f f ractions eluting in 1 M NaCl (Fraction s 42 and 45, lanes 1 and 2, r espectively) on SDS/PAGE revealed a prominent band of  40 kDa (Fig. 6C). These data further c onfirm that the additional factor rRLjunRP, p resent in rRNE-d, is o f  40 kDa. The interaction between rRLjunRP and RLjunRP appears to be very strong as both C1 and C2 complexes are observed in the s ame fraction. If rRLjunRP was weakly bound to RLjunRP, it would have dislodged at lower concentra- tions and only complex C1 would be observed in these fractions. When RLjunRP was purified from nRNE-d, it eluted at 2 M NaCl [19] whereas the factors from rRNE-d e luted a t slightly lower concentration of NaCl, i.e. 1 M (Fig.6A).This indicates that RLjunRP alone has higher affinity to the recognition c omplex than when it is complexed with t he additional factor in duced by partial hepatectomy. This is also su pported b y t he effect of NaCl on complex formation with nRNE-d and rRNE-d. Although a decrease in the complex formation with increasing salt concentration was seen with both t he extracts, nRNE-d retained the complex formationevenat250m M NaCl [19] whereas very little complex c ould be s een between factors present in rRNE-d and Jun-25 at this concentration ( Fig. 1D) . kDa 97 66 55 42 40 31 14 21 31 40 42 55 66 97 kDa 12 12 F A B Fig. 5. UV c rosslinking and S outh-Western blot analysis. (A) Deter- mination of th e mole cular mass of the complex between rRLjunRP– RLjunRP and the )148 to )124 region of c-jun by UV crosslinking. Complex between RLjunRP (lane 1) wit h it s c ognate sequence was formed un der standard cond itio ns using 100 lgofrRNE-dand5ng of labelled Jun-25 f ollowed by U V irradiation (2 · 60 mJ) in a UV crosslinker. D NA–protein complex was separated from free DNA by electrophoresis o n S DS /PAGE. Autoradiography revealed the p res- ence of complex (shown by arrows). Numbers represent protein molecular mass markers. F indicates free labelled Jun-25. (B) South- Western b lot analysis o f fraction r RNE-d with J un- 25. Fifty mic ro- grams of n RNE-d and rRNE-d were fractionated on SDS/PAGE (lanes 1 and 2), transferred onto a nitrocellulose sheet and probed with radiolabelled tetramer of Jun-25 oligonucleotide. The m olecular ma ss (kDa)ofthemarkersisshownontheleftside. FT 2 5 5 0.05 0.05 0.1 0.2 0.25 0.3 0.5 1.0 2.0 8 18 10 15 20 20 25 25 Fraction 30 30 38 42 52 35 40 45 50 1.5 0.5 1 1 C2 C1 kDa 210 134 82 40 32 ML12 L Absorbance 280 nm 0 P 1 P 2 (0.5M) (1.0M) A B C Fig. 6. Affin ity purification of factors interacting with the )148 to )124 region of c-jun from regenerating ra t liver. (A) Spectrophotometric elution p rofile: rR NE-d was subjected to sequence-specific affi nity column chroma tography and a ll the f ractions obtained were analysed spectrophotometrically. Absorbance a t 280 nm was measured a nd plotted. (B) Assessment of c omplex formation ability of eluted f rac- tions from DNA affinity column. P resence o f factors in different fractions ob tained b y a ffin ity ch romatography w as checked using EMSA with labelled )148 to )124 oligonucleotide fragment of c-jun.L represents EM SA re action with the lo aded f raction and the num bers on top represent the fractio n numbers. The nu mbers at the bottom represent the salt co ncentration in t he re spective fraction. ( C) S DS/ PAGE analysis of fractions positive for the complex formation with Jun-25. The fractionated nuclear extract, rRNE-d fraction (L) and the peak fractions number 42 (showing D NA binding ability in EMSA) and 45 eluting in 1 M NaCl were subjected to S DS/PAGE a nd silver stained (la nes 1 and 2, respectively ). M represents the m id-ran ge molecular m ass m arkers. 4898 S. Ohri et al.(Eur. J. Biochem. 271) Ó FEBS 2004 2D gel electrophoresis confirms the presence of rRLjunRP in addition to RLjunRP in regenerating rat liver EMSA and U V crosslinking data have indicated the presence of an additional factor-rRLjunRP in rRNE-d in addition to RLjunRP. Because only a single band of  40 kDa could be seen in South-Western b lot analysis, affinity purified factors f rom nRNE-d a nd rRNE-d were analyzed by 2D gel electrophoresis to verify the presence of rRLjunRP. As evident from Fig. 7A, a single spot at  40 kDa positio n was observed w ith RLjunRP s uggesting that it binds to Jun-25 as a homodimer. In affinity purified rRLjunRP, two s pots t ailing e ach o ther were visualized at the affinity purified RLjunRP position (indicated by an arrowhead). In addition, two s pots close to each other a t a slightly lower position than t hat of R LjunRP and to wards acidic pI (indicated by an arrow, Fig. 7B) were also observed. Thus, both RLjunRP a nd rRLjunRP in regen- erating liver appear to have two isoforms. These isoforms could a rise due to differential phosphorylation or any other post-translational modification of t he factors. Thus, the 2D- PAGE data clearly d emonstrate t hat RLjunRP b inds to Jun-25 as a homodimer (complex C 1) and an additional protein having app roximately t he same molecular mass but different pI value complexes with C 1 to give a heavier complex ( C2) i n the case of affi nity pur ified factor(s) from regenerating rat liver. Discussion In eukaryotes, expression of g enes is differentially regulated in response to a complex set of environmental and developmental cues. The stable association of multiple transcription factors with eukaryotic genes has been described in vitro and in vivo. The significance of such stable interactions is that, i n many cells, a stable pattern of gene activity is maintained for long periods of time and, in the case of t ermin ally differentiated cells, until cell death. However, for genes like c-jun, that require their transcrip- tional activity t o b e m odulated, the transient association and dissociation of transcription factor s is a dvantageous. c-jun belongs to a class of cellular g enes, t ermed e arly response or immediate early response genes, which are characterized by a rapid and transient activation of transcription in response to growth stimulus. Expression of c-jun is positively autoregulated by AP-1 [3,43]. However, sites further upstream o f the AP-1 site may play an important role in transcriptional regulation of c-jun [15,44]. A positive r egulatory trans-acting factor, RLjunRP, in rat liver has b een identified that interacts with the )148 to )124 region of c-jun [19]. The present investigation led to the identification of yet another factor, rRLjunRP in rat liver induced in response to p artial hepatectomy t hat interacts with RLjunRP complexed with the above region, indicating the key role this element m ay play in differential regulation of c-jun tran scription at different stages of h epatic regener- ation. Further, it is noted that although c-jun expression is maximum at 8 h after surgery, the new factor rRLjunRP appeared as early a s 2 h post surgery and i ts appearance coincided w ith the reported increase in c-jun mRNA levels in rat liver [33]. Thus, the interaction of this f actor involved in C2 formation may, i n p art, be attributed to the increased c-jun mRNA levels after p artial hepatectomy. The p resence of a diffused  40 kDa protein–DNA adduct i n UV crosslinking studies and e xtended protection i n DNase I footprinting analysis using regenerating liver nuclear extract indicate that rRLjunRP can weakly interact with the 3¢ of the )148 to )124 region of c-jun. However, this interaction was not found to be absolutely essential f or its interaction with RLjunRP c omplexed with Jun-25. The interaction of rRLjunRP with t he 3 ¢ end of Jun-25 might be stabilizing i ts interaction with RLjunRP bound to Jun-25. Extended protection could also be due to the larger protein complex in regenerating liver bo und to the t arget site. An increase in the C 1 complex formation can also be seen 2 h after partial hepatectomy, indicating in creased RLjunRP concentrations. RLjunRP that binds to the )148 and )124 region even in resting liver appears to b e involved in controlling both b asal and inducible transcrip- tion of c-jun. rRLjunRP that appears in response to partial hepatectomy is likely to play the key regulatory role in vivo in modulating c-jun expression in response to partial kDa IEF IEF SDS SDS kDa 134 82 40 32 18 7 210 134 82 40 32 18 7 BA 210 Fig. 7. 2D electrophoresis of affinity purified factors from nRNE-d and rRNE-d. Affinity purified proteins interacting with the )148 to )124 region of c-jun from nRNE-d (A) a nd rRNE-d (B) were s eparated in the first dimension by IEF using ampholytes pH 3–10 (from le ft to right). The second dimension was SD S(12%)/ PAGE followed by detection by silve r staining. Arrowhead points to the single spot of RLjunRP in (A). A rrowhead and arrow in (B) indicate the two spots c orresponding to R LjunRp a n d r RLjunRP, respectiv ely. The insets show 1.5· magnification of the spots. Ó FEBS 2004 Expression of c-jun in regenerating rat liver (Eur. J. Biochem. 271) 4899 hepatectomy. RLjunRP concentrations appear to be important for d ifferential c-jun expression. The activation of c-jun expression by binding of rRLjunRP to RLjunRP complexed to the target site is transient. Availability of abundant RLjunRP, involved in the formation of complex C1, a llows r RLjunRP to b ring about maximal activation of c-jun transcription. A similar relationship in t he concentra- tions of factors 5S RNA gene-specific transcription factor IIIA (TFIIIA) and the 5S RNA gene-specific transcription factor II IC (TFIIIC), involved in the regulation of 5S RNA in developing Xenopus oocytes has been reported ( [45], reviewed in [46]). The binding of TFIIIC and activation of 5S RNA is facilitated by an increase in TFIIIA concentra- tion. Without TFIIIA being bound, TFIIIC cannot recog- nize a 5S RNA gene specifically [47]. Like TFIIIC, rRLjunRP cannot b ind t o t he cis-acting e lement present within the )148 to )124 region a nd only e levates transcrip- tion from t he c-jun promoter by its interaction with RLjunRP occupying the element present within the )148 to )124 region of c-jun. T he fact that the rRLjunRP– RLjunRP–DNA complex has lower affinity than RLjunRP–DNA complex, suggests the transient role that rRLjunRP must play in the activation of c-jun transcription. Thus, t hese factors i nteracting with the )148 to )124 region of c-jun are involved i n differential expression o f c-jun in liver cells that are induced to proliferate. Persistence of complex C2 even with the extracts prepared from animals t reated with CHX s uggests that the factor rRLjunRp pre-exists in the cytosol. Upon growth stimulus by partial hepatectomy, it possibly undergoes s ome modification(s) resulting in its transloca- tion from the cytosol to the nucleus. However, the appearance of complex C2 only at 2 h post surgery indeed indicates that a cascade of s ignalling events m ight be occurring, preceding to the translocation o f rRLjunRP from cytosol t o nucleus, a nd converting its i nactive form to an active one. The activation of rRLjunRP is independent of protein synthesis, which suggests that modification of a pre-existing molecule is sufficient for its activation as has been reported for nuclear factor-jB (NF-jB), interleukin-6 dependent DNA b inding protein (IL-6 D BP), AP-1 and NF-jun [ 20,48,49]. Large scale purification of rRLjunRP for further characterization and cDNA cloning of the gene encoding the same are under p rogress t o help in understanding its structural and functional aspects. Presence of a nuclear factor NF-jun, recognizing the )139 to )129 region o f c-jun only in rapidly pro liferating cells is reported by B rach et al. [20]. Its p resence was not detectable in nonproliferating diploid lung fibroblasts, b lood mono- cytes, granulocytes or resting T-cells. Our studies with rat liver indicate that although, like NF-jun, pre-existing rRLjunRP is t ranslocated i n response t o s ignals transduced after p artial hepatectomy, it binds t o RLjunRP, a factor also present in normal liver, precomplexed with this element, and facilitates c-jun transcription. rRLjunRP is different from N F-jun; this is evident from t he fact that it is an  40 k Da protein that binds to the RLjunRP homodimer of  80 kDa giving r ise to a n  120 kDa DNA–protein adduct, whereas NF-jun is reported to f orm DNA–protein adducts of 55 and 125 kDa. This study thus provides an insight into one of the many molecular m echanisms that c ould b e involved i n differential gene regulation of c-jun expression in quiescent and proliferating r at liver. The role of the )148 t o )124 r egion of c-jun in transcriptional regulation of c-jun in rat liver is established and two f actors, RLjunRP and rRLjunRP present in normal [19] and proliferating liver, which recognize this element have been identified. The f actors binding to this region are in addition to the already known regulatory factors that mediate induction response to growth stimu lus. B ased on these results, a hypothetical model for regulation of c-jun expression mediated by the )148 to )124 region in normal and regenerating liver by these factors is proposed (Fig. 8). According to this, RLjunRP is involved in controlling both basal and inducible transcription of c-jun. I nduction of rRLjunRP upon partial hepatectomy apparently medi ates the interaction between RLjunRP and the factors of the i nitiation m achinery to form more actively transcribing initiation complexes. Signal transduction leading to differential phosphorylation o f factors a fter partial hepatectomy could i n p art m odulate the activity o f these factors. Differential recognition o f this region by these factors indicates the role this element may play in regulating c-jun expression. Unlike other response elements, namely those t hat m ediate induction by gluco- corticoid hormone [50,51], the cis-acting element present within the )14 8 to )124 region of c-jun has c onsiderable basal activity, by virtue of being bound by the positive regulator RLjunRP in resting liver prior t o s timulation by partial hepatectomy. Recognition of the same region by NF-jun, which is present only in p roliferating c ells, does indeed indicate that the transcriptional r egulation of c-jun is very complex, differing from one cell type to another and involves different cell and tissue specific factor(s) that b ind to their cognate recognition sequences bringing about modulated c-jun expression. -124 -148 -148 -124 2xnRLjunRP rRLjunRP P? + 1 + 1 Initiation Complex A B Fig. 8. Sch ematic model for the ac tivation of transcription o f c-jun in normal and regenerating rat liver b y factors interacting with the )148 to )124regionofc-ju n. (A) RLjunRP dimer is prebound to the )148 to )12 4 region of c-jun in normal liver. ( B) Partial hepatecomy r esults in the t ranslocation of rRLjunRP to the nucleus which then facilitates the interaction of transactivating domains with the facto rs of the initiation complex that re sults in more prod uctive initiation complexes. 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Interaction of an  40 kDa protein from regenerating rat liver with the )148 to )124 region of c-jun complexed with RLjunRP coincides with enhanced c-jun expression. regenerating rat liver with the )148 to )124 region of c-jun In order to establish w hether the factors p resent in rRNE-d bind to the )148 to )124 region of