The 148 to 124 region of c- jun interacts with a positiveregulatory factor in rat liver and enhances transcription Dipali Sharma*, Sujata Ohri and Aparna Dixit Gene Regulation Laboratory
Trang 1The )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 be40 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 complexprocesses 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] Inhibitransactiva-tion 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)
Trang 2Regulation 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)1nuclear
protein (RNE-d) at 30C for 30 min Transcripts extracted
with phenol/chloroform/isoamylalcohol (25 : 24 : 1) were
precipitated with ethanol and separated on a 6%
acryl-amide, 8Murea 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, AseI and EcoRI restriction sites were included in the forward and reverse primers, respectively PCR amplified fragments digested with AseI and EcoRI 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)1penicillin and
100 lgÆmL)1streptomycin at 37C in a humidified atmos-phere containing 5% CO2
Transfection assay CHO cells were plated at a density of
2· 105 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 lg either 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 37C in a 5% CO2 incubator for an additional 48 h Medium was again removed and the cells were rinsed with NaCl/Pifollowed by an incubation in 500 lL lysis buffer (100 mM Tris/HCl pH 7.4, 0.15M NaCl, 1.5 mM magne-sium acetate, 0.5% NP-40) at 37C 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-32P-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
Trang 3a)] were obtained from Rama Biotechnologies (Hyderabad,
India) Annealed oligonucleotide (Jun)25) was labelled by
end filling using Klenow fragment and [a-32P]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 ( 104c.p.m.), in a reaction mixture containing
1· binding buffer (10 mMTris/HCl pH 7.5, 50 mMNaCl,
2.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 0.1%
Triton-X-100, 5% glycerol) in a final reaction volume
of 40 lL at 30C for 30 min (unless otherwise stated) The
complexwas immediately loaded on a pre-electrophoresed
6% nondenaturing polyacrylamide gel and electrophoresed
in 1· Tris/glycine buffer (0.192M glycine, 25 mM Tris/
HCl pH 8.3) at 11 VÆcm)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 37C [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 4C
The membrane strip containing the sample was cut and
incubated in denaturing solution (6Mguanidine/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 3M, 1.5M, 0.75M, 0.38M
and 0.185Mwith 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
(106c.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-32P]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 mM potassium phosphate pH 8.0 The oligonucleotide-affinity resin thus prepared was collected on a sintered glass funnel, washed with 200 mL H2O and 100 mL 1M ethanolamine/HCl
pH 8.0 The oligonucleotide-affinity resin was finally sus-pended in 14 mL 1M ethanolamine/HCl All procedures were carried out at 4C 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 0.1MNaCl Fraction RNE-d (10 mg) was then incuba-ted for 10 min on ice with fragmenincuba-ted calf thymus DNA at
100 ngÆlg)1protein to block nonspecific binding followed
by incubation with the resin in a 15-mL tube with end-over-end mixing for 30 min at 4C 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
Trang 4titrates 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-p123jun-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 complexformation was observed The factor(s) involved in the complexformation 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 complexformation 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 complexformation was completely abolished when RNE-d was preincubated with unlabelled Jun)25 whereas no effect on the complexformation was observed when a 100- to 200-fold excess of nonspecific DNA was used for competition, indicating the specificity of complex formation The complexformation did not take place in the
Fig 2 Effect of )148 to )124 region on c-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-p123jun-eGFP along with 1 lg of pSV-bgal plasmid Cells transfected with 2.5 lg pEGFP-N1 and 1 lg pSV-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)
effect of the )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
molecular mass markers /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.
Trang 5presence 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
complexwill 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 1, 10 , 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 mM(Fig 4A, lanes 1–5) At
500 mM (lane 6), there was a decrease in the complex formation The fact that RLjunRP retained its binding activity even in the presence of 0.5MNaCl 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 complexformation 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 mM EDTA in the binding reaction resulted in a slight decrease in complexformation (lane 3) and no complexwas observed in the presence of
150 mMEDTA (lane 4) It is likely that in the presence of 50
or 100 mM 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 complexformation When the EDTA concentration is raised to 150 mM(Fig 4B, lane 4), all of these ions are chelated and no complexformation 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 MgCl2required for complex formation was then titrated (Fig 4C) Complexformation could be seen in the presence of 1 mM MgCl2 (lane 1) Binding was found to be maximal in the presence of 2.5 mM
MgCl2(lane 2)
Studies on the effect of temperature (Fig 4D) on complex formation revealed that the factors present in RNE-d formed the complexeven at temperature as low as 0C
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
complexwith 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 lg of
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 (lane 1), 10 lg
(lane 2), 20 lg (lane 3) and 40 lg (lane 4) fragmented calf thymus DNA.
Trang 6(lane 1) No significant change in complexformation was
observed untill 30C (lanes 2–6) However, very little
complexformation occurred when EMSA was carried out
at 45C (lane 7) and no complexwas formed at 55 C
onwards Unlike TATA binding protein that becomes
totally inactivated within 15 min of heat treatment at 47C
[34], junRP retains its DNA-binding activity, although at a relatively low level, even when the binding reaction was carried out at 45C 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 dephosphophospho-rylated 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 complexwas 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 complexshows 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 complexwith 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 0.1Mand 0.2MNaCl (Fig 6A) contained nonspecific DNA binding proteins, as these fractions did not show any complexformation 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 lg fractionated nuclear
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) Complexbetween
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 complexformed between RNE-d treated with heat inactivated
alkaline phosphatase (10 U) and labelled Jun )25.
Trang 7)124 region of c-jun eluted in the 2.0M NaCl fraction
(fractions 38–42) as evident from the formation of retarded
complexwith labelled)148 to )124 region of c-jun All of
the proteins that do not interact with the )148 to )124
region of c-jun, may nonspecifically bind to the affinity
matrixand 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 complexformation) 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-through fraction (F) and peak fraction number 38 (P) showing DNA 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 complexbetween junRP and the
)148 to )124 region of c-jun by UV cross-linking Complexbetween
RLjunRP (lane 1) with its cognate sequence was formed under
standard conditions using 100 lg RNE-d and 1 ng )48 to )124 region
of c-jun followed by UV irradiation (254 nm) for 15 min
DNA–pro-tein complexwas 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.
Trang 8region) 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 complexat 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 The animal work included in this paper had the approval
of Institutional Animal Ethics Committee, JNU (IAEC-JNU Project
Code no 27/1999).
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