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

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: or
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,

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

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

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

)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-

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

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

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

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

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

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

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)

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

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

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)

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

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

(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

-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. Differ-
ential phosphorylation ( P?) may p lay a key ro le in m odulating th e
activities of these factors. The transcription start site is denoted by +1.
4900 S. Ohri et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Acknowledgements
Council of Scientific and Industrial Research (CSIR), India is duly
acknowledged for t he Senior Research Fellowships to S .O. and D.S.
The animal work included in this paper had the approval of
Institutional Animal Ethic s Committee, J. N. U. (IAEC- JNU Proje ct
Code no. 27/1999).
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