From the Departments of Cancer Biology,
** Biochemistry, and
Medicine and the
§ Comprehensive Cancer Center, Wake Forest University School
of Medicine, Winston-Salem, North Carolina 27157 and the
Fels
Institute for Cancer Research and Molecular Biology, Temple University
School of Medicine, Philadelphia, Pennsylvania 19140
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ABSTRACT |
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We previously identified a major enhancer of the
mouse ferritin H gene (FER-1) that is central to repression of the
ferritin H gene by the adenovirus E1A oncogene (Tsuji, Y., Akebi, N.,
Lam, T. K., Nakabeppu, Y., Torti, S. V., and Torti, F. M. (1995) Mol. Cell. Biol. 15, 5152-5164). To dissect the
molecular mechanism of transcriptional regulation of ferritin H, E1A
mutants were tested for their ability to repress FER-1 enhancer
activity using cotransfection with ferritin H-chloramphenicol
acetyltransferase (CAT) reporter constructs. Here we report that
p300/CBP transcriptional adaptor proteins are involved in the
regulation of ferritin H transcription through the FER-1 enhancer
element. Thus, E1A mutants that failed to bind p300/CBP lost the
ability to repress FER-1, whereas mutants of E1A that abrogated its
interaction with Rb, p107, or p130 were fully functional in
transcriptional repression. Transfection with E1A did not affect
endogenous p300/CBP levels, suggesting that repression of FER-1 by E1A
is not due to repression of p300/CBP synthesis, but to E1A and p300/CBP
interaction. In addition, we have demonstrated that transfection of a
p300 expression plasmid significantly activated ferritin H-CAT
containing the FER-1 enhancer, but had a marginal effect on ferritin
H-CAT with FER-1 deleted. Furthermore, both wild-type p300 and a p300
mutant that failed to bind E1A but retained an adaptor function
restored FER-1 enhancer activity repressed by E1A. Sodium butyrate, an inhibitor of histone deacetylase, mimicked p300/CBP function in activation of ferritin H-CAT and elevation of endogenous ferritin H mRNA, suggesting that the histone acetyltransferase activity of
p300/CBP or its associated proteins may contribute to the
activation of ferritin H transcription. Recruitment of these broadly
active transcriptional adaptor proteins for ferritin H synthesis may represent an important mechanism by which changes in iron metabolism are coordinated with other cellular responses mediated by p300/CBP.
Ferritin is the major cellular iron-binding protein in eucaryotic
cells. It is composed of 24 subunits of two types (termed ferritin H
and ferritin L) that are assembled in various ratios in different
tissue and disease states, including inflammation and cancer (reviewed
in Refs. 1 and 2). Iron is a potent regulator of ferritin synthesis. In
the past decade, a number of elegant studies have shown that iron
coordinately regulates both the H and L subunits of ferritin at a
post-transcriptional level through the interaction of an
iron-responsive element with its binding proteins (reviewed in Ref.
3).
We and others have shown that ferritin is also subject to
transcriptional regulation. Transcription of the ferritin H gene is
preferentially modulated by cytokines (4, 5), hormones (6), oncogenes
(7, 8), and some inducers of cell differentiation (9). The preferential
alteration of ferritin H synthesis in response to these stimuli may be
an adaptive cellular response since ferritin H appears to be
responsible for more rapid iron uptake than ferritin L (10) and may
therefore protect cells from oxidative stress mediated by iron. In
fact, it has been reported that oxidative stress stimulates ferritin H
and L synthesis at both transcriptional and post-transcriptional levels
(11). Some studies have demonstrated that ferritin synthesis is
elevated in tumor tissues compared with their normal counterparts
(12-15), and elevation of serum ferritin has also been reported in
various cancer patients (16), although little is known about the
molecular mechanism and biological meaning of the increase in ferritin
associated with malignancy. Recent studies have suggested that ferritin
H may function as an autocrine growth factor for some tumors (17, 18).
An appreciation of mechanisms regulating ferritin synthesis is critical
to understanding these responses.
We have previously reported that the adenovirus E1A oncogene
selectively represses transcription of the ferritin H gene in mouse
NIH3T3 fibroblasts (8). Reduced ferritin transcription in
E1A-expressing cells may contribute to their enhanced sensitivity to
tumor necrosis factor cytotoxicity (19). Repression of ferritin H by
E1A is also consonant with numerous reports showing that E1A can
repress markers of cell differentiation (20).
Extensive studies on the mechanism of action of E1A have revealed that
many of its pleiotropic effects on gene transcription are mediated by
interaction with important and broadly active intracellular regulators.
These include members of the Rb1 tumor suppressor family
(p105Rb, p107, and p130) as well as p300 and CBP
(CREB-binding
protein). p300 and CBP are functionally related
transcriptional coactivators with intrinsic histone acetylase activity
that link key transcription factors (including nuclear hormone
receptors, MyoD, Fos, Jun, CREB, and p53) to the basal transcriptional
machinery (reviewed in Ref. 21). It has been demonstrated that p300
plays a role in cell cycle control, inhibiting exit from the
G0/G1 phases of the cell cycle and stimulating
differentiation (22-25). p300/CBP thus functions as a nodal point in
mediating cellular responses to a broad range of intrinsic and
extrinsic stimuli (reviewed in Ref. 21).
We have identified an E1A-responsive element (termed FER-1) in the
5'-flanking region of the mouse ferritin H gene (26). FER-1 serves as a
basal enhancer of the mouse ferritin H gene. FER-1 comprises two
elements, an AP1-like sequence followed by a dyad symmetry element,
both of which are essential for maximal FER-1 enhancer activity (26).
Very recently, we identified transcription factors that directly bind
to the FER-1 element: JunD, FosB, and ATF1 (bound to the AP1-like
sequences) and Sp1 and Sp3 (bound to the C-rich sequences in the dyad)
(27). Our discovery of the regulation of ferritin H by E1A allowed us
to further dissect and explore the relationship between binding of
these transcription factors and functional modulation of ferritin H
synthesis. To study the possible involvement of E1A-associated proteins
in the regulation of ferritin H transcription via FER-1, we utilized E1A mutant proteins defective in their ability to associate with selected E1A target proteins. Here we demonstrate that 1) the functional region of E1A containing the p300/CBP-binding domain contributes to the repression of endogenous ferritin H mRNA; 2) E1A
point mutants defective in the p300/CBP-binding domain fail to repress
ferritin H transcription; 3) p300 is able to restore ferritin H
enhancer activity repressed by E1A; 4) in the absence of E1A, p300 and
CBP are able to activate FER-1 enhancer activity; and 5) sodium
butyrate, a histone deacetylase inhibitor, is able to mimic the
p300/CBP effect in terms of the activation of endogenous ferritin H
mRNA synthesis as well as ferritin H-CAT. These results suggest
that p300/CBP is an additional component that regulates the
transcription of the mouse ferritin H gene via the FER-1 element.
Cell Culture--
Mouse NIH3T3 fibroblasts were cultured at
37 °C in a humidified 5% CO2 atmosphere in high-glucose
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum (Gemini Bioproducts, Calabasas, CA). Stable NIH3T3
transfectants expressing E1A or E1A mutant proteins (pools of >100
independent clones) were cultured as described previously (19).
Plasmids--
All CAT reporter constructs used in this study
have been described previously (26). Briefly, expression of CAT in
pBluescript KS( DNA Transfection and CAT Assay--
Transient DNA transfection
into NIH3T3 cells was performed by the calcium phosphate precipitation
method as described previously (19) with the following minor
modifications. Cells were plated in duplicate at a density of 3.5 × 105/60-mm dish containing 4 ml of the culture medium.
After incubation for 16-24 h, 400 µl of calcium phosphate/DNA
precipitate solution containing 5 µg of a CAT reporter plasmid plus a
total 5 µg of p12SE1A, the human p300 plasmid, or the mouse CBP
plasmid was added to the culture and incubated at 37 °C in 5%
CO2 for 16-24 h. Cells were then washed twice with
phosphate-buffered saline, followed by further incubation for 16-24 h
in culture medium. CAT assays were carried out as described previously
(8). Sodium butyrate was purchased from Sigma.
Western and Northern Blots--
Expression of endogenous
p300/CBP and various E1A mutants transiently transfected into NIH3T3
cells was estimated by Western blotting as described previously (31)
using the same cell extract as used in measuring CAT activity. An
anti-p300 monoclonal antibody (NM11) that reacts with both p300 and CBP
(32) was used to detect p300/CBP proteins. The anti-E1A antibody M58
was a generous gift from E. Harlow (Harvard Medical School). Ferritin
mRNA was analyzed by Northern blotting as described previously
(8).
Repression of Ferritin H by E1A Requires an Intact p300-binding
Domain--
We previously demonstrated that the E1A oncogene
preferentially represses the H subunit of the ferritin gene in NIH3T3
cells (8). To assess the involvement of functional domains of E1A in
ferritin H repression, we measured the mRNA level of ferritin in
NIH3T3 cells that were stably transfected with intact E1A or E1A
mutants in which the E1A region containing conserved region 1 (
To further investigate the domain of E1A required for transcriptional
repression of the ferritin H gene, a series of E1A mutants defective in
association with either p300 or the Rb family proteins (Rb, p107, or
p130) were cotransfected with p300/CBP Activates Ferritin H Transcription--
p300 was
initially identified as an E1A-associated protein (34, 35). Subsequent
studies have demonstrated that p300 has a structural homology to CBP
(36) as well as a functional similarity to CBP (37, 38) as a
coactivator of transcription factors such as CREB and AP1 (39, 40). We
previously identified an element (termed FER-1) as responsible for
transcriptional repression of the mouse ferritin H gene by E1A (26).
FER-1 is a 37-base pair composite element that binds AP1 and Sp1 family
members and functions as a basal enhancer of the mouse ferritin H gene
through the cooperative action of these two families of transcription factors (27). These results and the data obtained in Fig. 2, taken
together with evidence for the interaction between p300/CBP and some
members of the AP1 family (41, 42), prompted us to test whether or not
p300 and CBP contribute to ferritin H enhancer activity through FER-1.
To test this possibility, we first carried out cotransfection
experiments using a p300 or CBP expression plasmid and p300-dependent Activation of Ferritin H Is Mediated by
the FER-1 Element--
To assess the involvement of the FER-1 element
in ferritin H enhancer activation by p300, we then performed
cotransfection of a p300 expression plasmid with ferritin H-CAT that
lacks FER-1 ( E1A Represses Ferritin H by Targeting p300--
We then asked
whether E1A repressed ferritin H transcription by targeting p300. To
this end, a FER-1-containing ferritin H-CAT plasmid was cotransfected
with an E1A expression plasmid together with either wild-type p300 or a
p300 deletion mutant (p300del30) that is defective for interaction with
E1A, but is active as an adaptor (28). CAT activity driven by the
FER-1-containing ferritin H promoter was ~60% repressed by 12SE1A
(Fig. 5). Cotransfection of wild-type
p300 resulted in ~80% restoration of E1A-mediated repression of CAT
activity, and p300del30 completely restored the CAT activity repressed
by E1A. A control vector (pCMV) had no effect (Fig. 5). The fact that
p300del30 efficiently antagonized E1A-mediated ferritin H repression
(p300del30 cannot associate with E1A, but retains a transcriptional
adaptor function) suggests that the CAT activity restored by p300
protein is not simply due to sequestration of E1A, but rather to
transcriptional activation of the ferritin H enhancer by p300.
Inhibitor of Histone Deacetylase Augments Ferritin H
Transcription--
It has recently been demonstrated that p300 and CBP
both bind to the histone acetylase PCAF (49) and possess intrinsic
histone acetyltransferase activity (43). To test the importance of
histone acetylation in transcriptional activation of the ferritin H
gene, sodium butyrate, a histone deacetylase inhibitor, was employed to
investigate whether sodium butyrate can mimic p300/CBP function in
ferritin H transcription. NIH3T3 cells were transfected with a CAT
reporter plasmid driven by the ferritin H promoter/enhancer, followed
by treatment with a different concentration of sodium butyrate. As
shown in Fig. 6, sodium butyrate
increased CAT activity up to 2-fold in a dose-dependent
fashion, an increase comparable to the 2-3-fold activation of ferritin
H-CAT by p300 or CBP (Fig. 3). We also tested the effect of sodium
butyrate on endogenous ferritin H mRNA expression by Northern
blotting. These experiments demonstrated that ferritin H mRNA was
induced 2.4-fold by 2 mM sodium butyrate and 3.0-fold by 10 mM sodium butyrate after 24 h (Fig. 6). These results
indicate that conditions that block histone deacetylation activate
ferritin H transcription and suggest that the histone acetyltransferase
activity of p300/CBP or its associated proteins may contribute to its
activation of the FER-1 enhancer and elevation of ferritin H
transcription.
We previously observed that E1A preferentially represses
transcription of the ferritin H gene in mouse NIH3T3 fibroblasts, resulting in an alteration of the H/L subunit ratio of ferritin (8). To
understand the molecular mechanisms of transcriptional regulation of
the ferritin H gene and its repression by the E1A oncogene, we used
deletion and mutational analyses to investigate an E1A-responsive
element in the mouse ferritin H gene. These studies defined a 37-base
pair element (termed FER-1) as both the E1A-responsive element and a
basal enhancer of the mouse ferritin H gene (26). FER-1, located 4.1 kilobases upstream from the transcription initiation site, comprises an
AP1-like element followed by an element with dyad symmetry (26). Our
recent studies have identified the nuclear factors bound to these two
elements of FER-1: the AP1-like element has the ability to bind JunD,
FosB, and ATF1, and the C-rich sequences in the dyad symmetry can bind Sp1 and Sp3 (27). Using recombinant ATF1 and Sp1, we have demonstrated that these transcription factors bind simultaneously to each element of
FER-1 (27), regulating ferritin H transcription by binding directly to
FER-1 sequences.
In this study, we have identified p300/CBP as an additional component
required for FER-1 enhancer activity. Thus, E1A mutants that did not
have a p300-binding region including conserved region 1 failed to
repress the endogenous ferritin H gene (Fig. 1). Furthermore, in CAT
reporter assays, E1A point mutants lacking the ability to bind p300
failed to repress FER-1, whereas E1A mutants defective in interaction
with members of the Rb family retained the ability to repress FER-1
(Fig. 2A). Since endogenous p300/CBP levels were not
affected by E1A expression (Fig. 2B), the mechanism of
E1A-mediated repression of FER-1 enhancer activity is not due to
down-regulation of p300/CBP synthesis by E1A, but to E1A and p300/CBP interaction.
Overall, the transcriptional activity of the ferritin H promoter
depends in a complex way on the AP1, Sp1, and p300/CBP transcription factor families. For example, although the Sp1/Sp3-binding component of
FER-1 does not by itself confer enhancer activity, it is required for
the enhancer activity of the AP1-like element of FER-1 to be expressed
(26). In contrast, cotransfection experiments have demonstrated that
FosB and JunD directly activate ferritin H transcription. Thus,
cotransfection of FosB, JunD, and ferritin H-CAT in F9 cells (which do
not contain appreciable endogenous levels of AP1 (44)) led to an
~6-fold enhancement of ferritin H-CAT activity (27). As described
here, p300/CBP also plays a role in ferritin H transcription, augmenting ferritin H-CAT activity 2-3-fold in NIH3T3 cells (Fig. 3)
and BNL CL.2 cells.2 This
effect of p300/CBP on ferritin H transcription is within the range
reported for the effects of p300/CBP on other enhancers (2-3-fold)
(23, 45, 46). Furthermore, indirect evidence suggests that the
phosphorylation status of p300 may influence its ability to function as
a coactivator in ferritin H transcription. Thus, in undifferentiated F9
cells, p300 is poorly phosphorylated and possesses weak coactivator
activity; however, both phosphorylation and coactivator activity
increase following differentiation of F9 cells (47, 48). We observed
that cotransfection of p300 and ferritin H-CAT into F9 cells resulted
in a minimal augmentation of ferritin H-CAT activity over that seen
with AP1 alone,2 despite the demonstrated functionality of
the FER-1 element in F9 cells (27). This suggests that in cellular
contexts in which p300 is poorly phosphorylated, the effect of p300 on
ferritin H transcription may be blunted. It will be interesting to test this hypothesis by assessing the relationship between p300
phosphorylation and FER-1 enhancer activity during the differentiation
of F9 cells. Taken together, these results suggest that the activity
and abundance of Sp1, AP1/ATF1, and p300/CBP transcription factor
families collectively determine the activity of the FER-1 element of
ferritin H in a complex and cell type-dependent fashion.
What is the role of p300/CBP in the FER-1 complex? Recent work has
suggested that p300 is more than a simple adaptor between DNA-binding
proteins and transcription initiation factors. Thus, p300/CBP was shown
to possess intrinsic histone acetyltransferase activity (43) as well as
to associate with PCAF, a protein that also functions in histone
acetylation (49). Since transcriptionally active chromatin is
hyperacetylated (reviewed in Ref. 50), p300/CBP-mediated histone
acetylation may function to facilitate access of transcription factors
to the target sequences of DNA normally compacted in chromatin. Our
experiments provide two lines of evidence that are consistent with a
role for p300/CBP-mediated histone acetylation in FER-1 function. The
first derives from the observation that E1A competes with PCAF for p300
interaction since they share an overlapping binding domain on the p300
protein (E1A binds amino acids 1572-1818 of p300 (28), and PCAF binds
amino acids 1801-1851 of p300 (49)). In our experiments, a p300 mutant
lacking the E1A-binding domain (p300del30, deletion of amino acids
1737-1809 of p300) was able not only to activate ferritin H-CAT (Fig.
3), but to restore E1A-mediated repression of ferritin H-CAT (Fig. 5).
We also examined the behavior of another p300 mutant, p300del33. This
mutant contains a deletion encompassing 70% of the PCAF-binding region
(amino acids 1737-1836) and is predicted to have little or no
interaction with PCAF (49). In contrast to p300del30, p300del33 was
unable to substantially augment ferritin H-CAT expression,2
as was observed in the study of functional collaboration of p300 and
C/EBP Based on the results obtained in this study, we suggest the model
depicted in Fig. 7 for transcriptional
regulation of the mouse ferritin H gene. We speculate that
p300/CBP-mediated transcriptional activation of the ferritin H gene may
be achieved, at least in part, by tethering the FER-1-binding complex
to the basal transcription regulatory region (Fig. 7). This idea is
supported by two observations. First, p300/CBP can associate with
AP1/CREB family members including ATF1 (reviewed in Ref. 21), which
were identified as components of the FER-1-binding complex; second,
p300 and CBP were found to be components of the TATA-binding protein
complexes (32). Interactions at FER-1 may thus serve to target p300/CBP
and its associated histone acetylase activity to the ferritin H
promoter. Taken together with our prior results demonstrating a
reduction in factors bound to the AP1-like element of FER-1 in the
presence of E1A (26), this model suggests at least two mechanisms of E1A-mediated transcriptional repression of ferritin H. One is that E1A
reduces the ability of factors to bind to the AP1-like element (26), as
has been reported for repression of the AP1 site in the collagenase
gene by E1A (53). The other is that E1A removes p300 or CBP from the
protein complexes of FER-1 and the TATA box. Both mechanisms may
collaborate to decrease ferritin H transcription.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
)
4.8kbFHCAT is driven by the 5'-flanking region of
the mouse ferritin H gene from nucleotides
4819 to +24, containing
the FER-1 enhancer element. pBluescript KS(
)
4kbAP1FHCAT
transcription is driven by nucleotides
4060 to +24 of the mouse
ferritin H gene, containing an AP1(NF-E2) site, but not FER-1.
pBluescript KS(
)
4kbAP1+
FHCAT transcription is driven by
nucleotides
4109 to +24 of the mouse ferritin H gene, containing
FER-1. The human p300 expression plasmids CMV
p300 and
CMV
p300del30 (deletion of amino acids 1737-1809 in the E1A-binding
region of p300) were kindly provided by R. Eckner (University of
Zurich, Zurich, Switzerland) (28). The mouse CBP expression plasmid
pRc/RSV-mCBP-HA was a generous gift from R. Goodman (Vollum Institute,
Portland, OR) (29). The E1A plasmids used in this study have been
described elsewhere (24, 30).
RESULTS
23-107) or conserved regions 1 and 2 (
23-150) was deleted. Mutants of E1A containing conserved region 1 deletions are impaired in
binding to p300/CBP; conserved region 2 deletions interfere with
binding to the pocket proteins p105Rb, p107, and p130 (reviewed in Ref.
33). Total RNA isolated from control vector (Hyg and HygSR
)- or E1A
(intact E1A,
23-107, and
23-150)-transfected cells was analyzed
by Northern blotting after successive hybridization with cDNA
probes for ferritin H, ferritin L, and
-actin. As we reported
previously, expression of intact E1A repressed the mRNA for the H
subunit of ferritin in the absence of any effect on the mRNA for
the L subunit of ferritin or
-actin (Fig.
1). In contrast, deletion of the E1A
region containing conserved region 1 (
23-107, two independent cell
pools (A and B)) or conserved regions 1 and 2 (
23-150) completely
impaired the ability of E1A to repress ferritin H. The expression level
of these E1A mutant proteins was higher than that of intact E1A protein
(data not shown) (19). These results suggest that the functional region of E1A containing the p300-binding domain and conserved region 1 contributes to the repression of ferritin H.
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Fig. 1.
Functional domains of E1A are required for
repression of ferritin H. Total RNA was isolated from cells stably
transfected with control vectors (Hyg and HygSR ), 13SE1A (intact
E1A), an E1A mutant with amino acids 23-107 deleted (
23-107, two
independently isolated pools A and B), or an E1A mutant with amino
acids 23-150 deleted (
23-150). 10 µg of each RNA was subjected
to Northern blot analysis and sequentially hybridized with ferritin H
(top), ferritin L (middle), and
-actin
(bottom) cDNAs (8).
4.8kbFHCAT, our largest ferritin H
5'-fragment fused to the CAT gene, into NIH3T3 cells (Fig.
2A). The E1A mutants that
retained the ability to associate with all these cellular proteins
(DA21, LP49, EV55, and
81-120) repressed ferritin H enhancer
activity as efficiently as wild-type E1A (12Swt). Comparable repression
of the ferritin H enhancer was seen by E1A mutants blocked in the
ability to interact with Rb and p130 (YH47) or Rb, p107, and p130
(YH47/928). In contrast, mutations that interfered with p300
interaction, but not with the Rb family proteins (
2-36,
15-35,
and RG2), significantly reduced the ability of E1A to repress ferritin
H. Loss of ferritin H repression was similarly observed following
cotransfection of
4.8kbFHCAT with E1A mutants containing two point
mutations (RG2/928) that abrogate the interaction of E1A with both p300
and Rb family members. Similar expression levels of all E1A proteins
were detected by Western blotting (Fig. 2B). Endogenous p300
levels were not altered by E1A transfection (Fig. 2B),
suggesting that repression of the ferritin H enhancer by E1A is not due
to repression of p300 synthesis by E1A, but rather is dependent on the
interaction of E1A with p300.
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Fig. 2.
E1A mutants defective in p300 binding lose
the ability to repress ferritin H transcription. A, 5 µg of pUC18 or 5 µg of each E1A plasmid (except 12Swt, YH47, or
YH47/928, for which 3 µg of each E1A plasmid plus 2 µg of pUC18
were utilized to attain expression levels comparable to those exhibited
by other E1A mutants) was cotransfected with 5 µg of
pBluescript 4.8kbFHCAT into NIH3T3 cells. After 36-48 h, CAT activity
was assayed, and the radioactive spots on thin-layer chromatography
plates were quantitated by a phosphoimage analyzer. Percent acetylation
of chloramphenicol in extracts from cells transfected with pUC18 was
defined as 100%. DNA transfection was carried out in duplicate in each
experiment, and the results of five independent experiments and
standard errors are shown. The abilities of E1A mutants to interact
with p300, p105Rb, p107, and p130 are shown at the top (+,
interaction,
, no interaction) according to previous studies (24,
30). B, levels of endogenous p300/CBP (top) and
transfected E1A proteins (bottom) were simultaneously
measured by Western blotting using anti-p300/CBP antibody NM11 and
anti-E1A antibody M58, respectively. E1A mutant
81-120 protein was
not detected by anti-E1A antibody M58 since it lacks the M58 epitope,
but an expression level similar to that of 12 S wild-type E1A was
confirmed by Western blotting using anti-E1A antibody M73 (data not
shown).
4.8kbFHCAT. As
shown in Fig. 3, both p300 and CBP activated ferritin H transcription in a dose-dependent
manner. Activation was also seen with p300del30, a mutant of p300 that retains transcriptional adaptor activity (28). These functional assays
thus demonstrate that p300/CBP activates the ferritin H promoter.
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Fig. 3.
p300 and CBP activate ferritin H
transcription. 5 µg of 4.8kbFHCAT was cotransfected with 5 µg of CMV vector, 4 µg of CMV vector plus 1 µg of CMVp300, 5 µg
of CMVp300, or 5 µg of CMVp300del30 (left) and 5 µg of
Rc/RSV vector, 4 µg of Rc/RSV vector plus 1 µg of Rc/RSV-CBP, 3 µg of Rc/RSV vector plus 2 µg of Rc/RSV-CBP, or 5 µg of
Rc/RSV-CBP (right) into NIH3T3 cells. After 36-48 h, CAT
activity was measured, and the radioactive spots on thin-layer
chromatography plates were quantitated by a phosphoimage analyzer.
Percent acetylation of chloramphenicol in extracts from cells
transfected with 5 µg of control vector was defined as 100%. DNA
transfection was carried out in duplicate in each experiment, and the
results of four (left) and five (right)
independent experiments and standard errors are shown.
4kbAP1) or contains FER-1 (
4kbAP1+
). p300 by itself
significantly activated FER-1(+)FHCAT in a dose-dependent
manner (Fig. 4). In contrast, only a weak
activation of CAT by p300 was observed in FER-1(
)FHCAT (this weak
activation may be due to the existence of another potential
p300-targeted sequence on the mouse ferritin H gene; see
"Discussion"). These results suggest that the p300 adaptor protein
activates the ferritin H enhancer via the FER-1 element.
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Fig. 4.
p300 requires the FER-1 element for maximum
activation of the ferritin H gene. 5 µg of 4kbAP1FHCAT
(
FER-1) or
4kbAP1+
(+FER-1) was
cotransfected with 5 µg of CMV vector (
), 4 µg of CMV vector
plus 1 µg of CMVp300 (
), or 5 µg of CMVp300 (
) into NIH3T3
cells. After 36-48 h, CAT activity was measured, and the radioactive
spots on thin-layer chromatography plates were quantitated by a
phosphoimage analyzer. Percent acetylation of chloramphenicol in
extracts from cells transfected with 5 µg of CMV vector (
) and 5 µg of
4kbAP1+
(+FER-1) was defined as 100%. DNA
transfection was carried out in duplicate in each experiment, and the
results of six independent experiments and standard errors are
shown.
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Fig. 5.
p300 restores E1A-mediated ferritin H
repression. 5 µg of 4.8kbFHCAT was cotransfected with 5 µg
of pUC18, 3 µg of pUC18 plus 2 µg of p12SE1A, 3 µg of CMV vector
plus 2 µg of p12SE1A, 3 µg of CMVp300 plus 2 µg of p12SE1A, or 3 µg of CMVp300del30 plus 2 µg of p12SE1A into NIH3T3 cells. After
36-48 h, CAT activity was measured, and the radioactive spots on
thin-layer chromatography plates were quantitated by a phosphoimage
analyzer. Percent acetylation of chloramphenicol in extracts from cells
transfected with 5 µg of pUC18 was defined as 100%. DNA transfection
was carried out in duplicate in each experiment, and the results of
four independent experiments and standard errors are shown.
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Fig. 6.
Sodium butyrate activates transcription of
the ferritin H gene. Top, NIH3T3 cells were transfected
with 10 µg of 4.8kbFHCAT. After 36-40 h, cells were treated with
different concentrations of sodium butyrate for 9 h. CAT assays
were performed, and acetylated and nonacetylated forms of
chloramphenicol were quantitated by phosphoimage analysis. Percent
acetylation of chloramphenicol in extracts from cells without treatment
was defined as 100%. DNA transfection was carried out in duplicate in
each experiment, and the results of three independent experiments and
standard errors are shown. Bottom, NIH3T3 cells were treated
with 2 and 10 mM sodium butyrate for 24 h. Total RNA
was isolated, and 10 µg of RNA was subjected to Northern blot
hybridization with mouse ferritin H cDNA. Equivalent amounts of RNA
loading and transfer to membrane were confirmed by ethidium bromide
staining (data not shown).
DISCUSSION
(CCAAT-box enhancer binding protein
) (51). These results
suggest that activation of ferritin H by p300/CBP may require histone
acetylation and, in particular, association p300/CBP with PCAF. The
second line of evidence supporting the idea that histone acetylation
plays an important role in ferritin H transcription is the observation
that the histone deacetylase inhibitor sodium butyrate can mimic
p300/CBP as an activator of the endogenous ferritin H gene as well as
ferritin H-CAT (Fig. 6). These results are consistent with previous
observations (52).
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Fig. 7.
Model of FER-1-mediated transcriptional
regulation of the mouse ferritin H gene. FER-1 is composed of an
Sp1-like and an AP1-like element, to which Sp1/Sp3 and FosB/JunD/ATF1
bind. p300/CBP may tether this complex to the basal enhancer complex
(TBC), and its histone acetyltransferase activity may also
be involved in the activation of FER-1. E1A reduces the binding of
factors to the AP1-like element of FER-1. E1A interacts with p300/CBP,
a coactivator of FER-1, inhibiting the adaptor function of p300/CBP and
leading to repression of ferritin H transcription. kb,
kilobases.
Our results suggest that p300 plays an important role in ferritin H
transcriptional activity through its interaction with FER-1. While this
work was in progress, another potential target of p300/CBP in the
ferritin H gene was identified. Bevilacqua et al. (54)
reported the presence of a cyclic AMP-responsive element (B-site, 62
to
45 nucleotides upstream from the transcription initiation site) in
the human ferritin H gene. They suggested that cAMP-mediated induction
of human ferritin H transcription is inhibited by E1A through
sequestration of p300/CBP. The B-site does not contain a canonical
cAMP-responsive element sequence and was reported to bind a protein
complex containing a 120-kDa protein and p300 (54). This element
overlaps an NF-Y-binding region containing a CCAAT box in inverse
orientation, which is important in ferritin H induction during monocyte
to macrophage differentiation (55). Comparison of mouse and human
proximal 5'-flanking sequences reveals 78% conservation in the B-site
and complete conservation of the inverse CCAAT box (for mouse ferritin H DNA 5'-sequences (56)). Although this region does not function as a
strong enhancer in the mouse ferritin H gene,2 it may have
weak enhancer activity, and FER-1(
)FHCAT used in Fig. 4, which
contains the B-site/CCAAT box, may have been slightly activated by
p300. Thus, p300 may influence multiple regulatory elements in the
ferritin H gene. However, our results indicate that p300/CBP activates
transcription of the mouse ferritin H gene primarily through the FER-1
enhancer element. These results also suggest that similar mechanisms of
E1A-mediated transcriptional repression of ferritin H are operative in
both mouse and human genes, despite differences in functional roles and
binding complexes between the FER-1 element and the B-site.
What is the biological implication of the involvement of p300/CBP in the FER-1 enhancer activity of the ferritin H gene? One possibility is that p300/CBP may, at least in part, play a role in determining tissue-specific expression of ferritin H. FER-1 lies within the 180-nucleotide SacI-BglII fragment identified as responsible for the up-regulation of ferritin H in erythroid cells induced to differentiate with N,N'-hexamethylene-bis-acetamide (9). It is unclear whether the FER-1-binding complex (containing AP1 and Sp1 family members as well as p300/CBP) is involved in N,N'-hexamethylene-bis-acetamide-dependent induction of ferritin H in erythroid cells. However, it has been reported that cooperative interaction between AP1 and Sp1 family members may underlie myeloid-specific expression of the leukocyte integrin gene CD11c (57), suggesting that tissue-specific alterations in the constellation of proteins binding to FER-1 may similarly contribute to tissue-specific regulation of ferritin H in other cell types, including erythroid cells. In conjunction with transcription factors directly bound to FER-1, p300/CBP may also modulate ferritin H transcription during cell immortalization. It will be important to dissect the potentially changing composition of the FER-1-binding complex during these processes.
In summary, we have demonstrated the involvement of p300/CBP in the
basal enhancer activity of the mouse ferritin H gene via the FER-1
element, indicating that at least three families of factors (AP1/ATF,
Sp1/Sp3, and p300/CBP) regulate ferritin H transcription through the
FER-1 element. Environmental or intracellular events that target each
of these, individually or collectively, may be expected to modulate
ferritin H transcription. These molecular observations may help to
explain the response of ferritin H to diverse cellular signals.
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ACKNOWLEDGEMENTS |
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We thank Drs. R. Eckner, R. Goodman, and E. Harlow for generously providing p300 expression plasmids, a CBP expression plasmid, and anti-E1A antibody, respectively.
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FOOTNOTES |
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* This work was supported by Grant DK-42412 from the National Institutes of Health. Phosphoimaging analysis was performed in a facility supported by Grant CA12197 from the National Institutes of Health and by Grant 9510-IDG-1006 from the North Carolina Biotechnology Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27157. Tel.: 336-716-0232; Fax: 336-716-0255; E-mail: ytsuji{at}wfubmc.edu.
2 Y. Tsuji, E. Moran, S. V. Torti, and F. M. Torti, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: Rb, the retinoblastoma susceptibility gene; PCAF, p300/cBP-associated factor; CREB, cAMP-responsive element-binding protein; CAT chloramphenicol acetyltransferase, CMV, cytomegalovirus.
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REFERENCES |
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