(Received for publication, April 21, 1997)
From the § Dipartimento di Biochimica e Biotecnologie
Mediche, Università degli Studi di Napoli "Federico II," Via
S. Pansini 5, I-80131 Napoli, Italy, the ¶ Dipartimento di
Medicina Sperimentale e Clinica, Università degli Studi di Reggio
Calabria, Via T. Campanella, I-88100 Catanzaro, Italy, and the
Dipartimento di Biologia e Patologia Cellulare e Molecolare,
C.E.O.S.-CNR, Università degli Studi di Napoli "Federico
II," 80131 Napoli, Italy
Transcription of the H ferritin gene in
vivo is stimulated by cAMP and repressed by the E1A oncoprotein.
We report here the identification of the cis-element in the human
promoter responsive to both cAMP- and E1A-mediated signals. This
promoter region is included between positions 62 to
45 and binds a
approximate 120-kDa transcription factor called Bbf. Bbf forms a
complex in vivo with the coactivator molecules p300 and
CBP. Recombinant E1A protein reduces the formation of these complexes.
In vivo overexpression of p300 in HeLa cells reverses the
E1A-mediated inhibition of the ferritin promoter transcription driven
by Bbf. These data suggest the existence of a common mechanism for the cAMP activation and the E1A-mediated repression of H ferritin transcription.
The genes coding for the heavy and light ferritin subunits are expressed in a wide variety of organisms and, within an organism, in all cell types (1). Their expression is modulated by several factors, such as iron (2), hormones (3), drugs (4), and cytokines (5), which lead to the synthesis of multiple ferritin isoforms with different H to L ratio, called isoferritins.
Regulation of ferritin gene expression is exerted at translational and
transcriptional levels. Iron interferes with the binding of the
translational repressor (iron responsive element-binding protein) with
the target sequence (iron responsive element) in the 5-untranslated
region of ferritin transcripts (for review, see Ref. 6). Hormones and
drugs modulate transcription of ferritin genes. Transactivation of the
H promoter has been reported in C6 glioma cells treated with
insulin-like growth factor-I (7), in Friend erythroleukemia cells
treated with hemin (8), in differentiating Caco-2 cells (9), in CL3
neuroepithelial cells exposed to G418 (4), and in FRTL5 rat thyroid
cells treated with cAMP (10). Conversely, the gene product of the
adenovirus, E1A, inhibits ferritin expression (11).
The molecular dissection of H ferritin promoter indicates that the
relevant regulatory sequence elements are located in a 200-base pair
segment at 5 end of the transcription start site of the human gene
(12). Recently we have mapped a DNA sequence, the B-site, located in
the
62/
45 region of the human H gene, which acts as a positive
regulatory element. Deletion analysis of this region has shown that a
transacting factor, which binds the B site, is required for the
transcription of ferritin gene (12). Cells undergoing in
vitro differentiation or treated with translation inhibitors,
actively transcribe ferritin gene (9, 4). Under these conditions, the
activity of the B site binding factor
(Bbf),1 assayed by transient
transfection with target promoter or by DNA binding assays,
specifically increases in the nucleus.
Here we show that Bbf, a protein of approximately 120 kDa, is required for cAMP induction of the ferritin promoter. Bbf does not bind the consensus CRE sequence and does not react with antibodies versus CREB or ATF1. Furthermore, Bbf binds in vivo the transcriptional adaptors CBP and p300. The product of adenovirus E1A specifically competes the formation of the p300-Bbf complex. Transient or stable expression of E1A inhibits the transcription driven from the ferritin promoter. In the same cells, overexpression of p300 completely reverses E1A inhibition of ferritin transcription.
We propose that Bbf mediates cAMP induction of ferritin promoter and that the mechanism of transcriptional repression of the H ferritin gene by E1A is caused by the competition of E1A with Bbf for p300 binding, resulting in the disruption of the active transcriptional complex on the ferritin promoter.
HeLa cells were cultured as monolayers in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with 10% (v/v) fetal calf serum (Sigma), 100 units/liter penicillin (Hyclone). Cells were grown at 37 °C in a 5% CO2 atmosphere.
DNA Transfections and CAT AssaysThe H promoter/CAT
constructs 5-H A/M, 5
-H H/A, and 5
-H H/M have been previously
described (12). The Bbf-TATAA construct contains a double-stranded
B-site oligonucleotide (5
-CGGCGCTGATTGGCCGGG-GCGGGC-3
), in the
correct 5
to 3
orientation, cloned in the SmaI site of the
5
-H H/M construct upstream the ferritin TATAA box. The
Bbfmut-TATAA construct was obtained by inserting a mutated
B-site oligonucleotide (5
-CGGCTCTTACAGGCCGTTGCGAGC-3
),
in the correct 5
to 3
orientation, in the SmaI site of the
5
-H H/M construct. The pSVEK and pSVEKmut are expression vectors
carrying PKA-specific inhibitor tagged with a nuclear localization
signal under the control of SV40
promoter.2 The CRE-CAT
construct was a gift of Dr. P. Sassone-Corsi (13). For each experiment,
5 pmol of plasmid DNA were transfected into cultured HeLa cells by the
calcium phosphate coprecipitation method (14). To correct for
variations in DNA uptake and transfection efficiency, 5 µg of a
plasmid carrying the luciferase gene under the control of the
cytomegalovirus enhancer were included. For the cAMP treatment,
10
6 M 8-bromoadenosine 3
:5
-cyclic
monophosphate (Sigma) was added directly for the indicated times. CAT
and luciferase assays were performed as described (4, 15). The values
reported in the figures are the mean of three independent transfection
experiments.
Nuclear extracts were prepared
from HeLa cells as previously described (4). The
oligonucleotides used for EMSAs were: CTF-site 5-TTATTTTGGATTGAAGCCAATATGATAA- 3; CRE-site
5
-GTTCCGCCCAGT-GACGTAGGAAGTC-3
. To study DNA-protein
interaction on the B-site, the
100 to +1 region from the human H
promoter, terminally labeled, was used. EMSAs and competition assays
were performed as described previously (9). Anti-CREB antibody (Santa
Cruz, X-12) was assayed as supershifting reagent; the antibody was
added to the nuclear extract and incubated for 1 h on ice before
the addition of the labeled probes.
0.5 ng of terminally labeled B-site oligo
(5-CGGCGCTGATTGGCCGGGGCGGGC-3
) were incubated in a final volume of 20 µl, with 25 or 50 µg of HeLa nuclear extracts in the presence of 3 µg of poly(dI-dC) (Pharmacia) for 30 min at room temperature. After incubation, the samples were UV irradiated for 2 min at a wavelength of
254 nm and then loaded on a 5% polyacrylamide gel. The retarded bands
corresponding to the DNA-protein complexes were excised from the gel
and loaded on a 8% SDS-polyacrylamide gel electrophoresis.
Whole cell extracts were prepared as described (16). Immunoprecipitations, deoxycholate elutions, and gel retardation assays were performed as described in Ref. 17 with modifications. Briefly, 300 µg of HeLa whole cell extracts were incubated with anti-p300 (05-222 UBI) antibody or with non-immune antisera for 1 h at 4 °C in a buffer containing 10 mM Hepes (pH 7.6), 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM sodium fluoride, 0.5 mM sodium orthovanadate (Na3VO4), 1 mg/ml leupeptine, 1 mg/ml aprotinin. The samples were further incubated for 1 h at 4 °C with protein A-Sepharose. The beads were collected by centrifugation, washed four times with the incubation buffer, and finally incubated 20 min on ice with 10 µl of incubation buffer, 1% deoxycholate. Immunoprecipitates were collected and assayed by EMSA with the radioactive probes.
Alternatively, whole cell extracts from wild type HeLa cells were mixed with recombinant E1A protein or with recombinant BMV (tobacco mosaic virus, TNT Promega) protein 30 min at 4 °C prior to immunoprecipitation with anti-p300 antibodies.
cAMP stimulates the transcription of H ferritin gene
(3, 10). To analyze the promoter elements responsive to cAMP, we used
several plasmid fusions carrying segments of ferritin promoter upstream
the chloramphenicol acetyltransferase gene (CAT) (12). The 5-H A/M
construct (
160 to +1) contains the positive cis-elements A and B
necessary for the expression of H ferritin in vivo. The A
element is the target for the transcription factor Sp1, while the B
element is recognized by a nuclear protein, which we named Bbf. The
5
-H H/A construct (
100 to +1) retains only the B element; the 5
-H
H/M construct contains only the TATAA box and the start of
transcription of the H gene (
50 to +1).
HeLa cells were transiently transfected with these plasmids and
stimulated with cAMP. CAT activity was measured and normalized for the
transfection efficiency by including in each transfection the
luciferase gene driven by the cytomegalovirus promoter (CMV-Luc). Fig.
1 (panel A) shows that the
segment of the promoter between 100 and
50 from the transcription
start site was essential for cAMP-induced transcription. The presence
of the Sp1 site in the 5
-H A/M construct increased the basal activity
of the promoter (4, 12), and partly masked the cAMP-induced
transcription. Deletion of the
100 to
50 region inhibited
significantly both the basal and cAMP induced transcription of the
ferritin promoter. The region
100 to
50 contains the B site, which
by footprint and EMSA appears to bind only the Bbf complex. To
determine if this site is responsible for cAMP induction of ferritin
promoter, we synthesized a 20-base pair oligonucleotide spanning the
wild-type Bbf binding sequence and an equivalent mutated version of
this site, and fused these oligonucleotides to the minimal promoter element 5
-H H/M. Note that the mutated version of the oligonucleotide, tested by EMSA, did not bind Bbf (data not shown). The resulting constructs have been named Bbf-TATAA and Bbfmut-TATAA,
respectively. Panel A of Fig. 1 shows that the B
oligonucleotide fused to the ferritin TATAA box transactivates the
basal transcriptional activity of the resulting construct (compare the
activities of constructs 5
-H H/A, 5
-H H/M, and Bbf-TATAA) and confers
induction of transcription by cAMP. The Bbfmut-TATAA
construct shows a basal activity comparable to that given by the TATAA
box only and does not respond to cAMP stimulation. Time course of the
cAMP-induced transcription in cells expressing the
100/+1 promoter
indicated that the maximal response was achieved within 1-2 h of
continuous stimulation with cAMP (Fig. 1, panel B). The
specificity of cAMP-induced transcription was tested by including in
the transfection mixture a plasmid expressing the PKA-specific
inhibitor tagged with a nuclear localization signal derived from SV40
LT antigen (pSVEK). cAMP-induced CAT expression driven by the ferritin
promoter was completely inhibited by PKA-specific inhibitor, but not by
its mutated version (pSVEKmut) (Fig. 1, panel C). As
positive control a CAT fusion driven by the CRE element derived from
the somatostatin promoter was included (Fig. 1, panel C).
The kinetics of induction of CAT activity was very similar to that of
ferritin mRNA accumulation stimulated in vivo by cAMP
(data not shown). These data indicate that the region of ferritin
promoter between
100 to
50, where Bbf binds, contains the sequence
element responsible for cAMP induction of transcription.
Bbf Binding Was Not Stimulated by cAMP
Inspection of the DNA
sequence in the 100 to
50 region of ferritin promoter did not
indicate the presence of a classical or spurious CRE site or a
palindrome resembling the target of ATF/CREB family (for review, see
Ref. 18). DNase I footprinting analysis of this region with nuclear
extracts from different cell types (4, 12) demonstrated that a single
cis-element (B box) was recognized by the transcription factor Bbf. We
determined the specificity of Bbf binding to its DNA element in cells
stimulated with cAMP. Panel A of Fig.
2 shows that Bbf binding was not
stimulated by cAMP and was not competed by a canonical CRE
oligonucleotide. We have also analyzed the DNA-protein complexes at the
CRE and Bbf sites in the presence of anti-CREB or anti-ATF1 antibodies. Bbf binding was not affected by the inclusion of the antibody (CREB) in
the binding reaction; under the same conditions CRE-binding complexes
were supershifted by the specific CREB antibody (Fig. 2, panel
B). The same results have been obtained with an anti-ATF1 antibody
(data not shown).
To identify more precisely Bbf, we cross-linked protein-DNA complexes to labeled B oligonucleotide and visualized the labeled band by SDS-polyacrylamide gel electrophoresis (Fig. 2, panel C). A single specific band of approximately 120 kDa was detected. This band was specifically removed by excess of specific unlabeled oligonucleotide (Fig. 2, panel C, and data not shown).
Bbf Interacts with the Coactivator p300Recently, it has been shown that the nuclear proteins CBP and p300 interact with CREB to mediate cAMP induction of transcription. CBP and p300 are highly homologous (92%) and are thought to target CREB and other trans-acting factors to RNA polymerase initiation complex, by binding TFIIB (19).
We have explored the possibility that Bbf might use the same mechanism.
To this end, we have immunoprecipitated HeLa total proteins with
specific anti-p300 antibodies, eluted the immunoprecipitated proteins
with deoxycholate, and analyzed the presence of Bbf by EMSA.
Panel A of Fig. 3 shows that
Bbf is present in the p300 immunocomplexes; CREB also was in the same
immunocomplexes (Fig. 3, panel B). We have tested whether
other transacting factors, such as CTF1 (Fig. 3, panel C) or
NFB (not shown), were also forming complexes with p300. The results
indicate that neither of the aforementioned factors interacted with
p300. Since cAMP does not stimulate the binding of BBf to the promoter,
we have tested whether BBF binding to p300 was stimulated by cAMP. Fig. 3, panel D, shows that the binding of BBF1 to p300 is
markedly stimulated in cAMP-treated extracts. At present we cannot
determine if the phosphorylation of p300, BBF1, or both stimulates the
formation of the complex.
p300 is highly homologous to the protein CBP in the CREB-binding region (19). It is possible that Bbf could react with CBP as well. We have immunoprecipitated the HeLa whole cell extracts with specific anti-CBP antibody, and tested for the presence of Bbf. Similarly to p300, CBP immunoprecipitates contained Bbf (data not shown). These data indicate that Bbf interacts with p300 and CBP in vivo.
E1A Interacts with p300 and Reduces the Formation of the Complex Bbf-p300p300 has been isolated as E1A binding nuclear protein
(20). Since E1A represses ferritin transcription (11), we wished to
determine whether E1A competed with the formation of Bbf-p300 complex.
To this end, total extracts from HeLa cells were mixed with recombinant
E1A protein prior to immunoprecipitation with anti-p300 antibodies. The
immunoprecipitate was analyzed for Bbf binding activity by EMSA, as
described above. Bbf-DNA retarded complex was inhibited when
recombinant E1A was added to the extracts (Fig.
4). This inhibition was specific for E1A
protein, since another recombinant protein (BMV) did not significantly
reduce Bbf concentration in the p300 immunoprecipitates (Fig. 4). This result indicates that E1A competes with Bbf for the binding to p300.
p300 Rescues E1A-inhibited Ferritin Transcription
The
mechanism of the E1A inhibition of ferritin transcription might be
caused by competition of E1A with Bbf for p300 binding, as suggested by
Fig. 4. To explore this possibility, we transiently expressed E1A
carrying plasmids (12 S and 13 S) in several cell lines (HeLa, Chinese
hamster ovary, and thyroid cells) with the 100/+1 ferritin-CAT
construct (5
-H/HA). Fig. 5 shows that
the transcription of the CAT gene, driven by the ferritin promoter, is
markedly inhibited by coexpression of E1A gene. Bbf appears to be
the target of E1A repression, since the minimal ferritin promoter
construct (5
-H H/M) is not affected by E1A expression.
Transient transfection of a p300-expressing plasmid in normal or in cAMP-treated HeLa cells slightly stimulated ferritin/CAT expression. Conversely, when p300 was co-transfected with E1A, the ferritin/CAT expression was restored at normal levels. Under the same conditions, p300 expression completely reversed the E1A inhibition of CAT transcription driven by CRE promoter (data not shown). The same results have been obtained with cells stably expressing E1A or transformed by adenovirus (data not shown). These data suggest that E1A reduces ferritin transcription by titrating p300 and inhibiting the assembly of productive p300 complexes with Bbf.
Expression of ferritin is controlled by intracellular (such as iron) and by extracellular (hormones, growth factors) signals (2, 3, 7). The expression of the protein is controlled at transcriptional, post-transcriptional, and translational levels (1). The multiplicity of the stimuli and the mechanisms governing the expression of the gene explain the variety of ferritin phenotypes exploited by the cell under different conditions.
Mechanism of cAMP-PKA Stimulation of Ferritin Gene TranscriptionH ferritin mRNA levels and transcription of the
gene in vivo are stimulated by cAMP (3, 10, 21, 22). The
identification of the primary target sequences responsive to cAMP
signaling in the H ferritin promoter has not been carried out
extensively. The human H ferritin promoter is a compact element, where
the basic and the upstream control elements are contained in about 170 nucleotides upstream the transcription start site (12). In the mouse
promoter it has been also described as an enhancer element located at
4.1 kilobases from the start of transcription (23). We have
restricted our analysis to the main promoter element, previously mapped
by mutagenesis (12). Two sites have been mapped in this region,
recognized by Sp1 (at
132) and Bbf (at
62), respectively. Bbf is a
factor not yet cloned, which by competition analysis and size does not
appear to be a known nuclear transacting protein (4, 12). The deletion
of the element recognized by Bbf completely abolishes both the basal
and the cAMP-induced transcription driven by the ferritin promoter.
Moreover, fusion of a B-site oligonucleotide to the ferritin minimal
promoter element confers cAMP stimulation of transcription. Bbf does
not seem to be a CREB or ATF1 factor, since it is not competed by the
specific oligonucleotides and its binding is not affected by anti-ATF1
or anti-CREB antibodies (Fig. 2). Note that we have used anti-CREB
antibodies which recognize the KID domain, a common structural motif
present in all members of the CREB family (18). However, Bbf shows all
the functional properties of a CREB protein (Fig. 2): its binding is
not sensitive to cAMP (Fig. 2), it is phosphorylated (data not shown),
and it binds the nuclear adaptor molecules, CBP and p300 (Fig. 3).
Definite answer to the question of Bbf identity has to await the
molecular cloning of the protein. We have noted that a sequence similar to the Bbf-binding site (5
-CGGCGCTGATTGGCCGGGGCGGGC-3
) is present in
the promoter of the human tryptophan hydroxylase gene and in the
promoter of the rat fatty acid synthase gene. This sequence has been
shown to be the target of cAMP induced transcription (24, 25). We
suggest that this DNA element identify a new class of transacting
factor(s) which mediate(s) cAMP-induction of transcription.
p300 and CBP are two high molecular weight proteins, resident in the nucleus, which have an intrinsic transcription activation function (26, 27). They bind TFIIB (19), as well as other transacting factors, such as CREB, Jun, and YY1 (27-29). The interaction with CREB and Jun is facilitated by phosphorylation of these proteins (19, 30). We have shown that also Bbf interacts in vivo with p300 and CBP (Fig. 3). We suggest that p300 and CBP function as molecular adaptors which bring together Bbf and the RNA polymerase transcription initiation complex. Since the DNA binding of Bbf is not stimulated by cAMP or by treatment with okadaic acid (data not shown), we suggest that Bbf is permanently located on the DNA and that cAMP signaling stimulates the assembly of the complex Bbf-p300. This is consistent with our finding (Fig. 3, panel D) that BBF-p300 complex is stimulated by cAMP. Preliminary data indicate that both p300 and Bbf are phosphorylated by PKA. At present we cannot determine whether cAMP phosphorylation of Bbf directly stimulates the formation of the complex.
E1A Represses Ferritin Transcription by Interfering with the Formation of the Complex Bbf-p300Ferritin composition is altered in some tumors (31, 32). The mechanisms underlying the ferritin phenotypes remains unknown. Adenovirus E1A expression in mouse cells induces preferential down-regulation of H ferritin transcription and results in the production of ferritin molecules with altered H-L ratio (11, 23). The data we have shown indicate that E1A protein reduces the formation of p300-Bbf complex in vitro (Fig. 4). This reduction parallels the inhibition of transcription in vivo by E1A (Fig. 5). Overexpression of p300 completely reverses the transcription block and restores ferritin expression (Fig. 5). In our conditions, we did not detect any significant stimulation of ferritin promoter, at variance with the CREB-mediated activation shown by others (26, 27). Note, that in the latter cases CREB was overexpressed (26, 27). This finding suggests that BBF1 is limiting in vivo and that p300 alone cannot stimulate ferritin transcription. E1A expression possibly titrates p300 and reduces the formation of the active complex; under these conditions, expression of exogenous p300 shifts the equilibrium from E1A-p300 to Bbf-p300 and reactivates ferritin transcription. At present we do not know if cAMP might alter the equilibrium between these complexes. We have evidence that ferritin transcription is not efficiently induced by cAMP in the presence of E1A. It is likely that the equilibrium between p300/Bbf and p300/E1A depends on the relative concentrations of active Bbf and E1A proteins.
Recently, it has been reported that an enhancer element, found at 4.1
kilobase from the start of transcription of the mouse H ferritin gene,
is the target of E1A that mediates the repression of ferritin
transcription (23). This sequence element located far upstream from the
promoter, relative to the region target of Bbf, contains an AP1-like
site. This site probably positively controls the ferritin transcription
induced by other extracellular stimuli. At this site we predict p300 or
CBP to play an essential role, since they can form complexes with Jun
and other components of the AP1 complex (28). E1A expression in
principle should disrupt both AP1 and Bbf complexes, resulting in the
complete inhibition of ferritin transcription.
We propose that the 4.1 kilobase enhancer is sensitive to
signal-targeted transcriptional activators (30), while the Bbf site is
induced by cAMP signaling. The interplay between cAMP and tyrosine
kinases signals in vivo regulates the transcription of H
ferritin gene.