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Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and MethodsReferences |
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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and 70Z/3 cells stimulated with lipopolysaccharide
(2)
. Autophosphorylation activity, which correlates with
increased substrate-directed activity, was later shown to be increased
in lymphocytes upon stimulation with mitogenic lectin in Chinese
hamster ovary cells with IL-1 and in fibroblasts with
platelet-derived growth factor-ß (1)
, as well as in
multiple organs of mice injected with epidermal growth factor, phorbol
12-myristate 13-acetate, or IL-1 (3)
. The murine
Mediator transcription complex, which contains homologues of the yeast
transcriptional regulators Med6, Med7, Rgr1, and Srb7, also contains a
protein that is homologous to human RING3 and its Drosophila
homologue female sterile (1)
homeotic (fsh)
(4)
. This association led us to speculate that RING3, as a
nuclear participant in signal transduction cascades, might have
important transcriptional targets. Apart from this circumstantial evidence implying a role in transcription, little is known about the mechanism of action of the RING3 protein (5) . The gene is localized to the class II major histocompatibility locus on human chromosome 6p21.3 and encodes a member of a family of bromodomain-containing human proteins that include BRDT (6) and ORFX (7) . There are reports of homologous genes in mice (8) , frogs (9) , and zebrafish (10) . fsh is a temperature-sensitive, maternal effect gene required at two stages of development, identified by the trithorax phenotype. fsh activates the trithorax locus and interacts genetically with the Antennapedia and Ultrabithorax complexes (11) . An fsh mutant called rancor (rnc) is embryonic lethal and affects head homeotic development (12) , probably through the btd (Sp1)-dependent regulation of cnc, a bZIP transcription factor related to mammalian NF-E2.4 Intriguingly, a human homologue of Trithorax, ALL-1 or MLL, is a putative transcription factor that is damaged in leukemias associated with 11q23 chromosome breaks (13-15) . By functional conservation with fsh, it is likely that RING3 contributes to the regulation of ALL-1 activity, i.e., improper signal transduction through RING3 and ALL-1 could lead to leukemia (1) .
Two redundant genes in yeast, BDF1 and BDF2, are homologous to RING3 and fsh primarily in their NH2-terminal bromodomains (1617) ; at least one of the two genes is required for yeast viability. Bdf1 and RING3 (5) are related to the product of the mammalian cell cycle gene CCG1 (mammalian TAFII250); both gene products have COOH-terminal acidic domains and intrinsic kinase activity (18) . Ccg1 and its yeast homologue Taf145 also possess histone acetyltransferase activity. Furthermore, the presence of two mutually related bromodomains in Ccg1, Bdf1 and Bdf2, RING3 and fsh suggests that this class of proteins may affect transcription through chromatin restructuring. Bromodomains (19) are commonly found in transcription factors (20) and the proteins that comprise chromatin-remodeling complexes (21-23) .
In the present study, we explore possible mechanisms that might link increased RING3 activity with cancer, first with experiments focused on transformation activity and time course of activation and then with transcriptional analysis of potential target genes of RING3. We demonstrate that RING3 exhibits some characteristics of a mitogenic, signal-transducing kinase and that it transactivates the promoters of important E2F-responsive genes that regulate the cell cycle. Furthermore, E2F copurifies with RING3 in protein complexes isolated from nuclear extract. E2F proteins are pivotal regulators of mammalian cell cycle progression and differentiation. In relationship with members of the Rb family of proteins, they control the transcriptional activation or repression of numerous genes; destabilization of these control mechanisms can result in apoptosis, reversal of differentiation, or cancer (2425) . These proposed links between RING3 and E2F-dependent transcription provoke further study of the relationship between chromatin-modifying complexes and cell cycle progression.
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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shows that foci did not form in either assay when cells were
transiently transfected with empty expression vector alone or with
constructs that encoded wild-type or the catalytically inactive (K574A)
form of RING3 (1)
. Activated ras alone was sufficient to
form foci, which is a well-known phenomenon (26)
. The
amount of transfected expression construct for activated ras was
titrated to give a minimal number of foci, and in this background,
expression of wild-type RING3 protein significantly increased the
number of foci formed. RING3 overexpressed with activated ras is
therefore capable of transformation. Expression of the K574A mutant
together with activated ras did not augment the number of foci. The
transforming capability of RING3 was therefore dependent on its kinase
activity in that the K574A mutant did not increase the number of foci
above levels with ras alone.
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(above), RING3 autophosphorylation activity remained elevated through
16 h of mitogenic stimulation but then fell by 20 h and
remained low throughout the remainder of the mitogenic program, even as
RING3-immunoreactive material accumulated (Fig. 1
, below). This
decrease in activity was well correlated in time with the onset of S
phase, which suggests that changes in RING3 activity are coupled to the
cell cycle.
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). Although RING3 increased the basal transcription of the E2F reporter,
the enhancerless reporter did not respond to RING3 or serum. These
results confirmed that RING3 can affect serum-responsive, E2F-dependent
transcriptional events but is probably not involved in cAMP-responsive
transcriptional events.
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). Activated ras alone was capable of transactivating each
promoter under these conditions, but the effect was dramatically
enhanced (3–6-fold) when activated ras and RING3 were present
together. Transfection into BALB/3T3 gave essentially the same results
(not shown). We conclude that RING3 and ras activities combine to
transactivate these promoters. MEKK, an effector of ras, was also
tested for its ability to transactivate the dhfr promoter
(Fig. 3B
). As in the previous experiment, neither empty
vector alone nor WT RING3 alone exhibited significant transactivation
of the dhfr promoter, whereas constitutively active MEKK
alone induced the dhfr reporter about 5-fold. Wild-type
RING3 and MEKK together synergized to give a 60-fold induction.
Activated Jun kinase and v-abl were incapable of transactivating the
dhfr promoter in a RING3-dependent fashion, demonstrating
specificity of the RING3 effect in the ras-MEKK pathway.
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). RING3 and MEKK together were no longer capable of
transactivating this promoter, which strongly suggested that
RING3-dependent transcriptional induction of the dhfr
promoter required the binding of E2F proteins. To confirm this
hypothesis, wild-type Rb protein was transiently overexpressed under
conditions of maximal RING3-dependent transactivation. Excess Rb
protein was predicted to reduce the "free" E2F available to
transactivate this promoter (242530)
, resulting in
decreased luciferase activity. As expected, RING3-dependent
transcription was attenuated as a dose-dependent function of
overexpressed Rb (Fig. 4B
). As a control for RING3 kinase
activity, the (K574A) point mutant of RING3 was substituted for the
wild-type enzyme (Fig. 4A
). This mutant did not
transactivate the wild-type or mutant dhfr reporters, either
alone, or with activated MEKK.
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90,000, which agrees with the
average apparent molecular weight of the "free" kinase as
determined by its apparent mobility during SDS-PAGE. The second
fraction had an average apparent molecular weight of
Mr 330,000 (Fig. 5
) suggesting that RING3 might participate in a complex with other
nuclear proteins. The amount of RING3 detected in the complex could be
reduced and the amount of free RING3 increased if chromatography was
conducted in the absence of 5 mM magnesium ATP
(results not shown), consistent with an ATP-dependent recruitment of
RING3 into the complex.
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shows an immunoblot of the column input (Lane
1), the eluate of the nonimmune IgG control column (Lane
2), and the eluate of a column composed of anti-RING3 antibody
(Lane 3). An E2F protein was detected among the proteins
that copurified from the anti-RING3 antibody column.
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SnaBI mutation (Fig. 6C
), which removed the COOH-terminal 93 amino acids from
this 754-amino acid protein as described previously (1)
.
E2F-containing complexes were not detected in the eluate of any
subsequent column composed of more extensively truncated RING3
derivatives. |
Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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In preliminary experiments, we observed that overexpressed RING3 cDNA
transforms NIH/3T3 fibroblasts in a context where activated ras is also
present (Table 1)
. This result is consistent with the earlier
observation that bacterially expressed, recombinant RING3 is inactive
and requires phosphorylation by a putative kinase kinase to become
active (1)
. Activated ras may provide this signal. In
later experiments, we isolated a clonal line of NIH/3T3 cells
transformed with wild-type RING3 and injected them into athymic nude
mice, which gave rise to tumors in 12 of 12 cases. However, a clonal
line derived from K574A RING3 did not cause tumors in any of six
cases.5
Taken together, these results suggest that increased RING3 kinase
activity is likely to be a cause, not a consequence, of oncogenic
transformation.
RING3 activation after serum stimulation of quiescent, synchronized
fibroblasts (Fig. 1)
suggests that it carries a mitogenic signal
(1-3)
. By these criteria, RING3 might be classed with the
mitogenic, signal-transducing kinases p70S6K,
pp90RSK, MAPK, and ERK, which are also
rasresponsive (2728)
. RING3 activity increases
until the G1-S transition, which supports a
hypothesis that RING3 is among the factors that link ras to E2F
(3233)
, Rb (34)
, and
G1-S progression. The cotransforming ability of
RING3 with activated ras, its time course of activation by serum, and
its nuclear localization in HeLa cells (1)
suggested it
might have transcriptional targets. We used transcriptional reporters
to explore this idea and demonstrated that in fibroblasts RING3
transactivates promoters that contain E2F sites, from which we develop
a hypothesis that RING3 promotes G1-S
progression.
A synthetic, consensus E2F enhancer responded to overexpressed RING3 and serum, whereas a synthetic, consensus CRE enhancer did not respond to overexpressed RING3, although a control with forskolin alone responded properly. These results established promoter specificity; they implied that RING3 might participate in a serum-induced mitogenic program mediated through E2F activity, consistent with observations that cAMP-dependent signal transduction is generally not involved in fibroblast proliferation (35) . Overexpressed RING3 transactivates the RING3-responsive reporter constructs under synchronized, serum-starved conditions and then serum-stimulated conditions, or in the continuous presence of serum. Under the latter condition, where the cells are not re-entering the cell cycle after a period of starvation, an additional upstream signal is required, such as activated ras or MEKK, an effector of ras. These observations support a hypothesis that RING3 participates in a mitogenic signal transduction pathway.
After demonstrating that promoters of the E2F-responsive cell cycle
genes cyclin D1, cyclin A, cyclin E,
and dhfr were also RING3 responsive, we focused our analysis
on the dhfr promoter, which is an important and well-studied
early player in E2F-mediated cell cycle progression. We successfully
showed that RING3-dependent transactivation requires catalytically
active RING3, E2F activity, and a functional E2F binding site. These
observations raise the possibility that RING3 functions in a nuclear
complex with E2Fs. We obtained evidence that RING3 is present in a
large multiprotein complex by size-exclusion chromatography of HeLa
nuclear extract (Fig. 5)
. We tested the idea that some of these
proteins might be E2Fs by anti-RING3 immunoaffinity chromatography or
RING3 protein affinity chromatography of nuclear extract, followed by
anti-E2F immunoblot of the eluates. We detected copurified E2F-1 and
E2F-2 protein in association with RING3 in these experiments. The
timing of this association of E2Fs with RING3 during the cell cycle is
a subject of our ongoing investigation.
There is evidence that signal transducing kinases such as Raf-1 can associate with Rb complexes after mitogenic stimulation and promote E2F-dependent G1-S progression (36) in a ras-dependent manner. A number of recent studies have explored E2F targets of the ras signal transduction pathway. In fibroblasts, the pathway typically originates with ligand activation of tyrosine kinase activity in a growth factor receptor or its deregulated activity in a tumor, e.g., the neu proto-oncogene (37) , followed by the activation of a ras/Raf-1/MAPK signaling cascade (37) . In the canonical pathway, this cascade leads to cyclin-dependent kinase 4/6 activation and the subsequent phosphorylation of Rb protein (38) , which alleviates Rb/E2F repression of key cell cycle genes (3940) . This derepression is a trigger event that initiates entry into S-phase (41) . This pathway can be bypassed by viral oncoproteins such as human papilloma virus E7 (42) and SV40 large T-antigen (43) that bind Rb directly, thereby liberating E2F without regard to the stage of the cell cycle and provoking abnormal growth. We speculate that, like Raf-1, RING3 might partition into Rb/E2F-containing nuclear complexes at critical moments in the mitogenic program and facilitate proper transcription of cell cycle genes. RING3 is normally constitutively localized to the nucleus of exponentially growing cells such as HeLa, however in serum-starved fibroblasts it is delocalized throughout the cell and can be induced to translocate to the nucleus upon serum stimulation.6 Its regulated nuclear translocation is consistent with function at a step proximal to E2F activity. The recent identification of a RING3-like protein in the murine Mediator complex (4) , where it associates with proteins that are homologous to the yeast transcriptional repressors Srb7 and Rgr1, and coactivator Med7, provides additional support for the hypothesis that RING3 functions at a transcriptional end point of mitogenic signal transduction.
Multiprotein complexes that contain RING3 and E2Fs are likely to be
implicated in cell cycle progression, given the similar time course of
RING3 and E2F mobilization. In subsequent experiments, we have been
unable to detect a direct interaction between in vitro
translated E2Fs and RING3 proteins, nor have we been able to
demonstrate a RING3 effect on DNA binding by electrophoretic mobility
shift assay with nuclear extract and a radiolabeled E2F probe. This
result is perhaps not surprising because RING3 does not possess the
protein motifs associated with chromatin-remodeling helicases or DNA
binding activities or with Rb-like "pocket proteins" that bind
directly to E2Fs. We speculate that RING3 probably exerts its effects
on already assembled transcription complexes, consistent with the lack
of effect of RING3 on enhancerless promoters (Fig. 2)
. We are currently
investigating whether RING3 phosphorylates E2F, and if so, whether this
phosphorylation affects its transcriptional activity. A recent
two-hybrid screen with yeast Taf67, a homologue of mammalian
TAFII55, identified Bdf1 and Bdf2 (the two yeast
homologues of RING3; 18
), which suggests that, in addition
to E2F, RING3 complexes may also include TAFs and histone
acetyltransferase functions.
Mammalian members of the RING3 family and its Drosophila homologue fsh contain two bromodomains of unknown function, although a link has been proposed (1) between RING3 and the bromodomain-containing protein ALL-1, a human leukemic homologue of the Drosophila developmental transcription factor encoded by the trithorax gene. Deregulation of a fsh/Trithorax pathway in flies, producing homeotic mutations, might be analogous to deregulation of a RING3/ALL-1 pathway in humans, producing leukemias. ALL-1 associates with chromatin remodeling complexes such as SWI/SNF (44) , which in turn associate with Rb (234546) . SWI/SNF also associates with mammalian brahma (2147) , which is homologous to a bromodomain protein of the Drosophila trithorax group. Mammalian brahma is necessary for Rb-dependent cell cycle arrest (23) , mediated through histone deacetylase repression of cell cycle genes (394849) . The rnc mutant of fsh controls head development in Drosophila through a homologue of Sp1 (12) ,4 which in mammals also interacts with Rb/E2F (5051) . A RING3/ALL-1 pathway might therefore influence E2Fdependent cell cycle events through SWI/SNF and its associated histone modification activities. In view of these functional links, we speculate that RING3 and fsh will also be identified in other bromodomain- or E2F-containing cocomplexes where it may be associated with transcriptional repression.
These findings of a novel kinase, which is activated upon mitogenic stimulation, transforms cells when overexpressed and transactivates cyclin gene expression through E2F sites, provide a new potential mechanism for modulation of mitogenic signals. We propose that RING3 is a member of the growing class of bromodomain-containing proteins that function in transcription complexes at the promoters of key E2F-dependent cell cycle genes. In concert with chromatin-modifying enzymes, these proteins enable the proper transcription of the genes for growth; deregulation of the RING3-dependent mitogenic signal transduction program may lead to destabilization of the cell cycle and neoplastic transformation.
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Materials and Methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Plasmid Constructs.
Expression vectors for RING3 were as described (1)
. Rb,
constitutively active MEKK, and c-Jun
NH2-terminal kinase kinase were from C.
Lange-Carter (National Jewish Center for Immunology and Respiratory
Medicine, Denver, CO), and v-abl was from R. Ren (Brandeis University,
Waltham, MA). The CRE and E2F reporters (Clontech) comprised
three copies or four copies, respectively, of a consensus CRE
(5'-TGACGTCA-3') or E2F-responsive element (5'-TTGGGCGCGTT-3') upstream
from the herpes simplex thymidine kinase promoter and the firefly
luciferase gene. The luciferase reporter for cyclin D1 (-963) was from
J. Nevins (Duke University Medical Center, Durham, NC), for cyclin A,
from J. M. Blanchard (Institut de Genetique Moleculaire, CNRS,
Montpellier, France), and for cyclin E, from P. Jansen-Durr
(Forschungsschwerpunkt Angewandte Tumorvirologie, Heidelberg,
Germany). The dhfr reporter with a wild-type E2F binding
site (5'-CGATTTCGCGCCAAA-3') and a mutated E2F binding site
(5'-CGGCCCTATATCAAA-3') were from J. Xiao (Boston University School of
Medicine, Boston, MA) and were confirmed by sequencing. Serial
truncations of recombinant histidine-tagged RING3 were constructed with
convenient restriction sites in RSETA (Invitrogen). Excised fragments
were bounded at the 3' end by EcoRI and at the 5' end by
SnaBI, BspMI, BalI,
HindIII, PstI, or AccI to give
recombinant proteins of length 660, 578, 426, 298, 218, or 179 amino
acids, respectively, not including the
NH2-terminal histidine tag.
Chromatography.
Size-exclusion chromatography was performed with Superose-6 media
packed in a Pharmacia LKB XK 16/50 column and developed with an LCC-500
Plus liquid chromatography system (Amersham Pharmacia). A sample of 0.5
ml was applied to a column of dimensions 52 x 1.8 cm and
chromatographed at 4°C and 0.1 ml/min in buffer A [50 mM
sodium chloride, 20 mM Tris-HCl (pH 7.4), 50 mM
disodium glycerol-2-phosphate, 10% glycerol, 5 mM
magnesium chloride, 5 mM disodium ATP, 2 mM
DTT, and the protease inhibitors phenylmethylsulfonyl fluoride,
pepstatin, aprotinin, and leupeptin]. Proteins were immunoprecipitated
with anti-RING3 polyclonal antibody (1)
and solubilized in
SDS sample buffer for 12% PAGE analysis. The column was calibrated
with blue dextran (2,000,000), thyroglobulin (667,000), ferritin
(440,000), catalase (232,000), aldolase (158,000), ovalbumin (44,000),
and myoglobin (18,800; Amersham Pharmacia) under the same conditions.
Autophosphorylation assay was performed as described previously
(1)
.
Rabbit polyclonal antibodies were raised against recombinant RING3 (1) and purified from antisera by protein A affinity chromatography. A rabbit antibody affinity column (1.0 ml) was constructed by coupling 0.3 mg of periodate-oxidized antibody to a hydrazide-activated polymer (AvidChrom cartridge; Sigma). The control column of nonimmune rabbit immunoglobulin (IgG) agarose was from Sigma. HeLa nuclear extract (1.0 ml) was passed through a buffer exchange column charged with ice-cold buffer B [50 mM ammonium bicarbonate (pH 7.4), 5 mM disodium ATP, 5 mM magnesium chloride, 1 mM 2-mercaptoethanol, and protease inhibitors] and then applied to the antibody affinity columns at 0.1 ml/min. The columns were washed with 20 ml of ice-cold buffer B and eluted with 10 ml of acidic elution buffer (50 mM acetic acid, pH 2.5) and then 10 ml of basic elution buffer (120 mM sodium hydroxide pH 11.5). Eluted fractions were pooled, and proteins were precipitated with tRNA carrier and 10% trichloroacetic acid, washed with ice-cold acetone, and subjected to SDS-PAGE as above.
Recombinant RING3 protein affinity chromatography was performed by binding 0.5 ml of HeLa nuclear extract (5 mg protein) and 0.1 mg wild-type histidine-tagged RING3 protein (1) to 1.0 ml of Ni-NTA agarose (Qiagen) in ice-cold buffer C [50 mM disodium glycerol-2-phosphate, 30 mM imidazole (pH 7.4), 5 mM magnesium chloride, 5 mM disodium ATP, 1 mM 2-mercaptoethanol, 0.1% Tween 20, and protease inhibitors]. Column flow-through and washes (20 ml of buffer C) were collected, and then the columns were eluted with 5 ml of ice-cold 1 M NaCl in buffer C. Eluted proteins were precipitated as above. For experiments with truncated RING3 proteins, equal numbers of moles of each derivative were applied to the columns.
For immunoblotting experiments, proteins were separated by SDS-PAGE and electroblotted to a PVDF membrane (Bio-Rad), which was blocked with 5% nonfat dry milk in 120 mM sodium chloride, 10 mM Tris-HCl (pH 8), and 0.05% Tween 20 for 1 h, and then probed overnight at 4°C with an anti-E2F rabbit polyclonal antibody that detects all known E2Fs (Santa Cruz H-111), anti-E2F-1 mouse monoclonal antibody (Santa Cruz KH95), or an anti-E2F-2 rabbit polyclonal antibody (Santa Cruz C-20) diluted 1:2000 in the same solution. Primary antibody was detected with antimouse or antirabbit IgG secondary antibody conjugated to horseradish peroxidase (Boehringer Mannheim), diluted 1:10,000. Secondary antibody was visualized with Renaissance chemiluminescence reagent plus (New England Nuclear) and XB-1 blue film (Kodak). Prestained protein markers were from Bio-Rad and Amersham Pharmacia.
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Acknowledgments |
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Footnotes |
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1 This work was supported by USPHS Grants CA75107
(to G. V. D.) and CA50459 (to D. V. F.) from the National Cancer
Institute. 
2 To whom requests for reprints should be
addressed, at Cancer Research Center, Boston University School of
Medicine, Room L910, 80 East Concord Street, Boston, MA 02118. Phone:
(617) 638-4175; Fax: (617) 638-5609; E-mail: gdenis{at}bu.edu
3 The abbreviations used are: IL, interleukin;
RING3, really interesting new gene 3; dhfr, dihydrofolate reductase;
MAPK, mitogen-activated protein kinase; MEKK, MAPK kinase kinase; CRE,
cAMP responsive element; PVDF, polyvinylidene difluoride.
4 B. Florence, personal communication.
5 G. V. Denis, unpublished data.
6 N. Guo, D. V. Faller, and G. V. Denis, J.
Cell Sci., in press.
Received for publication 2/15/00. Revision received 6/21/00. Accepted for publication 6/21/00.
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 R. J. Greenwald, J. R. Tumang, A. Sinha, N. Currier, R. D. Cardiff, T. L. Rothstein, D. V. Faller, and G. V. Denis E{micro}-BRD2 transgenic mice develop B-cell lymphoma and leukemia Blood, February 15, 2004; 103(4): 1475 - 1484. [Abstract] [Full Text] [PDF]
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) or
containing CRE (
) or E2F sites (dark gray, hatched)
and expression constructs for RING3 or empty vector control. Cells were
starved of serum overnight (-) and then stimulated with 10% serum
(for the E2F-reporter and enhancerless plasmid transfections) or 50
µM forskolin (for the CRE-reporter transfections)
overnight (+) and then harvested for assay of luciferase.
Bars, SE.
