Departments of Obstetrics and Gynecology (T.E.C., E.M.K., M.Y., A.N., D.J.W.) and Genetics and Development (D.J.W.), The Center for Reproductive Sciences (D.J.W.), The Institute of Human Nutrition (D.J.W.), and The Columbia Comprehensive Cancer Center (D.J.W.), Columbia University College of Physicians and Surgeons, New York, New York 10032; and Department of Biological Sciences (T.E.C.), Columbia University, New York, New York 10027
Address all correspondence and requests for reprints to: Debra J. Wolgemuth, Ph.D., Department of Genetics and Development, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032. E-mail: djw3{at}columbia.edu.
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ABSTRACT |
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INTRODUCTION |
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Previous studies examined the developmental regulation of expression of the Fsrg1 mRNA and the intracellular localization of the encoded protein in cultured cells (1). In addition to expression during embryogenesis, transcription of Fsrg1 was detected in the adult gonads and epithelia of reproductive tissues such as the prostate, epididymis, vas deferens in the male, and mammary gland, oviduct, uterus, and cervix in females (Ref. 1 and Trousdale, R., and D. J. Wolgemuth, in preparation). The nonuniform expression of Fsrg1 mRNA and presence of bromodomains in the encoded protein are consistent with a cell typespecific and possibly gene-specific function in transcription regulation.
Further evidence that Fsrg1 might play a role in developmental regulation of gene expression comes from copurification of one of the related mouse proteins, Fsrg4, with the RNA polymerase II (Pol II) mediator complex (10). Because mediator is a regulatory complex involved in the recruitment of the holoenzyme to the promoter by a DNA-bound activator, this report suggests that Fsrg1 may also have a role in transcription regulation, possibly making direct contact with the polymerase. Additional biochemical evidence for Fsrg1 function in regulation of Pol II transcription comes from studies of RING3, which is 95% identical in amino acid sequence to Fsrg1 (1). Overexpression of RING3 in cultured cells results in activation of cell cycle-regulatory genes, and this protein is found to copurify with the cell cycle-driving transcription factor E2F (9).
To gain insight into the function of this bromodomain protein and how its activity is regulated in these processes, we examined 1) the cellular specificity of expression of Fsrg1 mRNA and protein to obtain clues as to possible regulatory targets and physiological relevance, 2) the correlation of nuclear localization of the protein with proliferation and apoptosis in a hormonally modulated tissue system, and 3) the association of the protein with components of the transcriptional machinery and with transcriptionally active chromatin. We chose the pregnancy-induced, secreting mammary epithelia as a system in which to examine these issues. The proliferation and differentiation of mammary epithelium are under the control of steroid hormone receptors and their corresponding coactivators and corepressors, several of which are bromodomain-containing proteins (11, 12).
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RESULTS |
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Association of the Human Homolog of Fsrg1(RING3) with E2F and Components of Mediator Is Stimulated by Acetylated Histones
The binding of bromodomains specifically to histones with acetylated lysine side chains suggests that such contacts might alter the conformation of the bromodomain-containing protein, thereby affecting its association with other nuclear proteins (7, 8). To determine whether acetylated histones will affect interaction of Fsrg1/RING3 with other proteins and to determine whether Fsrg1, like Fsrg4, associates with subunits of Pol II mediator, a series of coimmunopreciptation experiments were performed using nuclear extracts from HeLa cells. To increase the efficiency of the precipitations, the cells were transfected with the RING3-HA construct mentioned above, increasing the pool of protein produced by the endogenous RING3 gene. A high-salt extraction procedure that yields extracts containing most of the nuclear proteins but not histones was used (Materials and Methods). To assay for the effects of acetylated histones on interactions between RING3 and other proteins, peptides corresponding to the amino termini of histones H3 and H4, with and without acetylated lysines, were added to the extracts before precipitation.
Previous studies using purification of protein complexes by column chromatography has shown that two of the six isotypes of the E2F protein, E2F-1 and E2F-2, are associated with RING3 in the nuclei of proliferating cells (9). IP with antibodies specific for E2F-2 brings down both the 120-kDa and 150-kDa RING3 bands in the presence of acetylated histone peptides, but not in the presence of nonacetylated peptides (Fig. 2A, top panel). The complex precipitated in the acetylhistone-containing reaction also includes the mediator subunits cyclin-dependent kinase 8 (Cdk8) (13) and thyroid receptor-associated protein 220 (TRAP220) (14), and the Pol II large subunit (Pol II ls) (Fig. 2A
, lower panels). As is the case with RING3, the mediator subunits and Pol II do not coimmunoprecipitate in the presence of nonacetylated histone peptides. These data support the observations of E2F-2-RING3 association by other investigators (9) and provide evidence that RING3 and Fsrg1 contact subunits of the Pol II mediator complex. A complementary assay for these protein-protein interactions was performed by immunoprecipitating RING3 from the nuclear extracts with anti-Fsrg1 serum, and then assaying for coprecipitation of the other proteins by immunoblot. E2F-2 is coprecipitated with RING3 specifically in the presence of the acetylated peptides. The same is true for Cdk8, TRAP220, and Pol II large subunit (Fig. 2B
). RING3 protein produced by the endogenous gene as well as the RING3-HA from the transfection construct is being assayed in these co-IP experiments because the anti-Fsrg1 can pull down both proteins (direct IP, shown in Fig. 1
). Detection of the 150-kDa band in the co-IPs verifies that protein from the endogenous gene is being assayed because this band is not produced by the RING3-HA construct (Fig. 1A
).
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To determine whether Fsrg1 is also expressed in the alveoli and whether it is variable in different physiological contexts, in situ hybridization on tissue samples from pregnant, lactating, and postlactation animals was performed. Fsrg1 mRNA was detected in the budding alveoli during pregnancy (Fig. 3A) and continued to be present as these epithelial rings expand to their maximum size during lactation (Fig. 3B
) and during postlactation involution (Fig. 3
, C and D). The signal is strongest in the budding alveoli.
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The presence of Fsrg1 in the nucleus of alveolar cells appears to be correlated with cell proliferation and the initiation of apoptosis. This correlation is strengthened by the observation that Ki-67, a nuclear protein expressed specifically in proliferating cells [during all phases of the cell cycle (23)], is detected in the nuclei of budding alveoli but not in the expanded alveolar rings during lactation (Fig. 4, D and H). Except for the fact that Ki-67 is not sequestered in the cytoplasm during lactation, this expression pattern corresponds to that observed for Fsrg1. The significance of both Fsrg1 (Fig. 4I
) and Ki-67 (Fig. 4L
) reappearing in the alveolar nuclei at 2 d post lactation as the cells are preparing for apoptosis is addressed in Discussion.
Fsrg1 Protein Localizes to the Euchromatin in the Mammary Gland Epithelial Cells
The DNA in the heterochromatin near the centromeres consists of predominantly A-T base pairs and is never transcribed, whereas the remainder of each chromosome, the euchromatin, contains the genes and averages 40% G-C base pairs (22). The DNA stain DAPI (4'6-diamidino-2-phenylindole) binds specifically to A-T base pairs, producing a much stronger signal on the heterochromatin than on the euchromatin (24). Immunostaining for a protein involved in Pol II transcription regulation is therefore expected to give a chromatin staining pattern that does not overlap the strongest DAPI signal. Consistent with the hypothesis that Fsrg1 has a role in transcription regulation, the nuclear antibody signal is restricted to the euchromatin; i.e. it does not overlap the DAPI signal (Fig. 4, A, I, and M).
To further assay whether the Fsrg1 nuclear distribution pattern in mammary epithelia is consistent with a role in transcription, Fsrg1 and Cdk8 were localized simultaneously. Cdk8 is the heterodimeric partner of cyclin C and a well characterized mediator subunit (13). A double immunofluorescence stain of alveoli from 2-d postlactation tissue shows a punctate nuclear pattern for Cdk8 (Fig. 5B), similar to that observed for Fsrg1 (Fig. 5C
), and a merge of the two signals demonstrates coincident localization of these proteins (Fig. 5D
). When any of the four proteins assayed in mammary epithelia is found in the nucleus, a punctate pattern, not overlapping the DAPI pattern, is seen. These proteins are associated with euchromatin in mammary epithelia as expected.
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The immunoblot results with the new anti-Fsrg1 antibody are consistent with results obtained with the previous antiserum (1), indicating specificity of antibody for Fsrg1, although the 150-kDa band is more prominent in the current experiments. A longer coding sequence, not represented in the cDNAs analyzed to date, may be present in RING3 and produce the 150-kDa band. Alternatively, this band might be due to formation of a covalent heterodimer of some of the RING3 molecules (that migrate at 120 kDa) with a 30-kDa protein. The 150-kDa band may be more prominent on the current immunoblots because proteinase activity was suppressed more efficiently by inhibitors in the extraction buffers.
The mediator complex is closely associated with Pol II and has a role in recruitment of the holoenzyme to promoters by activators. Mediator has been observed in yeast, Caenorhabditis elegans, Drosophila, and mouse, and mutations in some of the metazoan-specific subunits cause nonlethal phenotypes, implying cell type-specific functions (13, 14, 25, 26, 27). Our observations that the human RING3 appears to associate with the Cdk8 and TRAP220 mediator subunits, and the Pol II large subunit, in the nuclei of HeLa cells suggests that like Fsrg4, the mouse Fsrg1 functions by contacting mediator.
Evidence of a role for the Fsrg1 protein in cell cycle-regulated Pol II transcription comes from biochemical and cell culture expression studies of RING3. Overexpression of RING3 in transfected BALB/3T3 (mouse) cells results in activation of transcription of several cell cycle-regulatory genes, and column chromatography purification produces a 330-kDa complex containing RING3 and the cell cycle regulation transcription factor E2F-2 (9). E2F-2 is a sequence-specific DNA-binding activator. We have used the co-IP technique to verify the association between RING3 and E2F-2 reported by Denis and colleagues (9), and the presence of mediator subunits in the complexes immunoprecipitated by antibodies to either of these proteins provides insight into the mechanism by which RING3, and presumably Fsrg1, assists E2F-2 in activating transcription. The euchromatic localization of Fsrg1 that we have observed in the interphase nuclei of mammary epithelial cells is consistent with the hypothesized role of this protein in transcription regulation. The restricted expression of Fsrg1 mRNA and protein suggests cell type-specific and probably gene-specific functions, as has been reported for the various metazoan mediator subunits (13, 14, 25, 26, 27).
Bromodomains have been observed in components of the various complexes recruited to Pol II promoters as part of the process of transcription activation or repression, such as chromatin-remodeling enzymes, coactivators and corepressors, the basal transcription factor TFIID, which includes the TATA-binding protein, and the polymerase holoenzyme which includes the mediator complex (28). Of particular relevance to our studies on reproductive tissues is the presence of bromodomains in coactivators known to associate with steroid hormone receptors. The human homologs of the Drosophila brahma, hBRM and BRG1, associate with the glucocorticoid receptor (12, 29), and human hSNF2 functions with the estrogen and retinoic acid receptors (11). The finding that some histone acetyltransferases contain bromodomains gave rise to speculation that this motif might have the ability to bind acetylated histones. Because the addition of acetyl groups to lysine residues near the amino terminus of histones on Pol II promoters precedes recruitment of the multiprotein complexes mentioned above, it was suggested that the bromodomain might allow for tethering of histone acetyltransferases and the other complexes to the remodeled promoter. Support for this idea has been provided by the specific binding of peptides including the single bromodomain of the chromatin-remodeling enzyme p300/CBP-associated factor (7), and the double bromodomain of TAFII250 (8), to only the acetylated form of peptides corresponding to the amino termini of histones H3 and H4.
At least 43 bromodomain proteins have been identified, and most of these have only one copy of this 110-amino acid motif; however 16 of these, including the four mouse Fsrgs and their human homologs, have two copies (6). This defines a subclass of bromodomain proteins and suggests that the function of Fsrg1 may be more similar to that of the other Fsrgs than to other bromodomain proteins. Our observations that association of RING3 with E2F, mediator subunits, and Pol II is dependent on the presence of acetylated histone peptides suggests that in addition to tethering protein complexes to a remodeled promoter, the bromodomain-histone contact might have an allosteric effect that alters the conformation of the bromodomain protein in a way that strengthens interactions with other transcription-regulatory proteins. It is possible such an allosteric effect could involve the ET domain because it is known to be necessary for association of RING3 with E2F (9), and necessary and sufficient for association of yeast BDF1 with the TAFs of TFIID (4).
Regulation of nuclear import is known to control the activity of a number of sequence-specific DNA-binding proteins including NF-B, some steroid hormone receptors (30, 31), and the Drosophila heat-shock transcription factor HSF (32); however, we are not aware of any previous examples of such regulation for a putative coactivator/corepressor such as Fsrg1. The Fsrg1 protein is nuclear in the dividing cells of the budding alveoli during pregnancy but becomes restricted to the cytoplasm once the alveoli have become fully expanded, are secreting milk, and have exited the cell cycle. The active cell cycle in the budding alveoli is confirmed by the presence of Ki-67 in the nucleus, whereas the G0 status of alveolar cells during lactation is verified by the absence of this protein. It is possible that the redistribution of Fsrg1 is caused by the variation in hormone levels mentioned earlier. The absence of Fsrg1 signal in the alveolar nuclei during lactation is not due to lack of penetration of the antibody, because both CBP and HDAC1 were detected in some of these nuclei. Lack of expression of CBP and HDAC1 in 8090% of the cells during lactation is probably related to the exit from the cell cycle. Expression of HDAC1 in mouse T-cells in culture is dramatically reduced when these cells exit the cycle (21). The colocalization of Fsrg1 and the mediator subunit Cdk8 on euchromatin in 2-d postlactation alveoli, detected in the double immunostain experiment, is consistent with a transcription function for Fsrg1. The only inconsistency in a proposed transcriptional role for Fsrg1 is that 1020% of the alveolar cells have CBP and HDAC1 in their nucleus during lactation, but none show Fsrg1 in the nucleus.
The return of Fsrg1 and CBP to the nucleus in alveolar cells 2 d after the end of the lactation period may be related to the need for activation of genes involved in breaking down the basal lamina during involution and in the initiation of apoptosis. This theory is consistent with the increase in Fsrg1 mRNA abundance observed in the superior cervical ganglia just before these cells enter apoptosis in the 3-d postnatal rat (33). The apparent contradiction in the expression of a potential transcription regulator being linked to both cell proliferation and programmed cell death can be addressed as follows. There are several examples of genes that were first characterized as inducers of cell proliferation that have subsequently been found to be up-regulated as the program of apoptosis in initiated. One example is cyclin D1 in neuronal programmed cell death (33) and in mammary epithelial cells (34). The oncogne c-myc is also up-regulated in the alveoli specifically during the pregnancy growth phase and in the 2 d after the end of lactation when these cells are preparing for programmed death (18). The reappearance of the cell proliferation marker Ki-67 in the alveolar nuclei as they prepare for apoptosis may be another example of this dual role. The simultaneous presence of conflicting signals, some that promote the cell cycle and others that inhibit it, may trigger programmed cell death (33). It is possible that Fsrg1 has a role in both proliferation and induction of apoptosis in mammary epithelia, and that different levels of expression or variation in expression of other regulatory factors gives rise to these distinct phenomena. Support for our observations of regulated nuclear import of Fsrg1 is provided by report of similar regulation for the human homolog, RING3, in cultured cells. In this case, translocation to the nucleus occurs if the cells are dividing rapidly and serum is in the culture medium, but not in starved cells (35). Although this is not a developmental system, the correlation between nuclear localization and cell proliferation is consistent with our Fsrg1 results.
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MATERIALS AND METHODS |
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Antisera
Bacterial expression of recombinant Fsrg1 protein (residues 51651), antigen purification, and affinity-purification of the rabbit anti-Fsrg1 serum were performed as previously described (1). The serum was produced at Covance Laboratories, Inc. (Denver, PA). Rabbit antibodies against CBP and HDAC1, and the acetylated histone peptides (acH3: residues 718, lysine 14 acetylated; acH4: residues 220, lysine 8 acetylated) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit antibodies against Cdk8, TRAP220, and Pol II large subunit, the anti-E2F-2 monoclonal, and the nonacetylated histone peptides (H3: residues 120; H4: residues 118) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-Ki67 was from Novocastra (Burlingame, CA), and the monoclonal 12CA5 (anti-HA) was a gift of J. Kitajewski.
Nuclear Extracts and IP
Nuclear extracts were prepared from rapidly dividing untransfected HeLa cells or HeLa cells transfected with a construct containing the HA-tagged RING3 coding sequence downstream of the cytomegalovirus promoter (35). To boost RING3-HA expression, trichostatin A, a histone deacetylase inhibitor known to stimulate transcription from the cytomegalovirus promoter, was added to a concentration of 3 µM when the culture reached 80% confluency (24 h before harvesting). A 3-liter batch of cells, grown to a density of 5 x 108 cells/liter, was harvested in PBS, and cells were spun down and then resuspended in hypotonic buffer [10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; and 0.5 mM dithiothreitol (DTT)]. Cells were spun down again and resuspended in 5 volumes of fresh hypotonic buffer. The cells were incubated on ice for 10 min and then spun down at 2000 rpm in a Sorvall SS34 rotor. The cell pellet was resuspended in one volume of hypotonic buffer, transferred to a Dounce homogenizer, and homogenized until more than 90% of the cells were disrupted and nuclei released. The nuclei were then pelleted in a Sorvall SS34 at 6000 rpm for 20 min, resuspended in 5 ml of hypertonic buffer (20 mM HEPES, pH 7.9; 25% glycerol; 420 mM NaCl; 1.5 mM MgCl2; 0.2 mM EDTA; 0.5 mM DTT; and proteinase inhibitors: 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A), and incubated at 4 C on a rocker for 45 min to allow the high salt to extract proteins from the nuclei. The preparation was then dialyzed overnight against 20 mM HEPES, pH 7.9; 20% glycerol; 100 mM KCl; 0.2 mM EDTA; 0.5 mM DTT to reduce the salt concentration, and the nuclear membranes were then spun down for 5 min at 13,000 rpm in a minifuge. The supernatant, which contained 9 mg/ml of extracted nuclear proteins, was then used for IP.
For single IP reactions, 300 µg aliquots of nuclear extract were mixed with 5 µl of unpurified anti-Fsrg1 serum or mouse monoclonal 12CA5 (anti-HA), or 2 µg of anti-TRAP220 or anti-Pol II ls (both rabbit sera), and the sample was incubated at 4 C overnight. Immune complexes were then precipitated by addition of 400 µg of protein A-Sepharose to the reactions with rabbit antibody, or protein G-Sepharose to the reactions with mouse antibody, and incubation at 4 C with agitation for 6 h. Complexes were then spun down, washed in Trisbuffered saline, resuspended in sodium dodecyl sulfate gel loading buffer and boiled for 60 sec before electrophoresis.
For the co-IP reactions, trichostatin A was added to 300 nM followed by the histone peptides at 35 µM, and the mixture was incubated on ice for 60 min to allow nuclear proteins to bind the peptides (trichostatin was added first to prevent deacetylation of the peptides). Antibodies were then added as follows: 5 µl per reaction of unpurified anti-Fsrg1 serum or 2 µg of anti-E2F-2 (a mouse monoclonal), and the preparation was incubated at 4 C overnight. Immune complexes were then precipitated and prepared for electrophoresis as for the single IP reactions.
Immunostaining
Tissue sections were prepared and stained with primary antibodies as previously described (36). An Alexa Fluor 594-conjugated goat-antirabbit (Molecular Probes, Inc., Eugene, OR; excitation/emission 590/617 nm, red fluorescence), or a fluorescein isothiocyanate (FITC)-conjugated swine-antigoat (Roche Molecular Biochemicals, Indianapolis, IN), secondary antibody was used. The counterstain was DAPI. Images were produced with an LSM 510 NLO Multiphoton confocal microscope (Carl Zeiss, Thornwood, NY). A helium-neon laser (543 nm) produced the red Alexa Fluor signal, an argon laser (480 nm) produced the green FITC signal, and a titanium 2-photon laser (850 nm) produced the blue DAPI signal (converted to green in some of the images). An eight-section Z-series, at 0.8-µm intervals, was captured with each laser, the signals were merged, and a projection was made of each series.
In Situ Hybridization
Antisense RNA, labeled with 35S-UTP and synthesized with T7 polymerase from a cDNA fragment corresponding to the coding sequence between the two bromodomains of the Fsrg1 protein (nucleotides 16982340 of the sequence previously described in Ref. 1), was used to probe for the Fsrg1 transcript. Sense strand RNA, synthesized from the same template with T3 polymerase, was used as the negative control. Sections from paraffin-embedded tissue (5 µm thick) were hybridized with the probes and a signal was produced as previously described (37). The preparations were counterstained with hematoxylin and eosin, and then photographed using a combination of brightfield illumination and fluorescence, with an immunogold stain (green) filter.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Fsrg1 was approved by the Mouse Gene Nomenclature Committee as a temporary name. A new name for the Fsrg family and their human homologs, based on the bromo and ET domains, will eventually be assigned.
Abbreviations: BDF, Bromodomain factor; Cdk, cyclindependent kinase; CBP, CREB-binding protein; DAPI, 4',6-diamidino-2-phenylindole; DTT, dithiothreitol; E2F, E2 promoter binding factor; ET, extraterminal; FITC, fluorescein isothiocyanate; fsh, female steroile homeotic; Fsrg1, female sterile homeotic-related gene; HA, hemagglutinin; HDAC1, histone deacetylase; IP, immunoprecipitation; Pol II, RNA polymerase II; Pol II ls, Pol II large subunit; RING3, really interesting new gene 3; RING3-HA, hemagglutinin-tagged RING3; TAF, TATA binding protein-associated factor; TFIID, transcription factor IID; TRAP220, thyroid receptor-associated protein 220.
Received for publication December 20, 2001. Accepted for publication March 29, 2002.
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REFERENCES |
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