Reproductive Cycle Regulation of Nuclear Import, Euchromatic Localization, and Association with Components of Pol II Mediator of a Mammalian Double-Bromodomain Protein

Thomas E. Crowley, Emily M. Kaine, Manabu Yoshida, Anindita Nandi and Debra J. Wolgemuth

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIALS AND METHODS
 REFERENCES
 
Fsrg1 (female sterile homeotic-related gene 1) is the mouse homolog of the human RING3 protein, which has been shown to associate with the E2 promoter binding factor (E2F) transcription factor and to have a possible role in cell cycle-linked transcriptional regulation. The Fsrg1 protein is 60% identical in sequence to the RNA polymerase II mediator subunit Fsrg4, another member of this subfamily of double bromodomain-containing proteins that are homologs of Drosophila female sterile homeotic. Antibodies against murine Fsrg1 were generated and used in immunoblot and immunoprecipitation experiments to identify proteins interacting with Fsrg1 and RING3. In the presence of acetylated but not nonacetylated histone H3 and H4 peptides, RING3 was shown to interact with E2F, mediator components cyclin-dependent kinase 8 and thyroid receptor-associated protein 220, and the RNA polymerase II large subunit. Fsrg1 mRNA had been previously shown to be expressed at high levels in the epithelium of the adult mouse mammary gland. To determine the physiological relevance of these potential associations, we examined the patterns of expression of Fsrg1 mRNA and protein in the adult mammary epithelia during the reproductive cycle as the tissue is responding to estrogen, progesterone, and prolactin. Changes in the nuclear vs. cytoplasmic localization of Fsrg1 were observed and correlated with physiological changes in mammary gland function. The observations suggested that Fsrg1 may be involved in the transcriptional activities of genes involved in proliferation of the mammary epithelia during pregnancy and in orchestrating postlactation involution and apoptosis. Localization of Fsrg1 on euchromatin, the transcribed portion of the chromosomes, is consistent with its hypothesized function as a transcription regulator.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIALS AND METHODS
 REFERENCES
 
THE MOUSE GENE Fsrg1 (female sterile homeotic-related gene 1)1 was named for its sequence similarity to fsh (female sterile homeotic) of Drosophila (1). The homology is strongest in two internal regions of the protein coding sequence that correspond to the bromodomain motif and a segment at the carboxy-terminal end that is also present in some other bromodomain proteins and has been named the ET (extraterminal) domain. An important role for Fsrg1 in development was suggested by defects caused by mutant fsh alleles in Drosophila (2). The bromodomain is found in transcription-regulatory proteins in eukaryotes ranging from yeast to humans, including plants. The evolutionary conservation of the Fsrg1/fsh type of gene is shown by the presence of genes coding for proteins with the same arrangement of two bromodomains and an ET domain in yeast [bromodomain factors 1 and 2 (BDF1 and BDF2)] and in man (RING3) (1, 3, 4, 5, 6). The bromodomains of p300/CREB-binding protein (CBP)-associated factor (7) and TATA binding protein-associated factor II 250 (TAFII250) (8) have been shown to bind to histone peptides acetylated on lysine side chains, consistent with a role for these proteins in transcription regulation. Deletion of the RING3 ET domain inhibits this protein’s association with E2 promoter binding factor (E2F) (9), and the yeast BDF1 ET domain has been shown to be necessary and sufficient for the interaction of BDF1 with subunits of the basal transcription factor TFIID (transcription factor IID) (4).

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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIALS AND METHODS
 REFERENCES
 
Production of New Anti-Fsrg1 Serum and Test of Specificity
The anti-Fsrg1 used for experiments described previously (1) was not effective for immunostaining or immunoprecipitation (IP); therefore, a new antiserum has been generated (see Materials and Methods). The previous antibody consistently detected a strong 110-kDa band in extracts from tissues and cultured cells, and occasionally a weaker band at 150 kDa. The first test of the specificity of the new antibody was to repeat the assay for hemagglutinin-tagged RING3 (RING3-HA) in nuclear extract from transfected HeLa cells. The new antibody detects a single band in anti-Fsrg1 or anti-HA immunoprecipitates that we measure at 120 kDa (Fig. 1AGo). The discrepancy between this relative molecular mass and the 110 kDa previously reported (1) is likely due to the different sets of markers being used. The new antibody was then used to assay for the endogenous RING3 in nontransfected HeLa nuclear extract. A band at 120 kDa, comigrating with the RING3-HA band, is seen along with a 150-kDa band, and these two bands are immunoprecipitated by anti-Fsrg1 (Fig. 1BGo). Because the RING3-HA expression construct used in the current experiments is the same as that used previously (1), it provides an internal standard for the migration of the endogenous protein. The 150-kDa protein must include an amino acid sequence not present in the 120-kDa protein, because an anti-HA immunoprecipitate from a nuclear extract from RING3-HA-expressing HeLa cells gives only a 120-kDa band (Fig. 1AGo).



View larger version (38K):
[in this window]
[in a new window]
 
Figure 1. The New Anti-Fsrg1 Serum Precipitates and Detects the 120-kDa Epitope-Tagged RING3 from Transfected HeLa Nuclear Extract, and Also Precipitates and Detects Bands at 120 kDa and 150 kDa from Nontransfected HeLa Nuclear Extracts

A, Immunoblot, probed with anti-HA, of immunoprecipitates from 300-µg aliquots of nuclear extract from RING3-HA-transfected HeLa cells. The anti-Fsrg1 is a rabbit serum; therefore, protein A-Sepharose is used to pull down antibody-antigen complexes, and the two negative control lanes are precipitations with two batches of rabbit preimmune sera plus protein A. The anti-HA is a mouse monoclonal; therefore, protein G-Sepharose is used to pull down complexes, and the negative control lane is precipitation with rabbit preimmune plus protein G. RING-HA is precipitated by either of the two antisera and runs at 120 kDa. Only very weak signals are seen at this molecular mass in the control lanes. The weak band at 73 kDa in the first three lanes is probably due to nonspecific binding of protein A with a protein unrelated to Fsrg1/RING3, because it is equally strong in the negative control lanes. B, Immunoblot, probed with anti-Fsrg1, of whole nuclear extract from HeLa cells (70 µg), and IPs from 300-µg aliquots of HeLa nuclear extract. The anti-Pol II (large subunit) and anti-TRAP220 (mediator subunit) precipitations serve as negative controls to show the specificity of the anti-Fsrg1 precipitation. Only a very weak 150-kDa band is detected in these control lanes.

 
Recent results from our laboratory (Shang, E., and D. J. Wolgemuth, in preparation) and another group (5) have revealed genes coding for three proteins similar in sequence to Fsrg1 in mouse, and homologs for each of these three in the human genome. This raises the possibility that the 150-kDa band represents cross-reaction of anti-Fsrg1 with one of the other members of this family; in particular, the gene we refer to as Fsrg4 has been found to encode a protein larger than Fsrg1 (5) (this larger protein is designated MCAP in this article), and the portion of this longer sequence that can be aligned with the Fsrg1 sequence is 60% identical. To address this issue, some of the immunoblots shown in Fig. 1Go were reprobed with anti-Fsrg4. This antibody detected a 200-kDa band in mouse tissues and HeLa nuclear extract as observed previously (5) but did not detect the 120-kDa or 150-kDa bands seen with anti-Fsrg1 (data not shown). In addition, immunostaining of testis sections with each of these antibodies reveals a nonoverlapping pattern in discrete cell types (Salazar, G., and D. J. Wolgemuth, unpublished observations). The 150-kDa band is definitely not Fsrg4, and because the sequence identity between any pairwise combination of the four mouse Fsrg proteins or the four human homologs is approximately the same, i.e. 60%, it is unlikely that the 150-kDa band is Fsrg2 or Fsrg3.

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. 2AGo, 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. 2AGo, 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. 2BGo). 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. 1Go). 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. 1AGo).



View larger version (34K):
[in this window]
[in a new window]
 
Figure 2. Association of the Human Homolog of Fsrg1 with E2F, Pol II, and Mediator Is Stimulated by Acetylated Histone Peptides

Immunoprecipitates from aliquots of RING3-HA-transfected HeLa nuclear extract (300 µg), preincubated with nonacetylated peptides (H3 and H4) or acetylated peptides (acH3 and acH4), were assayed for co-IP by immunoblot. Positive control lanes contained 70 µg of unprecipitated nuclear extract. A, IP with anti-E2F-2, assay for co-IP with anti-Fsrg1, anti-Cdk8, anti-TRAP220, and anti-Pol II ls. The bands detected migrate at 120 kDa and 150 kDa (RING3), 60 kDa (Cdk8), 217 kDa (TRAP220), and 217 kDa (Pol II ls). B, IP with anti-Fsrg1, assay for co-IP with anti-E2F-2, anti-Cdk8, anti-TRAP220, and anti-Pol II ls. E2F-2 migrates at 65 kDa. The bottom panel in each column shows a probe for the protein directly contacted by the antibody used for IP (E2F-2 in column A and RING3 in column B), to verify that efficiency of the direct IP is the same regardless of which histone peptide is present. The band above E2F-2 in the bottom panel in column A (IgG hc) is due to cross-reaction of the secondary antibody used for detection on the immunoblot with the IgG heavy chain of the antibody used for IP.

 
Fsrg1 mRNA Is Expressed in Mammary Epithelia During Pregnancy, Lactation, and Involution, and Nuclear Import of the Protein Is Regulated
Fsrg1 had previously been reported to be expressed at high levels in the adult mouse mammary epithelium (1). The mammary epithelium undergoes dramatic morphological and physiological changes during pregnancy, lactation, and the postlactation involution process, providing a good system in which to examine expression and intracellular localization of a putative transcription regulator such as the Fsrg1 protein in vivo. A histological section of mammary gland from a virgin mouse shows predominantly fat cells with sparsely distributed, tubular milk duct epithelia. These ducts carry the milk to the nipple during lactation; however, it is the alveoli, a distinct type of epithelia that bud from the walls of the duct epithelia during pregnancy, that secrete the milk. By the end of the 18-d pregnancy period, the alveoli dominate the gland, having replaced most of the fat cells. A section of a gland at this point in the reproductive cycle shows the alveoli as a network of adjacent epithelial rings, and because the cell volume is greater than that of the duct epithelial cells, the nuclei in the alveoli are spaced farther apart than those in the ducts. This characteristic is particularly advantageous for examining nuclear vs. cytoplasmic localization of proteins. During the 21-d lactation period, most of the alveolar cells exit the cell cycle (15, 16), and the morphology of this tissue remains static while milk is being secreted. Between 2 and 5 d after the end of lactation, the basal lamina that has been maintaining the shape of the alveolar epithelium is broken down by secreted proteinases, resulting in the involution of these structures, concominant with the entry of the alveolar cells into programmed cell death (apoptosis). Fat cells reappear to fill the void left by the loss of alveoli, and the gland eventually returns to the virgin morphology. Hormones play a key role in these changes in the alveoli, with the pregnancy specific prolactin and progesterone, in addition to estrogen, stimulating the proliferation, whereas a rapid drop in progesterone and estrogen at parturition allows prolactin to induce lactation. Secretion of the extracellular matrix protein tenascin is one of the signals that triggers involution and apoptosis (16, 17, 18, 19).

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. 3AGo) and continued to be present as these epithelial rings expand to their maximum size during lactation (Fig. 3BGo) and during postlactation involution (Fig. 3Go, C and D). The signal is strongest in the budding alveoli.



View larger version (89K):
[in this window]
[in a new window]
 
Figure 3. Fsrg1 mRNA Is Expressed in Mammary Alveoli Throughout the Reproductive Cycle

In situ hybridization with a radioactive antisense RNA probe was used to detect the message in budding alveoli from d 12 of pregnancy (A), fully expanded alveolar rings from d 12 of lactation (B), expanded rings at 1 d post lactation (C), and involuting epithelia at 2 d post lactation (D). A lighting combination that produces a green fluorescent hybridization signal and shows the blue nuclear counterstain (hematoxylin) and red cytoplasmic counterstain (eosin) was used for photography. Hybridizations to similar sections with a sense strand probe produced much less signal than any of the preparations shown here.

 
To determine whether the Fsrg1 message is translated in the alveolar cells during the reproductive cycle and to assay for regulation of intracellular localization of Fsrg1 protein, immunofluorescent staining was performed on tissue sections similar to those described above for the in situ hybridizations. As the alveoli are budding during pregnancy, a punctate nuclear signal is seen for Fsrg1 (Fig. 4AGo). When the alveoli become fully expanded into rings and lactation begins, Fsrg1 continues to be expressed in all the cells; however, it is sequestered in the cytoplasm (Fig. 4EGo). Two days after lactation has ceased, Fsrg1 returns to the nucleus in the alveolar cells (Fig. 4IGo) and remains there as involution proceeds (Fig. 4MGo).



View larger version (69K):
[in this window]
[in a new window]
 
Figure 4. Fsrg1 Protein Is Expressed in Mammary Alveoli Throughout the Reproductive Cycle but Restricted to the Cytoplasm During Lactation

Immunostaining of alveoli at d 12 of pregnancy (A–D), d 12 of lactation (E–H), d 2 post lactation (I–L), and d 5 post lactation (M–P). Primary antibody was anti-Fsrg1 (A, E, I, and M), anti-CBP (B, F, J, and N), anti-HDAC1 (C, G, K, and O), or anti-Ki-67 (D, H, L, and P), and in each case an Alexa Fluor 594-conjugated secondary antibody (red signal) was used. Preparations were counterstained with the heterochromatin-specific dye DAPI and the blue signal was converted to green to provide better contrast with the antibody signals.

 
To verify that the nuclear staining pattern observed for Fsrg1 in alveolar cells is, in fact, chromatin localization, and to determine the specificity of Fsrg1 nuclear import, alveoli at the various stages of the reproductive cycle were stained for the chromatin remodeling enzymes CREB-binding protein (CBP) (20) and histone deacetylase 1 (HDAC1) (21). Because CBP has histone acetyltransferase activity, CBP and HDAC1 have opposing functions, but transcription regulation of eukaryotic genomes involves a balance of these activities. Activation of a promoter requires acetylation of a subset of the lysines on the histones, whereas repression is affected by deacetylation of the majority of the histone lysines. Thus, both CBP and HDAC1 are expected to be present on chromatin in cells that are transcribing some genes while keeping others repressed (22). Like Fsrg1, CBP and HDAC1 are localized to the nucleus in a punctate pattern in all cells of the budding alveoli during pregnancy (Fig. 4Go, B and C); however, in the expanded rings during lactation, 80–90% of the cells no longer express either protein. In those cells that continue to express these proteins, the punctate nuclear pattern is still observed (Fig. 4Go, F and G). None of the alveolar cells show cytoplasmic sequestration of the chromatin remodeling enzymes as is observed for Fsrg1 during lactation; however, both CBP and HDAC1 reappear in all alveolar nuclei at the same timepoint as Fsrg1, i.e. 2 d post lactation (Fig. 4Go, J and K). Nuclear localization of CBP and HDAC1 continues as involution proceeds (Fig. 4Go, N and O).

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. 4Go, 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. 4IGo) and Ki-67 (Fig. 4LGo) 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. 4Go, 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. 5BGo), similar to that observed for Fsrg1 (Fig. 5CGo), and a merge of the two signals demonstrates coincident localization of these proteins (Fig. 5DGo). 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.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 5. Fsrg1 Colocalizes with Mediator Subunit Cdk8 on Euchromatin in Postlactation Alveoli

A portion of a mammary alveolus at 2 d post lactation was stained with anti-Fsrg1 as in Fig. 4Go (red signal), and with anti-Cdk8 [detected with an FITC-conjugated (green signal) secondary antibody]. The two antibody signals are shown in separate panels here: anti-Cdk8 (B) and anti-Fsrg1 (C). The heterochromatin-specific counterstain DAPI (A) is left in the natural blue, and the merge of all three signals (D) shows a yellow nuclear signal resulting from the overlap of the red Fsrg1 and green Cdk8 signals.

 
DISCUSSION
The presence of bromodomains in the Fsrg1 protein, its sequence similarity to a mediator subunit (Fsrg4), and the expression of Fsrg1 mRNA in reproductive epithelia known to respond to hormones provided several clues as to the function and regulation of this protein. These clues prompted experiments that have demonstrated association of Fsrg1/RING3 with E2F-2, mediator subunits, and Pol II in an acetylhistone-dependent manner. The association of Fsrg1 protein with E2F-2, the high level of Fsrg1 mRNA in the budding mammary alveoli during pregnancy, and the exclusion of the protein from the nucleus in the G0 alveolar cells during lactation are all consistent with a role for Fsrg1 in cell cycle progression. The return of Fsrg1 to the nucleus of mammary alveolar cells just before involution and apoptosis hints at a role in initiation of the programmed cell death pathway. In the cells that import Fsrg1 into the nucleus, this protein is localized to the transcribed portion of the chromatin, consistent with the role in transcription regulation predicted by the protein-protein interactions mentioned above.

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-{kappa}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 80–90% 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 10–20% 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIALS AND METHODS
 REFERENCES
 
Source of Cell Lines and Tissues
HeLa is a human cervical carcinoma cell line. The synthesis of the RING3-HA expression construct and introduction into HeLa cells was done previously in this laboratory (1). Mice were of the CD1 strain (Charles River Laboratories, Inc., Wilmington, MA) and were 2 months old at the beginning of pregnancy.

Antisera
Bacterial expression of recombinant Fsrg1 protein (residues 51–651), 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 7–18, lysine 14 acetylated; acH4: residues 2–20, 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 1–20; H4: residues 1–18) 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 1698–2340 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.


    ACKNOWLEDGMENTS
 
We thank Xiangyuan Wang for preparation of tissue sections, Enyuan Shang for comments on the manuscript, and Theresa Swayne for assistance with the confocal microscope.


    FOOTNOTES
 
This work was supported in part by grants from the Vidda Foundation and the NIH (PO1 DK-54057).

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. Back

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIALS AND METHODS
 REFERENCES
 

  1. Rhee K, Brunori M, Besset V, Trousdale R, Wolgemuth DJ 1998 Expression and potential role of Fsrg1, a murine bromodomain-containing homologue of the Drosophila gene female sterile homeotic. J Cell Sci 111:3541–3550[Abstract/Free Full Text]
  2. Haynes SR, Mozer BA, Bhatia-Dey N, Dawid IB 1989 The Drosophila fsh locus, a maternal effect homeotic gene, encodes apparent membrane proteins. Dev Biol 134:246–257[Medline]
  3. Haynes SR, Dollard C, Winston F, Beck S, Trowsdale J, Dawid IB 1992 The bromodomain: a conserved sequence found in human, Drosophila and yeast proteins. Nucleic Acids Res 20:2603[Medline]
  4. Matangkasombut O, Buratowski RM, Swilling NW, Buratowski S 2000 Bromodomian factor 1 corresponds to a missing piece of yeast TFIID. Genes Dev 14:951–962[Abstract/Free Full Text]
  5. Dey A, Ellenberg J, Farina A, Coleman AE, Maruyama T, Sciortino S, Lippincott-Schwarz J, Ozato K 2000 A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G2-to-M transition. Mol Cell Biol 20:6537–6549[Abstract/Free Full Text]
  6. Florence B, Faller DV 2001 You BET-CHA: a novel family of transcriptional regulators. Front Biosci 6:1008–1018
  7. Dhalluin C, Carlson JE, Zeng L, He C, Aggarwal AK, Zhou MM 1999 Structure and ligand of a histone acetyltransferase bromodomain. Nature 399:491–496[CrossRef][Medline]
  8. Jacobson RH, Ladurner AG, King DS, Tjian R 2000 Structure and function of a human TAFII250 double bromodomain module. Science 288:1422–1425[Abstract/Free Full Text]
  9. Denis GV, Vaziri C, Guo N, Faller DV 2000 RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F. Cell Growth Differ 11:417–424[Abstract/Free Full Text]
  10. Jiang YW, Veschambre P, Erdjument-Bromage H, Tempst P, Conaway JW, Conaway RC, Kornberg RD 1998 Mammalian mediator as an end-point of signal transduction pathways. Proc Natl Acad Sci USA 95:8538–8543[Abstract/Free Full Text]
  11. Chiba H, Muramatsu M, Nomoto A, Kato H 1994 Two human homologues of Saccharomyces cerevisiae SWI2/SNF2 and Drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the retinoic acid receptor. Nucleic Acids Res 22:1815–1820[Abstract]
  12. Fryer CJ, Archer TK 1998 Chromatin remodeling by the glucocorticoid receptor requires the BRG1 complex. Nature 393:88–91[CrossRef][Medline]
  13. Boyer TG, Martin MD, Lees E, Ricciardi RP, Berk AJ 1999 Mammalian Srb/mediator complex is targeted by adenovirus E1A protein. Nature 399:276–279[CrossRef][Medline]
  14. Ito M, Yuan CX, Okano HJ, Darnell RB, Roeder RG 2000 Involvement of the TRAP 220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action. Mol Cell 5:683–693[Medline]
  15. Gardner HP, Belka GK, Wertheim GBW, Hartman JL, Ha SI, Gimooty PA, Marquis ST, Chodosh LA 2000 Developmental role of the SNF1-related kinase Hunk in pregnancy-induced changes in the mammary gland. Development 127:4493–4509[Abstract/Free Full Text]
  16. Wiesen J, Werb Z 2000 Proteinases, cell cycle regulation, and apoptosis during mammary gland involution. Mol Reprod Dev 56:534–540[CrossRef][Medline]
  17. Topper YJ, Freeman CS 1980 Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049–1106[Free Full Text]
  18. Boudreau N, Werb Z, Bissell MJ 1996 Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle. Proc Natl Acad Sci USA 93:3509–3513[Abstract/Free Full Text]
  19. Lund LR, Bjorn SF, Sternlicht MD, Nielsen BS, Solberg H, Usher PA, Osterby R, Christensen IJ, Stephens RW, Bugge TH, Dano K, Werb Z 2000 Lactational competence and involution of the mouse mammary gland require plasminogen. Development 127:4481–4492[Abstract/Free Full Text]
  20. Goodman RH, Smolik S 2000 CBP/p300 in cell growth, transformation and development. Genes Dev 14:1553–1577[Free Full Text]
  21. Bartl S, Taplick J, Lagger G, Khier H, Kuchler K, Seiser C 1997 Identification of mouse histone deacetylase 1 as a growth factor-inducible gene. Mol Cell Biol 17:5033–5043[Abstract]
  22. Winston F, Allis CD 1999 The bromodomain: a chromatin-targeting module? Nat Struct Biol 6:601–604[CrossRef][Medline]
  23. Endl E, Gerdes J 2000 The Ki-67 protein: fascinating forms and an unknown function. Exp Cell Res 257:231–237[CrossRef][Medline]
  24. Schubeler D, Francastel C, Cimbora DM, Reik A, Martin DIK, Groudine M 2000 Nuclear localization and histone acetylation: a pathway for chromatin opening and transcriptional activation of the human ß-globin locus. Genes Dev 14:940–950[Abstract/Free Full Text]
  25. Myers LC, Gustafsson CM, Bushnell DA, Lui M, Erdjument-Bromage H, Tempst P, Kornberg RD 1998 The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain. Genes Dev 12:45–54[Abstract/Free Full Text]
  26. Zhang H, Emmons S 2000 A C. elegans mediator protein confers regulatory selectivity on lineage-specific expression of a transcription factor gene. Genes Dev 14:2161–2172[Abstract/Free Full Text]
  27. Boube M, Faucher C, Joulia L, Cribbs DL, Bourbon, HM 2000 Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification. Genes Dev 14:2906–2917[Abstract/Free Full Text]
  28. Jeanmougin F, Wurtz JM, LeDouarin B, Chambon P, Losson R 1997 The bromodomain revisited. Trends Biochem Sci 22:151–153[CrossRef][Medline]
  29. Muchardt C, Yaniv M 1993 A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor. EMBO J 12:4279–4290[Abstract]
  30. Kaffman A, O’Shea EK 1999 Regulation of nuclear localization: a key to a door. Annu Rev Cell Dev 15:291–339[CrossRef][Medline]
  31. Freedman LP 1999 Increasing the complexity of coactivation in nuclear receptor signaling. Cell 97:5–8[Medline]
  32. Wang Z, Lindquist S 1998 Developmentally regulated nuclear transport of transcription factors in Drosophila embryos enable the heat-shock response. Development 125:4841–4850[Abstract/Free Full Text]
  33. Wang S, DiBenedetto AJ, Pittman RN 1997 Genes induced in programmed cell death of neuronal PC12 cells and developing sympathetic neurons in vivo. Dev Biol 188:322–336[CrossRef][Medline]
  34. Han EKH, Begemann M, Sgambato A, Soh JW, Daki Y, Xing WQ, Liu W, Weinstein IB 1996 Increased expression of cyclin D1 in a murine mammary epithelial cell line induces p27kip1, inhibits growth and enhances apoptosis. Cell Growth Differ 7:699–710[Abstract]
  35. Guo N, Faller DV, Denis GV 2000 Activation-induced nuclear translocation of RING3. J Cell Sci 113:3085–3091[Abstract/Free Full Text]
  36. Zhang Q, Wang XY, Wolgemuth DJ 1999 Developmentally regulated expression of cyclin D3 and its potential in vivo interacting proteins during murine gametogenesis. Endocrinology 140:2790–2800[Abstract/Free Full Text]
  37. Rhee K, Wolgemuth DJ 1995 Cdk family genes are expressed not only in dividing but also in terminally differentiating mouse germ cells, suggesting their possible function during both cell division and differentiation. Dev Dyn 204:406–420[Medline]