Departments of Medicine and Cell Biology (J.F.E., M.A.K.-C., T.C.V., R.N.D.), University of Virginia Health System, Charlottesville, Virginia 22908-0578; and Metabolic Research Unit (F.S.), University of California, San Francisco, California 94143-0540
Address all correspondence and requests for reprints to: Richard N. Day, Ph.D., Department of Medicine, Box 800578, University of Virginia Health System, Charlottesville, Virginia 22908-0578. E-mail: rnd2v{at}virginia.edu.
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ABSTRACT |
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INTRODUCTION |
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We have investigated how the subnuclear localization of transcription factors in anterior pituitary cells was correlated with their cooperative interactions in the regulation of prolactin (PRL) gene expression. The transcription of the PRL gene is restricted to lactotropes, making it an excellent model for cell type-specific gene regulation. The pituitary-specific homeodomain (HD) protein Pit-1, which is required for the development of the lactotrope, somatotrope, and thyrotrope cell lineages, is also necessary for the transcriptional regulation of the genes encoding the hormone products of these cell types. Because of its central role in the genesis of these pituitary cell types, patients that have inactivating mutations in Pit-1 develop combined pituitary hormone deficiency (CPHD), a disease characterized by the lack of the hormones produced by these cells (6, 7).
The promoter and enhancer regions of the PRL gene direct its pituitary cell-specific expression and contain multiple binding sites for Pit-1 (8). Pit-1, alone, is necessary, but not sufficient, for PRL gene transcription, and it is the interplay between Pit-1 and other gene-regulatory proteins that controls the expression of PRL (9). In this regard, several other transcription factors, including the CCAAT/enhancer binding protein- (C/EBP
), are known to participate in the regulation of PRL gene expression. C/EBP
is a member of the basic region-leucine zipper (B-ZIP) family of transcription factors (10) and controls the expression of genes involved in terminal differentiation and energy metabolism (10, 11). C/EBP
controls the transcription of both the PRL and GH genes by binding to promoter elements adjacent to critical binding sites for Pit-1 (12, 13, 14).
Here we investigated the role of intranuclear compartmentalization of Pit-1 and C/EBP in their cooperative activation of PRL gene transcription. Fluorescence imaging of pituitary cells expressing either C/EBP
or Pit-1 as fusions to green fluorescent protein (GFP) revealed that each had a distinct pattern of distribution relative to other nuclear markers. However, when coexpressed in the same cells as fusions to FP color variants, we observed that C/EBP
was recruited to the intranuclear sites occupied by Pit-1. Mutational studies indicated that the HD of Pit-1 was required for this recruitment activity. Further, we found that a point mutation in the Pit-1 HD also failed to recruit C/EBP
. This Pit-1 mutant, which acts as a dominant inhibitor of PRL gene expression and is associated with CPHD syndrome in humans, became associated with C/EBP
in regions of centromeric heterochromatin. Together these studies provide evidence for an organizational role of Pit-1 in directing other cooperating factors to particular intranuclear sites. The disruption of the organizational function of Pit-1 by the CPHD point mutation suggests that intranuclear location may be a critical determinant of transcriptional outcome. The ability of Pit-1, C/EBP
, and other factors involved in pituitary-specific gene expression to assemble at particular subnuclear sites may constitute an overlooked epigenetic component of the combinatorial code responsible for cell-specific gene transcription.
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RESULTS |
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To assess the function of the GFP-tagged C/EBP and Pit-1, we measured their ability to bind to appropriate DNA-response elements. Extracts were prepared from GHFT15 cells expressing C/EBP
and GFP-C/EBP
or from 3T3 -L1 cells expressing GFP-Pit-1. These protein extracts were then incubated with the indicated DNA-binding sites and subjected to EMSA. The results shown in Fig. 2C
demonstrated that C/EBP
(lane 1) or GFP-C/EBP
(lane 6) each formed a single DNA-protein complex with the consensus C/EBP
DNA-response element (16). Competition by 3- to 100-fold excess unlabeled oligonucleotide demonstrated that the DNA-protein complexes were probe-specific (Fig. 2C
, wedges). Incubation with an antibody directed against C/EBP
resulted in decreased mobility of the DNA-protein complex, confirming that C/EBP
was present in these complexes (Fig. 2C
, lanes 5 and 10). Similarly, GFP-Pit-1 was found to bind specifically to the PRL gene promoter 3P Pit-1 DNA-response element (Fig. 2D
). Consistent with previous studies of Pit-1 DNA-binding activity (8), we observed two distinct DNA-protein complexes formed by GFP-Pit-1 (arrows, Fig. 2D
), as well as a complex containing ETS-family proteins that interact with the 3P DNA element in conjunction with Pit-1 (17, 18). Competition with an excess of unlabeled 3P DNA (wedge, lanes 24) and an antibody directed against Pit-1 showed these complexes each contained GFP-Pit-1. These results demonstrated that the GFP tag did not alter the DNA binding characteristics of either C/EBP
or Pit-1.
Coincident Subnuclear Localization of Endogenous and GFP-Tagged C/EBP and Pit-1
C/EBP is present in rat pituitary cell lines that secrete GH and PRL, but is absent from the mouse pituitary progenitor GHFT15 cells, which do not express these hormones (14). We showed that the expression of exogenous C/EBP
in GHFT15 cells leads to activation of a cotransfected GH gene promoter (14) and PRL gene promoter (Fig. 1A
), and results in inhibition of cell proliferation (19). Because the GHFT15 cells do not express C/EBP
, we characterized the intranuclear positioning of endogenous C/EBP
in a different mouse cell line. Earlier studies showed that C/EBP
was expressed in mouse 3T3-L1 cells that were induced to differentiate into adipocytes, where it plays a key role in the adipogenic cascade (20, 21). Here, the endogenous C/EBP
was detected by immunohistochemical staining in the nuclei of 3T3-L1 cells that had differentiated to adipocytes by hormone treatment (Fig. 3A
). To provide a marker for chromatin structure, the cells were stained with the blue fluorescent DNA dye, Hoechst 33342 (H33342). The H33342 preferentially binds to the tracts of satellite DNA repeats located at centromere regions of interphase chromosomes in mouse cells (14, 22, 23). The results showed that the endogenous C/EBP
was highly concentrated in large foci in the nuclei of the induced 3T3-L1 cells (Fig. 3A
). Overlaying the red fluorescent C/EBP
image with the blue fluorescent H33342 image resulted in a purple-colored image (Fig. 3A
, merge) indicating that the distributions of C/EBP
and H33342-stained DNA were identical. No C/EBP
was detected in undifferentiated 3T3-L1 cells (data not shown). A very similar pattern of intranuclear distribution was observed for GFP-C/EBP
when expressed in the 3T3-L1 preadipocyte cells (Fig. 3A
, right panel).
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We then used fluorescence microscopy to visualize the distribution of these GFP-fusion proteins relative to chromatin in the nucleus of living GHFT15. As we reported previously (14, 24), GFP-C/EBP was concentrated in regions of centromeric chromatin that were preferentially stained with the H33342 dye (Fig. 3C
). There was also a significant amount of GFP-C/EBP
localized to regions outside of the heterochromatin foci stained with H33342 (Fig. 3C
, intensity profile). In contrast, when only the carboxy-terminal B-ZIP region of C/EBP
fused to GFP (GFP-C/EBP
244) was expressed in GHFT15 cells, it was predominately localized to the heterochromatin foci (Fig. 3D
, intensity profile). This result suggests that activities associated with the amino-terminal transactivation domain could function to direct C/EBP
into other nuclear domains. As was shown for the endogenous Pit-1 protein (Fig. 3B
), the expressed GFP-Pit-1 adopted a web-like pattern of nuclear distribution and did not concentrate in the regions of heterochromatin that were preferentially stained by H33342 (Fig. 3E
). Therefore, the intranuclear locations adopted by the GFP-tagged C/EBP
and Pit-1 fusion proteins faithfully mimicked the intranuclear locations of their endogenous counterparts.
Pit-1 Coexpression Dispersed C/EBP Away from Peri-Centromeric Heterochromatin
We then determined whether the coexpression of Pit-1 and C/EBP in the same cell might influence their individual patterns of intranuclear distribution. For these experiments we used the promyelocytic leukemia (PML) protein as a marker for nuclear structure. PML is a member of a family of proteins that form well defined 0.5-µm subnuclear structures called nuclear bodies (25). The sequence encoding PML was fused in-frame to that of the Discosoma sp. red fluorescent protein (RFP) (26). We demonstrated previously that the expressed PML-RFP localized to discrete nuclear bodies (24) and that it colocalized with endogenous PML in HeLa cells (not shown).
When GHFT15 cells were cotransfected with plasmids encoding GFP-Pit-1, blue fluorescent protein (BFP)-C/EBP, and PML-RFP, we observed that the C/EBP
was no longer localized to regions of centromeric heterochromatin, but was instead localized in a reticular pattern that was coincident with GFP-Pit-1 (Fig. 4A
). PML-RFP remained localized to the well defined nuclear bodies, arguing against a restructuring of the nucleus in these transfected cells. Importantly, not all factors that were coexpressed with BFP-C/EBP
influenced its subnuclear localization. When BFP-C/EBP
was coexpressed with a functional estrogen receptor
(ER
)-GFP fusion protein (human ER-GFP; Ref. 27), the two fusion proteins distributed independently, with little overlap in their subnuclear localization (Fig. 4B
). Again, the PML-RFP was localized to nuclear bodies, and these were distinct from the foci occupied by BFP-C/EBP
. Together, these results provide evidence for the specific recruitment of C/EBP
to the intranuclear sites occupied by the coexpressed Pit-1.
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The Pit-1 HD Is Required for the Recruitment of C/EBP from Heterochromatin
Because the conserved Pit-1 HD was shown to mediate interactions with other proteins (28, 29), we examined what effect a deletion within the HD had on recruitment of C/EBP. Deletion of the carboxy-terminal portion of the Pit-1 HD (amino acid residues 255291, Pit-1
255291) abolished the ability of the protein to bind to a Pit-1 DNA element from the PRL promoter (Fig. 5A
, right panel). Imaging cells expressing GFP-Pit-1
255291 revealed a diffuse pattern of intranuclear distribution distinct from the reticular pattern observed for the intact GFP-Pit-1 (Fig. 5A
). When GFP-Pit-1
255291 was coexpressed with BFP-C/EBP
, the BFP-C/EBP
was not relocalized by the truncated Pit-1 protein (Fig. 5B
). Instead, the GFP-Pit-1
255291 tended to accumulate in the sites occupied by BFP-C/EBP
(Fig. 5B
, merge). This result indicates that domains other than the Pit-1 HD may also interact with C/EBP
or other associated coregulatory factors, but that the recruitment activity of Pit-1 for C/EBP
required the intact HD.
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A mutant Pit-1 protein with substitution of alanine for arginine 271 (Pit-1R271A) was prepared, and the dominant inhibitory activity of this protein was determined. The results in Fig. 6 demonstrate the ability of Pit-1R271A to block the cooperative actions of Pit-1 with C/EBP
at the rPRL promoter in transfected HeLa cells (Fig. 6A
). A GFP fusion to Pit-1R271A was made to examine its intranuclear distribution and ability to interact with C/EBP
. The DNA-binding activity of this fusion protein was assessed by EMSA using extracts prepared from transfected HeLa cells. The results shown in Fig. 6B
demonstrated that GFP-Pit-1R271A mutant bound with high specificity to the PRL gene 1P Pit-1 DNA element, but only a single, specific DNA-protein complex was observed (arrow, Fig. 6B
). This complex was cleared from the reaction by incubation with an antibody that disrupts Pit-1 binding to DNA. These results are in accord with an earlier study indicating that the CPHD Pit-1R271W bound to Pit-1 DNA elements predominately as a monomer whereas wild-type Pit-1 bound both as a monomer and a dimer (31). As a further measure of function, reporter gene analysis demonstrated that GFP-Pit-1R271A retained the dominant inhibitory activity. When expressed in HeLa cells, GFP-Pit-1 strongly induced the luciferase reporter gene linked to the -204 rPRL promoter, and increasing concentrations of the plasmid encoding the GFP-Pit-1R271A inhibited this activity (Fig. 6C
). Western blot analysis (inset, Fig. 6C
) showed these GFP-tagged proteins were expressed equivalently after transfection of identical amounts of expression vector. Together, these results confirmed the dominant negative attributes of Pit-1 mutated at amino acid residue 271 and showed that mutation at this residue blocked the cooperative activation of the PRL promoter by Pit-1 and C/EBP
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DISCUSSION |
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Cooperative Interactions Between Pit-1 and C/EBP Induce PRL Transcription
Pit-1 cooperates with other transcription factors to regulate both the basal expression and hormonal regulation of the pituitary-specific PRL gene (9, 13, 17, 18, 28, 29, 35, 36, 37, 38). We previously demonstrated that Pit-1 and C/EBP cooperate in the regulation of GH transcription (12, 14). Similarly, Jacob and Stanley (13) observed the interaction of Pit-1 and C/EBP
at the PRL gene promoter. Here, we showed that expression of C/EBP
in pituitary GHFT15 cells, which have low levels of Pit-1 and are devoid of endogenous C/EBP
, could induce rPRL promoter activity (Fig. 1A
). This activity required the PRL promoter element between -97 to -91 bp (Fig. 1A
), a site implicated in earlier studies as necessary for both basal and hormone-stimulated promoter activities (13, 36, 37, 38). A functional interaction between C/EBP
and Pit-1 was shown in the nonpituitary human HeLa cells, where the coexpressed proteins cooperated in the activation of PRL transcription (Fig. 1B
). Together, our results suggest a cooperative interaction between C/EBP
and Pit-1, which may involve the physical association of these transcription factors. Our recent studies using fluorescence resonance energy transfer microscopy supports this view (39).
The Intranuclear Positioning of C/EBP
Many recent studies suggest that transcription factors and other regulatory proteins assemble in higher order transcriptional complexes at specific subnuclear sites (39A, 40, 41, 42, 43). We observed here that the endogenous C/EBP in differentiated 3T3-L1cells was preferentially localized to regions of centromeric heterochromatin (Fig. 3
). Similarly, we found GFP-C/EBP
localized to these same subnuclear sites when expressed in either mouse 3T3-L1 preadipocyte cells or mouse GHFT15 pituitary progenitor cells (Fig. 3
and Ref. 14). Our earlier studies demonstrated that the subnuclear distribution of coregulatory proteins, including the CREB binding protein, the TATA binding protein, and acetylated histone H3, was altered upon C/EBP
expression such that they relocalized to the centromeric heterochromatin domains occupied by C/EBP
(14 43A ). In addition, we have found that point mutants of C/EBP
, which fail to concentrate at the sites of centromeric heterochromatin, are more transcriptionally active (Bo Wu, R. N. D., and F. Schaufele, unpublished data). This observation favors a model in which C/EBP
may be sequestered at transcriptionally inactive heterochromatin (14). The C/EBP family of proteins are known to regulate cell proliferation and differentiation (10, 11). For example, Tang and Lane (20) showed that the centromeric localization of the endogenous C/EBP
in mouse adipocytes functioned to control cell proliferation during adipocyte terminal differentiation. The GHFT15 cells used in our studies have the phenotype of the progenitor of the somatolactotrope cell lineage (15), and the targeting of GFP-C/EBP
to centromeres may be indicative of their stage in pituitary differentiation. These results indicate that the targeting of C/EBP
to regions of centromeric heterochromatin may be intrinsic to its biological functions.
The centromeric heterochromatin in mammalian cells consists of arrays of tandemly repeated satellite DNA that is assembled into higher order chromatin structure (22). These long arrays of repeated satellite DNA are present in all mammalian cells, but mouse cells possess large blocks of highly condensed centromeric heterochromatin that is readily visualized by staining with Hoechst DNA dyes (22, 23). Our ability to detect the positioning of C/EBP at these subnuclear sites was possible because these regions are particularly well defined in mouse cells (44, 45). In this regard our observations of the positioning of C/EBP
relative to centromeric heterochromatin could be a property that is unique to mouse cells. However, recent studies indicate that the structure of the centromeres is highly conserved across species, particularly at the level of the protein components of the kinetochore (46, 47). For example, the evolutionarily conserved histone-like centromere protein A is involved in the assembly of centromeric DNA into higher order structure in a variety of different organisms (47). Furthermore, C/EBP
was shown to be a strong inhibitor of cell proliferation in both mouse and human cell lines (20, 49), and we observed previously that the expression of C/EBP
in pituitary GHFT15 cells also inhibited cell growth (19). This similar activity in cell lines from different species argues against a mouse cell-specific function of C/EBP
. The well defined centromeric regions that are visible in the mouse cell nucleus, however, have enabled us to study the role of C/EBP
subnuclear distribution in its transcriptional and antiproliferative activities (14, 19, 24).
C/EBP Is Recruited to the Intranuclear Sites Occupied by Pit-1
These earlier findings prompted us to investigate whether the discrete intranuclear positioning of C/EBP in the mouse pituitary cells could be influenced by Pit-1. We found that the low level of endogenous Pit-1 protein in GHFT15 cells was distributed in a reticular pattern throughout the nucleus, but was not concentrated at sites of centromeric heterochromatin (Fig. 3
). This same pattern of subnuclear distribution was observed for GFP-Pit-1 expressed in GHFT15 cells (Fig. 3
). We showed that when GFP-C/EBP
was expressed in the pituitary GHFT15 cells, it was incompletely localized to regions centromeric heterochromatin (Fig. 3A
). In contrast, we found that a truncated GFP-C/EBP
244 protein, containing only the B-ZIP DNA-binding domain, was almost exclusively localized to these sites (Fig. 3B
). This could indicate that C/EBP
244 has a higher affinity for heterochromatin. However, studies by others demonstrated that the DNA binding specificity and affinity for the B-ZIP domain alone were very similar to those of the full-length protein (50). Alternatively, our results might indicate that activities associated with the amino-terminal transactivation domain function to direct C/EBP
to regions outside the heterochromatin foci.
In this regard, we found that when GFP-Pit-1 and BFP-C/EBP were coexpressed in the same pituitary cells, C/EBP
adopted a pattern of intranuclear distribution that completely overlapped that of Pit-1 (Fig. 4
). The colocalization of C/EBP
with Pit-1 did not result from nonspecific interactions of the FPs, because a third coexpressed protein, PML-RFP, remained localized to separate nuclear bodies in these same cells (Fig. 4
). Moreover, when BFP-C/EBP
was coexpressed with the ER
-GFP, C/EBP
remained localized to foci independent of the ER
-GFP. We also observed in fixed cells that GFP-Pit-1 partially overlapped sites of active gene transcription marked by BrUTP-labeled nascent mRNAs. In contrast, we previously demonstrated that C/EBP
accumulated at heterochromatin foci that were relatively devoid of nascent mRNA transcripts. Together, these results suggest that the recruitment activities of Pit-1 for C/EBP
may play an organizational role that is essential for their cooperative activation of pituitary-specific PRL and GH gene transcription, and the developmental progression of the somatolactotrope progenitor cells.
The HD of Pit-1and the Recruitment of C/EBP
The specific organizational activity of Pit-1 for C/EBP was demonstrated by the effect of deletions and mutations in Pit-1. A truncation of Pit-1 that removed the HD and abolished its activity at both the GH (51) and PRL (52) promoters failed to redistribute C/EBP
from the regions of centromeric heterochromatin (Fig. 5
). Instead, when coexpressed with C/EBP
, there was a tendency for the truncated Pit-1 protein to associate with C/EBP
in regions of centromeric heterochromatin. This suggests that the Pit-1 HD deletion retained some ability to interact with C/EBP
, but that the recruitment activities of Pit-1 required the intact HD.
The HD forms half of a conserved bipartite DNA-binding motif (8, 31), and binding to specific DNA sites may be prerequisite for the recruitment of other interacting protein partners (35). We tested this hypothesis by using a Pit-1 protein mutated at the R271 residue, which lies outside the DNA-binding domain. The dominant inhibitory activity previously reported for Pit-1 R271 mutants is causative for the syndrome of CPHD (6, 7, 30). We found that the Pit-1R271A mutant acted as a dominant inhibitor of PRL gene transcription, preventing the cooperation between Pit-1 and C/EBP (Fig. 7
). The Pit-1R271A mutant bound specifically to Pit-1 DNA response elements predominately as a monomer. The intranuclear distribution of GFP-Pit-1R271A, however, was diffuse like that of the Pit-1 HD deletion, which did not bind to Pit-1 DNA elements (compare Figs. 5
and 7
). This indicated that binding to specific DNA elements alone was not sufficient to direct the subnuclear targeting of Pit-1. Further, when coexpressed with C/EBP
, the Pit-1 point mutant failed to recruit C/EBP
and instead became associated with C/EBP
in regions of centromeric heterochromatin. It is possible that disruption of the Pit-1 dimerization interface by mutation of R271 (31), which prevents the protein from assuming dimer conformations, also prevents the formation of other important protein-protein contacts, such as that observed here for C/EBP
. These observations suggest that the dominant inhibitory actions of the CPHD Pit-1 mutant could be related to its redirection into the transcriptionally inactive centromeric heterochromatin. This raises the possibility that other proteins associated in complexes with Pit-1 would similarly be directed to these transcriptionally silent subnuclear domains.
Nuclear Compartmentalization as a Regulator of Gene Expression Patterns
Together our results indicate that the location of transcription factors within the nucleus may be a critical epigenetic determinant for the formation of transcriptional complexes necessary for the regulation of specific genes. In this case, the positioning of transcription factor complexes in nuclear compartments associated with centromeric heterochromatin may function to silence specific genes. The interphase centromere is thought to position chromosome territories (53), and the silencing of transcriptional activity can occur when genes are located close to the heterochromatin (54). A recent analysis of the human ß-globin locus control region demonstrated that a transcriptional enhancer functions to maintain the gene at a distance from centromeric heterochromatin, thus preventing its silencing (55).
The pituitary cell-specific expression of the GH gene requires a locus control region positioned 15 kb upstream of the promoter, and Pit-1 binds to an array of DNA-elements within this region (56). It is possible that the location of Pit-1 and its cooperating factors within the nucleus plays a critical role in the positioning of this locus control region. Thus, the Pit-1 CPHD mutant shown here, which localized to centromeric chromatin when coexpressed with C/EBP, could function to position genes with suitable DNA elements near these regions of silencing. Indeed, other transcription factors are known to function as repressors through this type of mechanism. For example, during B cell development the zinc-finger protein Ikaros/Lyf-1 becomes localized to centromeres where it functions to recruit genes to be silenced during lymphocyte activation (57, 58, 59, 60). In addition, proteins with the Krüppel-associated box also function as transcriptional repressors, exerting their silencing activity by recruitment of other factors and target gene loci to the transcriptionally inert centromeric heterochromatin (61). Further studies using techniques such as fluorescence in situ hybridization and chromatin immunoprecipitation assay will be necessary to determine how the distribution of these protein complexes is related to the positioning and the transcriptional state of specific endogenous gene loci in pituitary cells.
Our observations of the colocalization of Pit-1 and C/EBP in the pituitary cell nucleus using conventional fluorescence microscopy are limited by the diffraction of light to a resolution of approximately 200 nm. Although these observations imply that C/EBP
and Pit-1 are associated, they do not conclusively establish this point. Our failure to detect a physical interaction between these proteins using more traditional biochemical approaches suggested either an indirect interaction or an interaction dependent upon conditions only present in the environment within the intact living cell. Significantly, we have used fluorescence resonance energy transfer microscopy to demonstrate that Pit-1 and C/EBP
are in close physical association in the living pituitary cell nucleus (39). Together with the studies reported here, we provide striking evidence that some transcription factors can specifically interact with, and direct, cooperating factors to particular sites in the nucleus, and that a mutation associated with human disease can dramatically alter the intranuclear targeting of these factors.
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MATERIALS AND METHODS |
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Western Blotting and EMSA
For Western blotting, GHFT15 cells were transfected with the indicated protein expression vector, and detergent lysates were prepared after 24 h as described previously (63). After electrophoresis and transfer to nitrocellulose membranes, the proteins were detected by incubation with antibody against C/EBP (sc-61, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:200 final dilution) or Pit-1 (sc-442; 1:5000 final dilution). This was followed by incubation with secondary antibody (horseradish peroxidase-conjugated goat antirabbit, Pierce Chemical Co., Rockford, IL) at a final dilution of 1:50,000. The membranes were then washed and incubated in enhanced chemiluminescence substrate (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 min and then exposed to film for 10 min as described previously (62).
EMSAs were performed on whole-cell extracts prepared from transiently transfected GHFT15 cells as described previously (18). Duplex oligonucleotides probes corresponding to either a consensus C/EBP binding site (16) or Pit-1 sites were (18):
CEBP RE 5'-GATCGAGCCCCATTGCGCAATCTATATTCG
PRL 1P 5'-CCTGATTACATGAATATTCATGAAGGTG
PRL 3P 5'-GGCTTCCTGAATATGAATAAGA
Each probe was prepared by end-labeling using [-32P] ATP and T4 polynucleotide kinase. Samples of cell extracts (10 µg) were added to the reaction mixtures assembled on ice. For competition studies, unlabeled duplex oligonucleotide was added in 3-, 30-, or 100-fold excess. Where indicated, antisera against the expressed protein (0.2 µl) was added to the reaction mixtures and incubated for 20 min at 4 C. Antibodies specific for Pit-1 that either super-shifted the DNA-protein complex (Geneka Biotechnology, Inc., Montréal, Canada) or disrupted DNA binding (63) were used in these studies. The reaction mixtures were transferred to tubes containing 25,00050,000 cpm of the end-labeled probe and incubated for 20 min at room temperature. The samples were then fractionated on prerun 6.0% polyacrylamide gels, followed by autoradiography as described previously (18).
Immunohistochemistry and Labeling of Nascent Transcripts
To induce differentiation to adipocytes, the 3T3-L1 cells were incubated in medium containing 10% FCS that was supplemented with 1 µg/ml insulin, 1 µM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine for 2 d, followed by incubation in medium containing 10% FCS with 1 µg/ml insulin (20). The nontransfected mouse pituitary GHFT15 cells and adipocyte cells were cultured on glass cover slips. Cells were maintained in culture 2448 h, and then fixed by incubation in 1.5% formaldehyde in PBS, and then processed for immunohistochemical detection. Endogenous C/EBP was detected in fixed adipocytes by incubation with a rabbit polyclonal C/EBP primary antibody (1:100 dilution of sc-61, Santa Cruz Biotechnology, Inc.) followed by incubation with an antirabbit rhodamine-conjugated secondary antibody. The endogenous Pit-1 was detected in fixed GHFT15 cells by incubation with a rabbit polyclonal Pit-1 antiserum (1:500 dilution), followed by incubation with antirabbit Texas red-conjugated secondary antibody. The antiserum to Pit-1 was described previously (63). The cover slips were washed and the fixed cells were stained with 0.2 µg/ml H33342 for 5 min and then rinsed. The cover slips were then mounted on slides using Prolong Antifade mounting media (Molecular Probes, Inc., Eugene, OR) and viewed by fluorescence microscopy using the appropriate filter sets.
Labeling of nascent mRNA transcripts was performed as previously described (4) except cells were exposed to BrUTP for 20 min. Briefly, transfected cells were grown on cover glasses for 24 h and then permeabilized with saponin. The cells were exposed to BrUTP for 20 min at 33 C to label nascent mRNA and then fixed in paraformaldehyde. After fixation, cells were washed and incubated overnight at 4 C with antibromouracil antibody (BMC9318, Roche Molecular Biochemicals, Indianapolis, IN; 1:100 final dilution). The next day cells were washed followed by detection with a Texas Red-conjugated secondary antibody. Cells were washed again and stained with H33342 at a concentration of 0.2 µg/ml, and the cover glasses were mounted using Vectashield (Vector Laboratories, Inc., Burlingame, CA). Fluorescent images were captured and processed as above for the live cell imaging, with the gray-level Texas red signal being assigned to the red channel of the red-green-blue image.
Microscopy and Image Analysis
Pituitary GHFT15 cells were transfected with between 3 and 30 µg of expression plasmid DNA encoding the proteins of interest fused to the fluorescent proteins, and inoculated into culture dishes containing 25-mm cover glasses. The cells were maintained in culture for 24 h as described above and then subjected to fluorescence microscopy as described previously (24). For experiments involving staining with H33342, the stain was added to a final concentration of 0.5 µg/ml approximately 20 min before imaging of living cells or at 0.2 µg/ml for 5 min to image fixed cells. The fluorescence images were acquired using an inverted IX-70 (Olympus Corp., Lake Success, NY) equipped with a 60x aqueous-immersion objective lens. The filter combinations were 485/22 nm excitation and 535/50 nm emission for GFP; 365/15 nm excitation and 460/50 nm emission for H33342 or BFP images, and a tetramethyl rhodamine isothiocyanate filter set for RFP and Texas red imaging (Chroma Technology Corp., Brattelboro, VT). Grayscale images with no saturated pixels were obtained using a cooled digital interline camera (Orca-200, Hamamatsu, Bridgewater, NJ). All images were collected at a similar gray-level intensity by controlling the excitation intensity using neutral density filtration, and by varying the on-camera integration time. ISEE software (Inovision Corp., Raleigh, NC) was used to background subtract and then convert the digital images to red-green-blue images. All image files were processed for presentation using Canvas 7.0 (Deneba Systems, Miami, FL).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Current address: Department of Biology, Austin College, Suite 61582, Sherman, Texas 75090.
Abbreviations: BFP, Blue fluorescent protein; BrUTP, bromouridine; C/EBP, CCAAT/enhancer binding protein-
; CPHD, combined pituitary hormone deficiency; B-ZIP, basic region-leucine zipper; ER, estrogen receptor; FCS, fetal calf serum; FP, fluorescent protein; GFP, green fluorescent protein; H33342, Hoechst 33342; HD, homeodomain; PML, promyelocytic leukemia protein; PRL, prolactin; RFP, red fluorescent protein; rPRL, rat prolactin.
Received for publication September 4, 2001. Accepted for publication October 28, 2002.
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REFERENCES |
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