Tudor and Nuclease-Like Domains Containing Protein p100 Function as Coactivators for Signal Transducer and Activator of Transcription 5
Kirsi Paukku,
Jie Yang and
Olli Silvennoinen
Haartman Institute (K.P., O.S.), Department of Virology, and Biomedicum Helsinki (K.P., O.S.), Programme for Developmental and Reproductive Biology, FIN-00014 University of Helsinki, Helsinki, Finland; Institute of Medical Technology (J.Y., O.S.), University of Tampere; and Department of Clinical Microbiology (O.S.), Tampere University Hospital, FIN-33014 Tampere, Finland
Address all correspondence and requests for reprints to: Olli Silvennoinen, Biomedicum Helsinki, Programme for Developmental and Reproductive Biology, Haartmaninkatu 8, P.O. Box 63, FIN-00014 University of Helsinki, Helsinki, Finland.
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ABSTRACT
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Signal transducer and activator of transcription 5 (Stat5) plays a critical role in prolactin (PRL)-induced transcription of several milk protein genes. Stat5-mediated gene regulation is modulated by cooperation of Stat5 with cell type- and promoter-specific transcription factors as well as by interaction with transcriptional coregulators. Recently, the expression of a tudor and staphylococcal nuclease-like domains containing protein p100 was found to be increased in mammary epithelial cells during lactation in response to lactogenic hormones. p100 was initially identified as a transcriptional coactivator of the Epstein-Barr virus nuclear antigen 2. In this study we investigated the potential role of p100 in PRL-induced Stat5-mediated transcriptional activation. PRL stimulation increased the p100 protein levels in HC11 mouse mammary epithelial cells. p100 did not affect the early activation events of Stat5, but p100 enhanced the Stat5-dependent transcriptional activation in HC11 cells. p100 associated with Stat5 both in vivo and in vitro, and the interaction was mediated by both the tudor and staphylococcal nuclease-like domains of p100. Together these results suggest that p100 functions as a transcriptional coactivator for Stat5-dependent gene regulation and the existence of a positive regulatory loop in PRL-induced transcription, in which PRL stabilizes p100 protein, which in turn can cooperate with Stat5 in transcriptional activation.
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INTRODUCTION
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SIGNAL TRANSDUCERS AND activators of transcription (Stats) represent a family of latent cytoplasmic transcription factors that are activated by various cytokines. Seven mammalian Stat proteins exist, and they all share a similar overall structure with central DNA binding domain, Src homology 2 (SH2) domain, and the C-terminal transactivation domain (TAD), which is the most divergent part among the Stats. Stat5 was originally identified as a transcription factor that regulates the ß-casein gene in response to prolactin (PRL) (1). Two forms of Stat5, Stat5A and Stat5B (2, 3), exist, and both are activated by, in addition to PRL, several other cytokines including GH, erythropoietin, granulocyte-macrophage colony-stimulating factor, thrombopoietin, IL-2, IL-3, IL-5, IL-7, IL-9, and IL-15 (4, 5, 6).
PRL is a primary factor required for the growth and terminal differentiation of mammary epithelial cells (7). Binding of PRL to its receptor at the cell surface induces receptor dimerization and activation of cytoplasmic Janus kinase 2, which phosphorylates the receptor on cytoplasmic tyrosine residues that provide docking sites for SH2 domain-containing proteins, including Stat5 (8). Janus kinase 2 phosphorylates Stat5 on a conserved C-terminal tyrosine residue, which interacts with the SH2 domain of another Stat5 molecule to form a dimer. Stat5 dimers then translocate to the nucleus where they bind to consensus DNA elements on cytokine-responsive promoters (1). Binding of Stat5 to the gene promoter is necessary for the induction of ß-casein, as well as for
s1-casein (9), whey acidic protein (10), and ß-lactoglobulin milk protein genes (11). The physiological function of Stat5 in PRL signaling is clearly demonstrated in the knockout models, where Stat5-null mice show a mammary phenotype similar to that of PRL receptor-deficient mice, exhibiting impaired differentiation of lobuloalveolar units and inability to lactate (12, 13, 14, 15, 16).
The molecular mechanisms that underlie Stat5-mediated transcription are not fully understood, but these mechanisms involve interactions and cooperation with both sequence-specific transcription factors as well as with transcriptional coregulators. In the regulation of the ß-casein gene promoter, the interaction between Stat5, glucocorticoid receptor (GR), and CCAATT/enhancer binding protein-ß (C/EBPß) results in synergistic activation of transcription (17, 18, 19). Stat5 has also been shown to interact with coregulator proteins such as p300/CBP[cAMP response element binding protein (CREB)-binding protein], N-Myc interactor, and a centrosomal P4.1-associated protein (20, 21, 22) that function as scaffold proteins for assembly of transcription complexes and in remodeling of chromatin structure. Recently, the expression of a tudor (TD) and staphylococcal nuclease-like (SN-like) domains containing coactivator protein p100 was found to be abundant in the nuclei of mammary epithelial cells (23). The protein levels of p100 increased in response to lactogenic hormones during lactation and correlated with the induction of ß-casein gene expression. The human p100 protein was initially identified as a transcriptional coactivator for the Epstein-Barr virus nuclear antigen 2 (EBNA-2) (24). In addition to binding to the acidic transactivation domain of EBNA-2, p100 has been found to interact with general transcription factor TFIIE, homeodomain-containing transcription factor c-Myb (25), and Pim-1 serine/threonine kinase (26). Furthermore, p100 was identified as a positive regulator for Stat6 in IL-4 signaling (27).
p100 protein levels are induced by lactogenic hormones, and the protein is present also in the cytosol, endoplasmic reticulum, and lipid droplets of milk-secreting cells (28), suggesting a role for p100 in lactation. These findings led us to investigate the possible function of p100 in the regulation of Stat5-dependent gene transcription. Our results show that PRL up-regulates p100 protein levels in HC11 cells, and that p100 interacts with Stat5 and enhances the Stat5-mediated transcriptional activation, thus indicating that p100 functions as a coactivator for Stat5.
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RESULTS
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p100 Protein Levels Are Up-Regulated by PRL Stimulation
The correlation between increased p100 protein levels and ß-casein gene expression (23) led us to consider whether p100 is involved in PRL-induced transcriptional activation of Stat5. HC11 mammary epithelial cells have been used to characterize molecular events involved in the regulation of milk protein gene expression. Treatment of HC11 cells with lactogenic hormones insulin, glucocorticoids, and PRL results in transcription of the ß-casein gene (29, 30). To this end, we generated HC11 cell lines stably expressing Flag-tagged p100 protein (HC11-p100 cells) or empty pCIneo vector expressing control cell lines (HC11-pCIneo). Two representative HC11-p100 clones were used for subsequent studies.
p100 protein levels are up-regulated by lactogenic hormones (28). It has been suggested that the modulation of p100 activity in mammary gland may occur through posttranscriptional control of expression, because the increase in p100 protein abundance occurs without a corresponding increase in p100 mRNA (23). However, the role of different lactogenic hormones in up-regulation of p100 protein has not been investigated. To study the regulation of the p100 protein expression in more detail, we stimulated HC11-p100 cells with dexamethasone (Dex) or PRL or both for different periods of time. Stimulated pCIneo cells were used as negative controls. The p100 protein levels increased after stimulation with lactogenic hormones, thus confirming that the HC11-p100 cell line behaved in a manner similar to that of lactating mammary epithelial cells (Fig. 1
). Stimulation with Dex alone did not affect the p100 protein levels at any time point, whereas PRL stimulation enhanced the amount of p100 protein after 3 h stimulation (Fig. 1
). Stimulation with both PRL and Dex did not further increase the p100 levels when compared with PRL stimulation. In conclusion, the up-regulation of p100 protein levels is mediated by PRL signaling, and Dex is not involved in this regulation.

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Fig. 1. p100 Protein Levels Are Up-Regulated by PRL
HC11-pCIneo cells or HC11-p100 cells were stimulated with Dex (top) or PRL (middle) or both (bottom panel) for times indicated. The total cell lysates were electrophoresed in SDS-PAGE and immunoblotted with anti-Flag antibody. The upper band corresponds to p100-Flag, whereas the lower band is unspecific.
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p100 Enhances the Transcriptional Activity of Stat5 in HC11 Cells
To investigate whether p100 is involved in the transcriptional regulation exerted by Stat5, reporter gene assays were carried out in different HC11 cell lines with ß-casein promoter-driven reporter genes. Cooperation between Stat5 and GR is required for ß-casein expression (17, 18) and, accordingly, stimulation of HC11-pCIneo cells with PRL resulted in only 2-fold induction, whereas costimulation with both PRL and Dex resulted in 8-fold induction of the ß-casein reporter gene (Fig. 2A
). Interestingly, in HC11-p100 cells stimulation with PRL and Dex resulted in 2-fold higher induction compared with the PRL+Dex-stimulated HC11-pCIneo cells. p100 expression did not affect the basal activity of the reporter gene in either cell line. Also transiently transfected p100 enhanced the induction of ß-casein promoter in a manner similar to HC11-p100 cells.

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Fig. 2. p100 Enhances the Transcriptional Activity of Stat5 in HC11 Cells
A, HC11-pCIneo cells (bars 1 and 2) and HC11-p100 cells (bar 3) were transfected with pZZ1 luciferase reporter construct (250 ng) together with pRLTK control vector (50 ng) and Stat5A expression vector (100 ng). Cells were left unstimulated (gray bars), stimulated with PRL (1, hatched bar) or with PRL and Dex (2 and 3, black bars). Parental HC11 cells were also transfected with pZZ1, pRLTK, and Stat5A vectors together with p100-Flag (250 ng). Cells were either left unstimulated or stimulated with PRL and Dex (4, black bar). The luciferase activity was measured as described in Materials and Methods. Shown is the mean from three independent experiments and the SEMs. Below is the Stat5 immunoblot showing the expression levels of Stat5. Parental HC11 cells were transfected with pSpi-luc2 luciferase reporter construct (250 ng) together with pRLTK control vector (50 ng) and Stat5A expression vector (100 ng) with or without expression vector encoding p100-Flag (250 ng) or CBP-hemagglutinin (250 ng), as indicated. After starvation, cells were left unstimulated (gray bars) or stimulated with PRL (black bars) (B) or with PRL and Dex (C). Immunoblots showing the expressions of Stat5 and p100-Flag are shown below both figures. HC11-pCIneo cells and HC11-p100 cells were transfected with C/EBPß-dependent reporter plasmid (250 ng) (D) or NF- B (250 ng) reporter construct (E) together with pRLTK control vector (50 ng). After starvation, cells were left unstimulated (gray bars) or stimulated with PRL and Dex (black bars).
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The half-palindromic GR binding sites in ß-casein gene promoter recruit GR molecules to the promoter (31, 32). We next wanted to study whether the effect of p100 on Stat5-mediated transcription was dependent on DNA binding of GR. Parental HC11 cells were transfected with Serine protease inhibitor (Spi) 2.1 reporter plasmid containing only the Stat5 response element along with expression construct encoding for p100-Flag, CBP-hemagglutinin, and Stat5A, as indicated. p300/CBP has been previously shown to function as a coactivator for Stat5 in PRL signaling (20, 33, 34), and CBP was therefore used as a positive control in this assay. Stimulation with PRL resulted in 5-fold induction of Stat5-dependent transcription, and the coexpression of p100 further enhanced this transcription at a similar level as CBP (1.5-fold; Fig. 2B
). However, stimulation with both PRL and Dex enhanced both the activity of the promoter as well as the stimulatory effect of p100 (Fig. 2C
). Since Spi-luc2 reporter construct lacks binding sites for GR, it is likely that the effect of Dex stimulation is mediated through physical protein-protein interaction between Stat5 and GR as previously reported (17, 35). Taken together, the enhancing effect of p100 on the transcriptional activity of Stat5 is not dependent on DNA binding of GR, but simultaneous activation of GR enhances the stimulatory effect of p100 possibly through Stat5-GR protein complex formation.
We wanted to further assess the specificity of p100 action by analyzing the effect of p100 on C/EBPß and NF-
B (nuclear factor-
B), which both bind to ß-casein gene promoter and regulate its expression. HC11-p100 cells and HC11-pCIneo cells were transfected with C/EBPß and NF-
B luciferase constructs. Both reporter genes were constitutively active in HC11 cells, and stimulation with PRL and Dex had no effect on their activities. Expression of p100 did not affect the activity of C/EBPß or NF-
B luciferase construct, as shown in Fig. 2
, D and E, thus indicating that the effect of p100 is targeted to Stat5 activation.
p100 Does Not Affect Tyrosine Phosphorylation of Stat5
The phosphorylation of a C-terminal tyrosine residue in Stats is a transient and tightly regulated process. GR is known to enhance the PRL-induced DNA binding and transcriptional activity of Stat5 by increasing its tyrosine phosphorylation probably through modulating the rate of dephosphorylation (36). To study whether p100 would modulate tyrosine phosphorylation of Stat5, HC11-p100 cells and HC11-pCIneo cells were stimulated with PRL and Dex for different periods of time, and endogenous Stat5A was immunoprecipitated from the cell lysates. Expression of p100 did not affect the level or kinetics of tyrosine phosphorylation of Stat5 (Fig. 3A
). Activation of Stat5 was analyzed also by EMSA using ß-casein oligonucleotide as a probe. Activation and deactivation of Stat5 occurred similarly in HC11-pCIneo and HC11-p100 cells (Fig. 3B
). These results thus indicate that p100 does not affect the early activation events or the rate of dephosphorylation of Stat5.

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Fig. 3. p100 Does Not Affect Tyrosine Phosphorylation of Stat5
A, HC11-pCIneo cells and HC11-p100 cells were stimulated with PRL and Dex for the times indicated. The lysates were subjected to immunoprecipitation with anti-Stat5A antibody. The immunoprecipitates and total cell lysates were electrophoresed in SDS-PAGE and immunoblotted with anti-phosphotyrosine (PY) (upper panel) or anti-Stat5A (lower panel) antibodies. B, Nuclear lysates were analyzed in the mobility shift assay using 33P-labeled ß-casein oligonucleotide as a probe and separated in 4.5% Tris-borate EDTA-PAGE, followed by autoradiography. As a control, one sample was supershifted with Stat5-specific antibody.
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Stat5 Interacts with p100 in Vivo
Since p100 had a stimulatory effect on the PRL-induced transcriptional activity of Stat5, we wanted to determine whether p100 functioned as a coactivator for Stat5. We investigated the potential interaction between p100 and Stat5 in coimmunoprecipitation experiments. HC11 cells were transfected with expression vector encoding for Stat5A with or without p100-Flag expression construct. After stimulation p100 was immunoprecipitated from the cell lysates with anti-Flag antibody. Stat5A was detected in immunoprecipitates only from cells expressing p100-Flag (Fig. 4A
). PRL stimulation did not affect the coimmunoprecipitation between Stat5 and p100.

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Fig. 4. Stat5 Interacts with p100 in HC11 and 293T Cells
(A) Expression vector encoding Stat5A (500 ng) was transfected alone or together with p100-Flag expression vector (1.5 µg) into HC11 cells. After starvation cells were left unstimulated or stimulated with PRL and Dex for 20 min, as indicated. p100 was immunoprecipitated from lysates with anti-Flag antibody. As a control, one sample was left without anti-Flag antibody. Bound proteins were subjected to SDS-PAGE, followed by immunoblotting with anti-Stat5A (top) or anti-Flag (middle) antibodies. Total cell lysates were also separated by SDS-PAGE and immunoblotted with anti-Stat5A antibody (bottom panel). Molecular masses of the protein standards are indicated on the left in kilodaltons. B, COS-7 cells were transfected with a vector encoding SH2 mutant of Stat5A (250 ng) with or without p100-Flag expression vector (1.5 µg), as indicated. The lysates were analyzed by immunoprecipitation using anti-Flag antibody. The immunoprecipitates and 5% of cell lysate were electrophoresed in SDS-PAGE and immunoblotted with anti-Stat5A (top) and anti-Flag (middle) antibodies. Total cell lysates were also separated by SDS-PAGE and immunoblotted with anti-Stat5A (bottom panel) antibody. C, 293T cells were transfected with expression vectors encoding p100-Flag (1.5 µg), TD-Flag (2 µg), or SN-Flag (1.5 µg) together with Stat5 expression vector (500 ng), as indicated. After immunoprecipitation with anti-Flag antibody, the bound proteins and 5% of cell lysate were electrophoresed in SDS-PAGE and immunoblotted with anti-Stat5A or anti-Flag antibodies. Total cell lysates were also separated by SDS-PAGE and immunoblotted with anti-Stat5A antibody.
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The SH2 domain mediates the interaction of Stat5 with the tyrosine-phosphorylated receptor and the dimerization of the phosphorylated Stat proteins. To study whether a functional SH2 domain of Stat5 is required for the association with p100, a vector encoding a SH2 mutant of Stat5A was transfected with or without vector encoding p100-Flag into COS-7 cells. p100-Flag was immunoprecipitated from cell lysates, and the associated Stat5 was detected by Stat5A immunoblotting. p100 was found to coimmunoprecipitate also with SH2 mutant of Stat5A (Fig. 4B
). Taken together, the interaction between p100 and Stat5 is independent of tyrosine phosphorylation or functional SH2 domain of Stat5.
To define the domains of p100 that mediate the interaction with Stat5, Flag-tagged expression constructs consisting of SN-like domains or TD domain of p100 were made. A vector encoding for Stat5A was transfected together with p100-Flag, SN-Flag, or TD-Flag expression construct into 293T cells. Full-length p100, SN-like domains, and TD domain were immunoprecipitated from total cell lysates with anti-Flag antibody, and Stat5 was detected by immunoblotting. As shown in Fig. 4C
, Stat5 was detected in full-length p100 but not in control immunoprecipitates. In addition, Stat5 was found to coimmunoprecipitate with both TD domain and SN-like domains of p100, suggesting that both regions mediate association with Stat5.
p100 Interacts with Stat5 in Vitro
To confirm the interaction between p100 and Stat5, we made glutathione-S-transferase (GST) fusion proteins of TD and SN-like domains of p100 and analyzed their interaction with Stat5 in vitro. GST, SN-GST, and TD-GST were expressed in bacteria and bound to glutathione-Sepharose beads, which were then incubated with cell lysates containing Stat5A. Stat5 binding was visualized by immunoblotting. As shown in Fig. 5A
, GST alone did not bind Stat5, while SN-GST and TD-GST both interacted with Stat5. Consistent with the in vivo results, these results indicate that both N- and C-terminal regions of p100 mediate the interaction. The mobilities of SN-GST and Stat5 are similar in the gel (Fig. 5B
) and the SN-GST protein slightly shifts the Stat5 band, resulting in seemingly smaller molecular weight of Stat5 in SN-GST lane compared with Stat5 bound to TD-GST.

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Fig. 5. Interaction of p100 and Stat5 in Vitro
A, COS-7 cells were transfected with expression vector encoding Stat5A. Cell lysates were incubated with either GST alone or with SN-GST or TD-GST. Binding results were subjected to SDS-PAGE and visualized with anti-Stat5A antibody. B, Samples of purified GST-proteins were run in the gel and stained with Coomassie Brilliant Blue. C, COS-7 cells were transfected with p100-Flag expression vector. Cell lysates were incubated with either GST alone or with Stat5A-TAD-GST. The bound proteins were resolved in SDS-PAGE and immunoblotted with anti-Flag antibody.
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The C-terminal TAD is the most divergent part among the Stats. TAD regions of different Stat proteins have been reported to interact with various coactivator proteins, and the Stat5-TAD has earlier been shown to associate with p300/CBP (20). To characterize whether Stat5-TAD interacts with p100, we expressed Stat5A-TAD as a GST fusion protein and used it for binding experiments. Stat5A-TAD was found to bind to p100 from cell lysate whereas no interaction was observed with GST control as shown in the anti-Flag immunoblot in Fig. 5C
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DISCUSSION
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The immediate signaling events, leading to tyrosine phosphorylation, dimerization, and DNA binding of Stat5, are relatively well characterized (1), but the molecular mechanisms of Stat5-mediated transcription are still largely elusive. In the present study we show that p100 protein functions as a transcriptional coactivator for Stat5.
p100 is an ubiquitously expressed protein, which has earlier been characterized as a transcriptional coregulator for EBNA-2, c-Myb, and Stat6. p100 is present in mammary epithelial cells (23), and the p100 protein levels increase in response to PRL during lactation and correlate with induction of ß-casein gene expression. We observed that p100 interacts with Stat5 both in vivo and in vitro. Association between p100 and Stat5 did not require tyrosine phosphorylation or functional SH2 domain of Stat5. Since p100 is detected in both the cytoplasm and nucleus, the interaction could therefore occur earlier in the cytoplasm and p100 and Stat5 could translocate to the nucleus as a complex. Phosphorylation of Stat is not required for their interactions with most of the coregulators, and interactions of Stat6 with p100, Stat5 with GR, and Stat1 and Stat3 with c-Jun, for example, are independent of tyrosine phosphorylation of Stats (27, 36, 37). The interaction between Stat5 TAD and p100 was observed with recombinant proteins and is likely to be a direct protein interaction, but we cannot definitely exclude the possibility of the presence of an adaptor protein or interaction of other region of Stat5. The association between Stat5 and p100 appeared to be of rather low affinity, and we were unable to detect the p100-Stat5 complex with endogenous proteins. However, the interaction between sequence-specific transcription factors and coregulators is generally rather weak. In the case of Stats, the previously identified interactions between Stats and coregulator proteins have been mainly detected in in vitro or in overexpression conditions (20, 21, 27, 38, 39, 40, 41, 42, 43, 44) with only a few exceptions, such as Stat3-nuclear coactivator 1 (NcoA-1) (45), and Stat5-centrosomal P4.1-associated protein (22) interactions, which have been detected also with endogenous proteins.
The p100 protein has an interesting domain structure consisting of SN-like and TD domains. Staphylococcal nucleases (SNs) are small calcium-dependent enzymes, which hydrolyze both DNA and RNA. SNs consist of two subdomains. The first subdomain belongs to the large oligonucleotide/oligosaccharide-binding (OB)-fold superfamily (46), and the second subdomain consists of two independently folded
-helices. The catalytic amino acids present in nucleases are missing in the SN-like domains of p100, suggesting that these repeated motifs could only serve to bind DNA without catalytic activity, as in many other OB-folds (47). It is currently still unclear whether p100 binds DNA. Instead, there is evidence supporting the idea that SN-like domains function as protein interaction domains. SN-like domains of p100 have been shown to contain EVES sequence. The EVES sequence of c-Myb mediates an inhibitory intramolecular interaction (25) and, in addition, the EVES sequence of p100 is able to interact with c-Myb and inhibit its activity. Furthermore, p100 interacts with Pim-1 serine/threonine kinase through a sequence located in SN-like domains, but different from EVES (26), indicating that there are several protein interaction motifs in SN-like domains of p100. In line with this, we found that the SN-like domains of p100 mediate interaction with Stat5.
We found also that the TD domain of p100 associates with Stat5 and can therefore function as a protein interaction domain. Interestingly, the interaction with Stat6 is mediated only by the SN-like domain of p100 (27). The interaction domains can vary, however, between members of a protein family. CBP, for example, binds to the TADs of all the Stats, even though there is virtually no homology between the various Stat TADs (39). Indeed, distinct domains of CBP are mediating the interactions to different Stats. In addition, in Stat1 CBP binds to two different regions, the Stat1-TAD and also the N-terminal region of Stat1 (38). The TD domain of p100 is a hybrid SN-like domain, in which the OB-fold is replaced by a domain that is found in multiple copies in the Drosophila melanogaster Tudor protein. TD domains are conserved protein modules of unknown function that are often present in proteins that associate with RNA (48). The determined structure of the TD domain of survival of motor neuron protein suggests, however, that TD domains function as protein interaction motifs and do not bind to RNA directly (49). In line with this hypothesis and our results, the TD domain of survival of motor neuron protein has been found to mediate interactions with a spliceosomal Sm core protein (49) and with a small nucleolar RNA-associated protein fibrillarin (50), and TD domains of tudor repeat associator with PCTAIRE 2 protein have been shown to associate with a Cdc2-related kinase PCTAIRE 2 in rat brain (51). Also the TD domain of ets-related gene-associated protein with a suppressor of variegation, enhancer of zest and trithorax (SET) domain histone methyltransferase associates with histone deacetylase 1 and histone deacetylase 2, and the transcription corepressors mSin3A and mSin3B (52). Thus, TD domains appear to be commonly used interaction domains in transcriptional regulation.
In line with studies performed with HC11 cells stably expressing ß-casein construct (30, 53, 54), in our assays the proper stimulation of ß-casein promoter also required activation of GR. The enhancing effect of p100 was not dependent on GR, but activation of GR enhanced the stimulatory effect of p100 and this effect was not dependent on DNA binding of GR. Stat5 and GR physically interact with each other, and this protein complex may mediate a more stable interaction surface with p100 than the relatively small TAD of Stat5 alone. C/EBPß activates ß-casein in synergy with Stat5 and GR, and several binding sites for C/EBP have been mapped in the ß-casein gene promoter (55). Also, NF-
B has a binding site in the ß-casein gene and NF-
B has been shown to negatively regulate ß-casein gene expression by inhibiting the activity of Stat5 (54). We showed that p100 had no effect on the transcriptional activity of C/EBPß or NF-
B, thus demonstrating the specificity of p100 on Stat5-mediated activity.
Transcriptional activity of Stat5 can be modulated by several mechanisms. The increased tyrosine phosphorylation and/or DNA binding can stimulate transcriptional activity, as has been observed in Stat5-GR cooperation (36). We did not observe any changes in the early steps of Stat5 activation by p100. Serine phosphorylation has been implicated in regulation of Stat activity, and phosphorylation of Stat1 and Stat3 on serine within their transactivation domain is needed for maximal interferon-induced transcriptional activation (56, 57, 58, 59, 60). In the Stat5-TAD, there are two proline-juxtaposed serine residues, which become phosphorylated in the mammary gland during gestation and lactation. Several serine/threonine kinases are activated upon PRL stimulation, but the kinase phosphorylating Stat5 is currently unknown (61). The interaction between p100 and Pim-1 raises the possibility that p100 could recruit Pim-1 to Stat5. However, serine phosphorylation of Stat5 in the mammary gland has an inhibitory effect on the transcriptional activity of Stat5 (61); therefore it is unlikely that the function of p100 would be to recruit a serine kinase to Stat5.
Modulation of chromatin structure by histone acetylation is critical for the activation of transcription, and several coactivators, such as p300/CBP, possess histone acetyltransferase activity. p100 has earlier been shown to be devoid of histone acetyltransferase activity (62), but coactivators can also function as bridging factors to the basal transcription machinery. p100 was found to increase the IL-4-induced transcriptional activity of Stat6, possibly by bridging Stat6 to RNA polymerase II (27). It is thus likely that p100 mediates a similar function in Stat5 by mediating interaction with the basal transcriptional machinery.
During the gestation-lactation cycle the high amount of p100 protein is associated with lactation; thus p100 is unique among the proteins implicated in the regulation of milk gene expression. The increase in p100 protein abundance during lactation is a common feature among mammals, and it is observed in the bovine, murine, and ovine mammary glands (23). The modulation of p100 activity in mammary gland probably results from posttranscriptional control of expression, because the increase of p100 protein occurs without a corresponding increase in p100 mRNA (23). We found that, among lactogenic hormones, PRL, but not glucocorticoids, was responsible for an increase in p100 protein levels. The physiological role of Stat5-p100 interaction in PRL signaling awaits confirmation by targeted gene deletion studies, but the costimulation of Stat5-mediated transcription by p100 and the PRL-induced increase in the p100 protein levels suggest the existence of a positive regulatory loop, where PRL stabilizes p100 protein, which in turn can cooperate with Stat5 in transcriptional activation.
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MATERIALS AND METHODS
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Reagents, Cell Culture, and Transfections
293T and COS-7 cells (American Type Culture Collection, ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Life Technologies, Inc., Gaithersburg, MD) and antibiotics. 293T and COS-7 cells were transfected with Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN) following the manufacturers instructions or by electroporation with a Bio-Rad gene pulser at 260V/960 µF, and lysed after 72 h. Mouse mammary epithelial cell line HC11 (29) was kind gift from L. A. Haldosén. Stable p100-Flag-expressing cells were generated by transfecting pCIneo-p100-Flag or empty pCIneo vector into HC11 cells, and stable clones were isolated with 600 µg/ml G418 (Sigma, St. Louis, MO). HC11 cells were cultured in RPMI 1640 with 10% FBS, 5 µg/ml insulin (Sigma, St. Louis, MO), 10 ng/ml epidermal growth factor (Sigma), 50 µg/ml Gentamycin (Biochrom KG, Berlin, Germany), L-glutamine, and antibiotics. Before hormone treatment, confluent HC11 cells were starved for 48 h in medium lacking epidermal growth factor, but containing 5 µg/ml insulin and 2% FBS. Cells were kept 2 d in confluence and stimulated in starvation media with 5 µg/ml PRL (Sigma) and 100 nM Dex. HC11 cells were transfected with Fugene 6 or with ExGen 500 (MBI Fermentas, Hanover, MD) reagent. The following antibodies were used: monoclonal antiphosphotyrosine antibody (clone 4G10, Upstate Biotechnology, Inc., Lake Placid, NY), monoclonal anti-Stat5A antibody (Zymed Laboratories, Inc., South San Francisco, CA), monoclonal anti-Flag M2 antibody (Sigma), and polyclonal anti-Stat5B antibodies (C-17x; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) recognizing both A and B isoforms of Stat5.
DNA Constructs
Expression vectors for wild-type mouse Stat5A, SH2 mutant (Arg618Leu) of mouse Stat5A, and luciferase reporter construct containing the Stat5 binding site from the promoter of the serine protease inhibitor gene 2.1 (pSpi-luc2) (63) were kind gifts from Dr. T. Wood. pSG5-p100-Flag, pSG5-SN-Flag (amino acids 1639 of p100), pSG5-TD-Flag (amino acids 640885 of p100) expression constructs, and GST fusion constructs SN-GST and TD-GST were made as described earlier (27). pCIneo-p100-Flag was generated by PCR using primers with C-terminal Flag sequence. The amino acid 721793 region of Stat5A was cloned into pGEX 4T-1 vector (Amersham Pharmacia Biotech, Arlington Heights, IL) to generate Stat5A-TAD-GST construct. All PCR products were sequenced. The (-344/+1)ß-casein luciferase reporter construct (pZZ1) containing the 344 nucleotides preceding the RNA initiation site from the promoter of ß-casein gene (1), C/EBPß luciferase reporter (64), NF-
B luciferase reporter construct (65), and the expression vector for CBP containing hemagglutinin tag (HA) in the C terminus (66) have been previously described.
Immunoprecipitation, GST Pull Down, and Western Blotting
HC11, 293T, and COS-7 cells were lysed in 50 mM Tris-HCl, (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 (NP-40), 20% glycerol, 1 mM Na3VO4, 50 mM NaF, supplemented with protease inhibitors. The cell lysates were centrifuged to remove cell debris. The protein concentrations were determined by using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA), with BSA as the standard. Equal amounts of cell lysates were used for immunoprecipitations. Cell lysates were incubated with specific antibodies at 4 C for 2 h, followed by incubation with protein-G-Sepharose (Amersham Pharmacia Biotech) at 4 C for 30 min. Immunoprecipitates were washed several times with washing buffer (lysis buffer with 200 mM NaCl), followed by elution with reducing Laemmli sample buffer.
Stat5A-TAD-GST, SN-GST, and TD-GST fusion proteins were purified according to manufacturers instructions (Amersham Pharmacia Biotech). For binding assays, fusion proteins were bound to glutathione Sepharose beads (Amersham Pharmacia Biotech) and incubated with COS-7 cell lysates in lysis buffer containing 50 mM Tris-HCl (pH 7.6), 0.5% NP-40, 150 mM NaCl, 0.1 mM EDTA, 20% glycerol, and protease inhibitors. After washes the bound proteins were eluted with reducing Laemmli sample buffer.
Cell lysates, immunoprecipitates, and GST pull downs were electrophoresed in SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). Immunodetection was performed using specific primary antibodies, biotinylated secondary antibodies (Dako A/S, Glostrup, Denmark), and streptavidin-biotin horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech), followed by enhanced chemiluminescence detection.
EMSA
Nuclear lysates from HC11 cells and mobility shift assays were done as described earlier (3) using [
-33P]ATP (Amersham Pharmacia Biotech)-labeled ß-casein (5'-AGATTTCTAGGAATTCAAATCC-3') oligonucleotides.
Luciferase Assay
Starved HC11 cells were transfected and after 6 h (ß-casein, NF-
B, and C/EPBß luciferase constructs) or 18 h (Spi-luc2) incubation with transfection mixture, cells were stimulated for 20 h (ß-casein, NF-
B, and C/EPBß) or 6 h (Spi-luc2). Cells were lysed and luciferase activities were measured using the Dual Luciferase Assay System (Promega Corp., Madison, WI). The luciferase activity was normalized to the activity of the cotransfected Renilla luciferase plasmid (pRLTK; Promega Corp.).
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ACKNOWLEDGMENTS
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We thank Drs. Elliot Kieff and Kara Carter for p100-Flag plasmid, Dr. Tim Wood for Stat5A and pSpi-luc2 plasmids, and Dr. Lars-Arne Haldosén for HC11 cells.
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FOOTNOTES
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This work was supported by the Academy of Finland, Emil Aaltonen Foundation, Maud Kuistila Foundation, Finnish Cultural Foundation, Finnish Cancer Organizations, Biomedicum Foundation, Instrumentarium Science Foundation, Ida Montin Foundation, Sigrid Juselius Foundation, and the Medical Research Fund of Tampere University Hospital.
Abbreviations: CBP, cAMP response element-binding protein (CREB)-binding protein; C/EBPß, CCAATT/enhancer binding protein-ß; Dex, dexamethasone; EBNA, Epstein-Barr virus nuclear antigen; FBS, fetal bovine serum; GR, glucocorticoid receptor; GST, glutathione-S-transferase; NF-
B, nuclear factor-
B; OB, oligonucleotide/oligosaccharide binding; PRL, prolactin; SH2, Src homology 2; SN, staphylococcal nuclease; Spi, serine protease inhibitor; Stat, signal transducer and activator of transcription; TAD, transactivation domain; TD, tudor.
Received for publication July 22, 2002.
Accepted for publication June 9, 2003.
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