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Molecular and Cellular Biology, March 2008, p. 1584-1595, Vol. 28, No. 5
0270-7306/08/$08.00+0     doi:10.1128/MCB.00421-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Corepressor CtBP and Nuclear Speckle Protein Pnn/DRS Differentially Modulate Transcription and Splicing of the E-Cadherin Gene

Roman Alpatov,¹ Yujiang Shi,³ Gustavo C. Munguba,¹ Babak Moghimi,² Jeong-Hoon Joo,¹ Jorg Bungert,² and Stephen P. Sugrue^1*

Department of Anatomy and Cell Biology,¹ Department of Biochemistry, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, Florida 32610,² Division of Endocrinology, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115³

Received 11 March 2007/ Returned for modification 9 April 2007/ Accepted 26 November 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CtBP is a transcriptional corepressor with tumorigenic potential that targets the promoter of the tumor suppressor gene E-cadherin. Pnn/DRS (Pnn) is a "nuclear speckle"-associated protein involved in mRNA processing as well as transcriptional regulation of E-cadherin via its binding to CtBP. Here, we show that CtBP can recruit Pnn to CtBP-associated complexes, resulting in Pnn-dependent chromatin remodeling at the E-cadherin promoter. In addition, CtBP and Pnn can differentially modulate E-cadherin mRNA splicing, with polymerase II serving as an interface in this event. Therefore, the Pnn/CtBP functional interplay represents a novel mechanism linking the corepressor CtBP and Pnn to the transcription-coupled mRNA splicing of a major tumor suppressor gene. Our findings implicate the existence of the molecular switches involved in tumorigenesis, which coordinate promoter-specific events and mRNA processing, by serving as bridging elements between the regulatory complexes both at gene promoters and within the mRNA splicing machineries.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Gene expression is a complex process involving coordinated events at the promoter regions and during pre-mRNA processing. A number of studies have demonstrated a connection between coactivator or corepressor complexes, operating at the gene promoters, as well as mRNA splicing (2, 3, 12, 60). Furthermore, the mechanism coupling of promoter-associated regulators to mRNA splicing complexes might depend on the promoter architecture and may involve RNA polymerase II (Pol II)-dependent specific interactions (3, 9, 10, 37, 53, 56).

CtBP is a transcriptional corepressor that silences the tumor suppressor gene E-cadherin, which is essential for epithelial cell-cell adhesion (62). CtBP associates with transcriptional repressor complexes involved in gene regulation in varied developmental and oncogenic contexts (7, 15, 17, 19, 22, 29, 30, 39, 40, 41, 46). CtBP is targeted to promoters via sequence-specific DNA-binding transcription factors and, in turn, contributes to gene silencing by recruiting complexes with histone methyltransferase, demethylase, and deacetylase activities (16, 33, 41, 49, 51, 54). For example, CtBP can be recruited to the E-cadherin gene promoter by the repressor ZEB (7, 8, 15, 16, 41, 51, 61). Similarly, the corepressor mSin3A has been demonstrated to interact with CtBP in vivo (23) and target the E-cadherin promoter via its interaction with Snail (38) and as part of the CoREST complex (14, 51).

A distinguishing structural feature of CtBP is a conserved NAD(H)-binding motif. NAD(H) binds to CtBP and regulates its function, thereby promoting tumor cell migration under hypoxic conditions (62). In addition, CtBP-mediated repression and the interaction with transcriptional proteins are regulated by levels of NAD(H), thereby linking CtBP activity to the local metabolic state (4, 13, 22, 26, 32, 55, 57, 61).

Pinin/DRS (Pnn/DRS) is a multifunctional protein that promotes epithelial adhesion properties (20, 35, 36, 47, 50, 52). Pnn also influences the expression of a number of tumor suppressor genes (48, 50). In the nucleus, Pnn interacts with multiple mRNA processing factors such as SRm300, SRp75, SRrp130, and RNPS1 and is involved in mRNA splicing (27, 44, 58, 63). Pnn also associates with the components of the basal transcriptional machinery, such as Mediator and CA150 (45, 53). We have shown that Pnn modulates E-cadherin promoter activity through its NADH-dependent interaction with the corepressor CtBP (1), thus supporting Pnn's role in transcription-specific mechanisms in the context of tumorigenesis.

Here, we investigated the potential mechanism by which the CtBP/Pnn functional interaction can influence E-cadherin gene expression. We demonstrate that CtBP can recruit Pnn to CtBP-associated silencing complexes, in turn resulting in Pnn-dependent effects on chromatin remodeling at the E-cadherin promoter. We also demonstrate that Pnn interacts with the transcriptionally competent form of Pol II and positively influences E-cadherin mRNA splicing, whereas CtBP negatively affects the splicing possible through its modulatory effect on the amount of initiation- and elongation-specific Pol II. These findings carry intriguing implications, that CtBP and Pnn may be involved in tumor progression through both promoter-dependent regulatory events and posttranscriptional mRNA processing.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell lines, cell culture, and transfections. Suspensory HeLa (sHeLa) cells stably expressing a Pnn-Flag-hemagglutinin (HA) fusion protein (HeLa Pnn-Flag-HA) or a CtBP-Flag-HA fusion protein (HeLa CtBP-Flag-HA) were designed as described previously (51). They expressed exogenous Pnn and CtBP proteins at the levels comparable to the levels of the wild-type proteins. Control HeLa cells contained the Flag-HA vector alone.

Cells were transfected by utilizing 3 µl of 1 mg/ml of 25-kDa branched polyethyleneimine (Sigma-Aldrich) per 1 µg of DNA. Polyethyleneimine and DNA were incubated in serum-free Dulbecco's modified Eagle's medium for 10 min in separate tubes. Following incubation, the contents of two tubes were combined, incubated for an additional 10 min, and applied onto cells.

Expression vectors. pCMV-CtBP1-Flag, expressing human CtBP1, and pcDNA3.1-Pnn-myc/His, expressing human Pnn (hPnn), were based on pCMV-Flag (Stratagene) and pcDNA3.1-myc/His (Invitrogen), respectively. The Pnn-His-glutathione S-transferase (GST) fusion construct was based on the Pet 42b(+) vector (Novagen). The ZEB1-myc vector was a gift from A. A. Postigo and D. C. Dean (Washington University). The mSin3A-myc vector was a gift from E. Seto (University of South Florida).

RNA Pol II antibodies. Mouse anti-RNA Pol II H14 antibody recognizes the phosphoserine 5 form of initiation-specific Pol II (Covance), mouse anti-RNA Pol II H5 antibody recognizes the phosphoserine 2 form of elongation-specific Pol II (Covance), mouse anti-RNA Pol II 8WG16 antibody recognizes the hypophosphorylated form of preinitiation Pol II (Covance), and rabbit N-20 antibody recognizes the N terminus of Pol II (Santa Cruz).

Coimmunoprecipitation and immunoblotting. HEK293 cells were transfected with 4 µg of the expression plasmid ZEB1-myc or mSin3A-myc and CtBP1-Flag in the presence or absence of 4 µg of Pnn-green fluorescent protein (GFP). Twenty-four hours posttransfection, cells were washed with cold phosphate-buffered saline and lysed in immunoprecipitation buffer (20 mM HEPES [pH 7.9], 200 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 10 mM KCl, 25% glycerol [vol/vol], 2 mM phenylmethylsulfonyl fluoride, and complete cocktail of protease inhibitors [Roche]).

Coimmunoprecipitations were performed using anti-Flag (Sigma-Aldrich) or anti-myc agarose beads (Sigma-Aldrich). Immunoprecipitates were eluted with sodium dodecyl sulfate (SDS) sample buffer and fractionated by electrophoresis on 8% SDS-polyacrylamide gel electrophoresis (PAGE) gels, followed by Western blotting.

ChIP assays. Chromatin immunoprecipitation (ChIP) was performed according to guidelines from Upstate Biotechnology. ChIPs were performed using Flag M2 agarose (Sigma-Aldrich) or the following antibodies: rabbit anti-acetylated histone H3 at lysine 9 (Upstate), rabbit anti-dimethylated histone 3 at lysine 9 (diMeH3K9) (Upstate), rabbit anti-acetylated histone H at lysine 14 (Upstate), N-20 anti-Pol II (Santa Cruz), and rabbit anti-CBP (Abcam). For the real-time PCRs, primers spanning the E-cadherin promoter region, the E-cadherin intragenic region, the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) promoter region, and the GAPDH intragenic region were utilized.

Isolation of the endogenous Pnn-Pol II complex. sHeLa nuclear (200 µl) lysates were brought up to 500 µl with equilibration buffer (20 mM HEPES [pH 7.9], 150 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 10 mM KCl, 25% glycerol [vol/vol]) and incubated with rabbit N-20 (Santa Cruz) or control immunoglobulin G (IgG) for 2 h, followed by a 1-h incubation with protein A-Sepharose Fast Flow (Amersham Biosciences). Beads were then washed with equilibration buffer and resuspended in SDS loading buffer. Proteins were resolved by 8% SDS-PAGE followed by Western blotting.

Pnn and RNA Pol II complex isolation. Nuclear extracts from sHeLa Pnn-Flag-HA cells expressed tagged Pnn at a ratio to the endogenous Pnn of 1:1 and were prepared as described previously (34). Nuclear extracts from 4 liters of cells were incubated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads (Sigma-Aldrich), followed by washes until no Pnn was released and elution with SDS loading buffer. The samples were subjected to SDS-PAGE followed by Western blotting.

Lysate-dependent kinase and binding reactions. Kinase reactions were performed as described previously (31), with modifications. Isolated nuclei were resuspended in kinase buffer (20 mM HEPES [pH 7.4], 50 mM NaCl, 10 mM MgCl₂, 0.02% NP-40, 1 mM dithiothreitol) and briefly sonicated. Lysates, which also served as the "input" for pull-down experiments, were centrifuged and applied onto recombinant C-terminal domain (CTD)-GST or Pnn-GST immobilized on glutathione agarose in the presence of 1 mM ATP. Reaction mixtures were incubated at 30°C for 1 h with shaking. Control reaction mixtures contained kinase buffer instead of nuclear lysates. Beads were washed three times in kinase buffer and resuspended in SDS buffer. The samples were subjected to SDS-PAGE followed by Western blotting.

Splicing reporters and splicing assays. The E-cadherin exon 4-intron-exon 5 segment was PCR amplified using Pfu polymerase (Stratagene) and genomic DNA as a template. Splicing reporter constructs were based on the pGL-3 basic luciferase reporter (Promega) carrying the –427 +53 E-cadherin basal promoter or the simian virus 40 (SV40) promoter. Luciferase cDNA was excised and replaced with the cassette, which included intact exon 4 (144 bp) and exon 5 (156 bp) of the E-cadherin gene linked by the native intronic sequence (124 bp). E-cadherin[E-cadEx4-Ex5] or SV40[E-cadEx4-Ex5] splicing reporters contained E-cadherin or SV40 promoters driving the E-cadherin exon 4-intron-exon 5 cassette, which was followed by the SV40 poly(A) signal sequence carried over with the pGL-3 backbone.

HEK293 cells were cotransfected with 500 ng of splicing reporters and 1 and 2 µg of Pnn expression vector or 1 µg of Pnn RNA interference (RNAi) short hairpin vector (20) or the GFP RNAi vector as a control. One microgram of the CtBP expression vector was used in the splicing experiments. The total amount of DNA was equalized using an unrelated vector. Twenty-four hours posttransfection, total RNA was isolated using a NucleoSpin kit (BD Biosciences). RNA was additionally treated with RQ1 DNase (Promega) for 1 h, followed by phenol-chloroform extraction. An equal amount of RNA was subjected to reverse transcription (RT) using a cMaster RT kit (Eppendorf) primed with an SV40-specific primer. The resulting cDNA was 26-cycle PCR amplified utilizing E-cadherin exon 4- and exon 5-specific primers. In the case of CD44 splicing assays, 3 µg of total RNA was subjected to RT, followed by 30 cycles of PCR utilizing primers specific for variant and constant exons. The linear amplification range of the PCR products was determined in pilot experiments. PCR products were resolved utilizing a 5% Tris-borate-EDTA-polyacrylamide Criterion gel system followed by staining for detection using SYBR green dye (Molecular Probes). Gels were scanned on a Typhoon 8600 apparatus (Amersham Pharmacia Biotech). The band intensities of the amplicons were quantified utilizing ImageQuant software (Molecular Dynamics). In addition, PCR products corresponding to spliced and unspliced E-cadherin message were excised from the gel and verified by sequencing. For endogenous E-cadherin splicing, 3 µg of total RNA from both wild type (wt/wt) mouse embryonic stem cells and Pnn^(3f/3f) (hypomorh) mouse embryonic stem (ES) cells was subjected to RT, followed by 30-cycle PCR to detect intronic and spliced E-cadherin signal and 25-cycle PCR to detect GAPDH message. PCRs were resolved on a 1.5% agarose gel and stained with ethidium bromide. The band intensities of the amplicons were quantified using ImageJ software.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pnn is recruited to repressor complexes in a CtBP-dependent manner. CtBP binds to transcriptional repressors and recruits chromatin-modifying enzymes, thereby achieving gene silencing (Fig. 1A). In order to investigate the mechanisms behind the Pnn-dependent modulation of CtBP-mediated silencing, we entertained two possible models, which could account for the Pnn-mediated derepression of the E-cadherin promoter (Fig. 1B and C). Pnn may sequester CtBP away from the CtBP-associated proteins, which target the E-cadherin promoter (Fig. 1C). Alternatively, Pnn may be brought to the silencing complexes via its interaction with CtBP and attenuate CtBP-mediated repression (Fig. 1B). To explore these possible outcomes, we focused on two transcriptional silencers, ZEB and mSin3A, which interact with CtBP and target the E-cadherin promoter.

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FIG. 1. Pnn is recruited to CtBP-associated repressor complexes. (A) CtBP bridges silencing machinery and its target gene promoters. (C) Pnn may sequester CtBP from the CtBP-associated repressors. (B) Alternatively, Pnn can be recruited to the repressor complex via CtBP. (D and E) HEK293 cells were cotransfected with vectors expressing ZEB1-myc (D) or mSin3A-myc (E) and CtBP1-Flag in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of hPnn-GFP. Immunoprecipitations (IP) were performed using Flag affinity agarose, followed by Western blotting using anti-Pnn-143, anti-myc, and anti-Flag antibodies. Exogenous hPnn-GFP is indicated by an arrowhead, and endogenous Pnn is indicated by an arrow. An increase in Pnn levels did not appreciably affect the amount of ZEB1 or mSin3A coprecipitated with CtBP (D and E, respectively). Exogenous and endogenous Pnn could be detected in CtBP-Flag immunoprecipitates, indicating that Pnn may be present in ZEB1/CtBP1 and mSin3A/CtBP1 complexes.

HEK 293 cells were cotransfected with vectors expressing either ZEB1-myc (Fig. 1D) or mSin3A-myc (Fig. 1E) along with CtBP1-Flag in either the absence (lanes 1 and 3) or presence (lanes 2 and 4) of hPnn-GFP vector. Subsequently, immunoprecipitations were performed using anti-Flag agarose. Immunoprecipitated material was then subjected to Western blotting using anti-Pnn-143, anti-myc, and anti-Flag antibodies. These experiments revealed that hPnn-GFP expression did not result in changes in the amount of ZEB1 or mSin3A associated with CtBP1, leading us to conclude that Pnn does not drastically affect the interaction of CtBP with either ZEB or mSin3A. Furthermore, both endogenous as well as exogenous Pnn were detected in the CtBP-Flag immunoprecipitates, suggesting that Pnn may be present in the CtBP-dependent silencing complexes (Fig. 1D and E).

Next, we determined whether the binding to CtBP is essential for Pnn's presence at the silencing complexes. HEK293 cells were cotransfected with ZEB1-myc (Fig. 2A) or mSin3A-myc (Fig. 2B) in the presence of RNAi vectors directed against either CtBP1 (Fig. 2B, lanes 2 and 4) or GFP (lanes 1 and 3) as a control. The transient transfection of the RNAi vector for CtBP1 resulted in an approximately 60% reduction in CtBP1 protein levels. Immunoprecipitations from the extracts of the RNAi-treated cells were performed using anti-myc agarose, followed by Western blotting using anti-Pnn-143 and anti-myc antibodies. These experiments revealed that endogenous Pnn was detected in anti-ZEB1-myc and anti-mSin3A-myc immunoprecipitates from control RNAi-treated cells. On the other hand, the amount of Pnn was significantly diminished in the anti-ZEB-myc and anti-mSin3A-myc immunoprecipitates from the extracts of cells treated with CtBP RNAi. These data are consistent with the contention that the association of Pnn with the ZEB1/CtBP and mSin3A/CtBP repressor complexes may be CtBP dependent, as depicted in Fig. 1B.

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FIG. 2. Pnn can be recruited to the ZEB1/CtBP and mSin3A/CtBP complexes via CtBP. HEK293 cells were cotransfected with ZEB1-myc (A) or mSin3A-myc (B) in the presence of the short hairpin-mediated RNAi (shRNAi) vector for CtBP1 (lanes 2 and 4) or GFP RNAi (lanes 1 and 3). Immunoprecipitations (IP) were performed using anti-myc agarose, followed by Western blotting using anti-Pnn-143 and anti-myc antibodies. The amounts of Pnn in ZEB1 and mSin3A precipitates were diminished in the presence of CtBP RNAi, indicating that the Pnn association with the ZEB1/CtBP and mSin3A/CtBP repressor complexes was CtBP dependent. The bottom panels of A and B illustrate the efficiencies of CtBP RNAi in each corresponding experiment.

Pnn can be present at the E-cadherin promoter. Because the expression of Pnn results in the enhanced activity of the E-cadherin promoter (1), we investigated whether or not Pnn can be detected at the promoter region and if the expression of Pnn can affect local chromatin modifications. HeLa cells, both control cells and those stably expressing Pnn-Flag-HA, were subjected to ChIPs utilizing anti-Flag agarose to precipitate chromatin associated with Pnn. The ChIPs were subsequently subjected to real-time quantitative PCR (qPCR) with E-cadherin promoter-specific primers or GAPDH promoter primers as controls (Fig. 3A and B, lane 1). These experiments revealed that Pnn was associated with the E-cadherin but not the GAPDH promoter. Next, HeLa Pnn-Flag-HA or control HeLa cells were subjected to quantitative ChIP (qChIP) analysis utilizing antibodies against CBP acetyltransferase and RNA Pol II, which are frequently enriched at the promoters of actively transcribed genes. We used antibodies against histone 3 acetylated at lysine 9 (AcH3K9) and against histone 3 acetylated at lysine 14 (AcH3K14), which are markers of transcriptionally active chromatin. We also used an antibody against dimethylated histone 3 at lysine 9 (diMeH3K9), which is a marker of silenced chromatin. E-cadherin promoter qChIPs from cells expressing Pnn-Flag-HA revealed enhanced H3K9 and H3K14 acetylation (Fig. 3A, lanes 4 and 5, respectively), an increased presence of CBP and Pol II (Fig. 3A, lanes 2 and 3, respectively), and a decrease in H3K9 dimethylation (Fig. 3A, lane 6) at the E-cadherin promoter compared to those from extracts from control HeLa cells. This was not observed in case of the GAPDH promoter. These results suggest that the presence of Pnn may indeed affect local chromatin modifications in the vicinity of the E-cadherin promoter. Therefore, the presence of Pnn at the E-cadherin promoter appears to positively influence the local chromatin modifications responsible for driving the promoter to a transcriptionally favorable state.

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FIG. 3. Pnn is present at the E-cadherin promoter, where it modulates the transcriptional state of chromatin. HeLa Pnn-Flag-HA cells or control HeLa cells were subjected to ChIP utilizing anti-Flag agarose, or antibodies against CBP, Pol II, AcH3K9, and AcH3K14 ChIPs were amplified using real-time PCR (qChIP) with either E-cadherin promoter (A)- or GAPDH promoter (B)-specific primers Pnn enrichment was detected at the E-cadherin promoter in HeLa Pnn-Flag-HA cells compared to the GAPDH promoter in the ChIP experiments utilizing anti-Flag agarose (lanes 1). In addition, HeLa Pnn-Flag-HA cells exhibited enrichment for CBP (A, lane 2), Pol II (A, lane 3), acetylated histone 3K9 (A, lane 4), and acetylated histone 3K14 (A, lane 5) at the E-cadherin promoter compared to control HeLa cells. The same degree of enrichment was not observed in the case of the GAPDH promoter (B). In contrast, the presence of diMeH3K9, a marker for silenced chromatin, was diminished in HeLa Pnn-Flag-HA cells compared to control HeLa cells. qChIP data are expressed as enrichment relative to the IgG control antibody. The enrichment in the case of HeLa Pnn-Flag-HA cells was expressed relative to the enrichment in the case of control HeLa cells, which was assigned a value of 1. Bars represent standard deviations of data from duplicate experiments.

Pnn can associate with transcriptionally competent Pol II. The observation that Pnn expression correlates with an increased presence of RNA Pol II at the E-cadherin promoter prompted us to investigate the potential involvement of Pnn in basal transcriptional machinery. In order to determine whether endogenous Pnn and Pol II can be coisolated, we utilized HeLa nuclear extracts to perform immunoprecipitations with anti-Pol II (N-20) antibody, followed by Western blotting with anti-Pol II (N-20) and Pnn-143 antibodies. Pnn was detected in the Pol II precipitates, indicating that these two components may indeed associate in vivo (Fig. 4A).

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FIG. 4. Pnn interacts with the transcriptionally competent pool of Pol II, which can be depleted by CtBP. (A) Coimmunoprecipitations from the HeLa nuclear extracts using anti-Pol II (N-20) antibody followed by Western blotting using N-20 and anti-Pnn-143 antibodies. Pnn and Pol II were found to be associated. (B) Nuclear extracts from the HeLa Pnn-Flag-HA cells were used for immunoprecipitation using anti-HA antibody followed by Western blotting for HA, the preinitiation form of Pol II (CTD), initiation-specific Pol II (Pol II P-Ser5), and elongation-specific Pol II (Pol II P-Ser2). Pnn was found to be associated with transcriptionally competent forms of Pol II but not with the preinitiation form of Pol II. (C) Western blotting of control HeLa or HeLa CtBP-HA nuclei resuspended in SDS buffer. N-20 antibody detected a lower amount of hyperphosphorylated Pol II (IIo) in HeLa CtBP Flag-HA nuclei. Amounts of initiation-specific Pol II and elongation-specific Pol II were also diminished in the nuclei of CtBP-expressing cells. Nuclear lamins served as a loading control. (D) CTD-GST and Pnn-GST were subjected to the lysate-depended kinase reaction using control HeLa or HeLa CtBP-Flag-HA nuclear lysates. CTD-GST (top) but not Pnn-GST (bottom) exhibited a less pronounced molecular weight shift in HeLa CtBP Flag-HA lysates due in part to diminished Ser-5 phosphorylation (middle). Quantitation of the band intensity was performed using Adobe Photoshop software. (E) CTD-GST pull-downs from the kinase reactions were screened for bound proteins (as described in Materials and Methods). There were fewer Pnn and SR proteins bound to CTD in HeLa CtBP Flag-HA extracts.

Next, we assessed if Pnn preferentially associates with a particular form of Pol II. Functionally unengaged (preinitiation) Pol II is hypophosphorylated at its CTD, while initiation-specific Pol II is phosphorylated predominantly at Ser-5 of the CTD, and elongation-specific Pol II is phosphorylated predominantly at Ser-2 of the CTD (11, 18, 28, 42, 43, 64). Nuclear extracts from the HeLa Pnn-Flag-HA cells were subjected to immunoprecipitation using anti-HA antibody, followed by Western blotting using anti-HA and antibodies directed against different forms of Pol II (Fig. 4B). Pnn immunoprecipitates contained both initiation- and elongation-specific Pol II but not the preinitiation form of Pol II. These data thus suggest that Pnn associates predominantly at the transcriptionally active RNA Pol II complexes.

CtBP can affect the Pol II phosphorylation status. Because Pnn interacts with both CtBP and Pol II, we wanted to determine whether or not CtBP can affect the Pnn/Pol II association. We performed coimmunoprecipitations using antibodies against initiation- and elongation-specific Pol II from the nuclear extracts of HeLa or HeLa CtBP-Flag-HA cells. However, we consistently found less phosphorylated Pol II in the precipitates from the HeLa CtBP-Flag-HA cells than in control HeLa cells (data not shown). Therefore, in order to determine whether CtBP can affect the degree of Pol II phosphorylation, we lysed the same number of nuclei from HeLa cells or HeLa CtBP-Flag-HA cells in SDS-containing buffer and subjected samples to SDS-PAGE. Interestingly, lysates of HeLa CtBP-Flag-HA nuclei contained moderately smaller amounts of initiation-specific Pol II and significantly less elongation-specific Pol II than did lysates of control HeLa nuclei (Fig. 4C). N-20 antibody against the N terminus of Pol II, which recognizes both hyperphosphorylated (IIo) and hypophosphorylated (IIa) forms of Pol II, demonstrated an increase in the ratio of hypophosphorylated to hyperphosphorylated forms of Pol II in HeLa CtBP-Flag-HA nuclei compared to control HeLa cell nuclei (Fig. 4C).

Next, we determined if CtBP has an effect on the phosphorylation status of the C-terminal domain of Pol II in vitro. We adopted the protocol based on the in vitro phosphorylation reaction of the C-terminal domain of Pol II, which utilizes the recombinant CTD as a substrate and nuclear extract as a source of kinase activity (31). We incubated recombinant CTD-GST and Pnn-GST, which served as substrates, with either HeLa or HeLa CtBP-Flag-HA nuclear extracts, which served as sources of kinase activity, in the presence of kinase buffer. We chose Pnn as a control substrate because it is a known SR-like phosphoprotein that carries a C-terminal stretch of serine residues, which are heavily phosphorylated on SR protein family members. Antibody against GST was used on Western blots to detect a CTD molecular weight shift after incubation with nuclear extracts due to lysate-dependent posttranslational modifications (Fig. 4D). Interestingly, incubation with HeLa CtBP-Flag-HA nuclear extracts resulted in a smaller shift of the CTD than that elicited by control HeLa nuclear extracts. Consistent with the SDS-PAGE gel shift, Western blots for CTD phosphorylated at Ser-5 detected less Ser-5 CTD phosphorylation subsequent to incubation with HeLa CtBP-Flag-HA nuclear extracts than did control HeLa nuclear extracts. Unfortunately, anti-phosphorylated Ser-2 antibody exhibited possible cross-reactivity to GST and was therefore omitted during the experiments. In contrast, when we utilized recombinant Pnn-GST as a substrate, we detected a more prominent molecular weight shift of Pnn in reactions utilizing HeLa CtBP-Flag-HA nuclear extracts than in those utilizing control HeLa nuclear extracts. These data suggest that CtBP might have a specific negative effect on the Pol II-CTD phosphorylation status.

Most interestingly, when CTD-bound material from the nuclear lysate-dependent kinase reactions was Western blotted for Pnn and members of the SR family of splicing factors, we found less Pnn and SR proteins associated with Pol II-CTD in HeLa CtBP-Flag-HA lysates than in control HeLa lysates (Fig. 4E). Because Pnn and SR proteins target predominantly phosphorylated CTD, it is tempting to speculate that CtBP negatively affects these interactions by either directly or indirectly inhibiting CTD phosphorylation.

CtBP and Pnn differentially affect mRNA splicing. Because Pnn is involved in pre-mRNA processing (27, 58), we considered the possibility that Pnn expression can impact the efficiency of E-cadherin mRNA splicing. To examine this, we created splicing reporter constructs, which carried the intact E-cadherin exon 4-intron-exon 5 cassette driven by either the E-cadherin promoter or the SV40 promoters (Fig. 5). HEK293 cells were cotransfected with splicing reporters along with increasing amounts of Pnn. Increasing the expression of Pnn resulted in a greater ratio of spliced to total mRNA when the reporter was driven by the E-cadherin promoter (Fig. 5A, left). However, no Pnn-induced increase of splicing was observed with SV40 promoter-driven constructs (Fig. 5A, right). Consistent with these observations, splicing assays conducted in the presence of Pnn RNAi demonstrated a reduction in the ratio of spliced to total mRNA with the E-cadherin-driven vectors (Fig. 5B, left) but not with SV40 promoter-containing constructs (Fig. 5B, right). These data suggest that Pnn may be capable of modulating E-cadherin mRNA splicing efficiency in a promoter-specific manner. Next, we wanted to determine if Pnn has an effect on endogenous E-cadherin splicing. For this purpose, we utilized mouse ES cells carrying the insertion of a neomycin resistance cassette into intron 8 of Pnn (3f/3f cells), which results in a hypomorphic knockdown of Pnn (approximately 90%) (21). Splicing assays were performed using RT-PCR, where unspliced message was detected using primers against E-cadherin intron 2, whereas total spliced E-cadherin message was detected using primers spanning the region between exons 4 and 8 (Fig. 5C). The 3f/3f cells exhibited lower levels of E-cadherin message than did wild-type (wt/wt) ES cells, supporting the role of Pnn in the regulation of E-cadherin expression. Furthermore, the 3f/3f cells displayed an accumulation of the intronic amplicon, which resulted in an increased ratio of unspliced to total mRNA message compared to wt/wt cells (Fig. 5C, bottom). These data suggest that in 3f/3f cells, splicing is hindered compared to that in wt/wt cells. For cotranscriptional splicing to occur, splicing factors are recruited to the processive RNA Pol II as it progresses along the transcribed gene. Thus, we determined whether Pnn is present within the E-cadherin open reading frame. HeLa Pnn-Flag-HA cells were subjected to ChIP utilizing anti-Flag affinity agarose followed by qPCR using E-cadherin intragenic primers or GAPDH intragenic primers as a control (Fig. 5D). Pnn was enriched in the E-cadherin intragenic region but not in the GAPDH intragenic region. These data, together with Pnn/Pol II interaction studies, support Pnn's role in E-cadherin mRNA processing, possibly through its interaction with transcribing Pol II.

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FIG. 5. Pnn can modulate E-cadherin mRNA splicing efficiency. (A) HEK293 cells were cotransfected with E-cadherin[E-cadEx4-Ex5] (left) or SV40[E-cadEx4-Ex5] (right) splicing reporters and increasing amounts of Pnn. The splicing efficiency was calculated as a ratio of intensities of the amplicons corresponding to the spliced mRNA to the sum of unspliced and spliced mRNA (total mRNA). Increasing Pnn expression resulted in an increased ratio of spliced mRNA to total mRNA in the context of the E-cadherin promoter but not the SV40 promoter. (B) Transfection of the Pnn RNAi vector resulted in a decreased ratio of spliced mRNA to total mRNA in the context of the E-cadherin promoter but not the SV40 promoter. Bars represent standard deviations of data from three independent experiments. (C) Mouse ES cells carrying an insertion of the neomycin resistance cassette into intron 8 of Pnn (3f/3f cells) or wild-type ES cells (wt/wt) were utilized as the source of RNA for splicing assays using RT-PCR. Unspliced message was detected using primers against E-cadherin intron 2, and total spliced E-cadherin message was detected using primers spanning the region between exons 4 and 8 (top). Splicing efficiency was calculated as a ratio of the intensity of the intronic amplicon to that of total spliced E-cadherin message (bottom). The ratio in the case of wt/wt cells was assigned a value of 1. 3f/3f cells exhibited an increase in the ratio of the intronic amplicon to total E-cadherin message compared to wt/wt cells, suggesting that in 3f/3f cells, E-cadherin splicing is inhibited. Bars represent standard deviations of data from duplicate experiments. (D) HeLa Pnn-Flag-HA cells were subjected to ChIP utilizing anti-Flag agarose followed by qPCR using E-cadherin intragenic primers or GAPDH intragenic primers as a control. Pnn enrichment was detected in the E-cadherin intragenic region but not the GAPDH intragenic region. The enrichment was expressed relative to the IgG control antibody, which was assigned a value of 1. Bars represent standard deviations of data from three experiments.

Because CtBP can negatively affect the amount of elongation-specific Pol II as well as decrease the binding of positive regulators of splicing, such as Pnn and SR proteins, to Pol II-CTD, we next sought to determine whether CtBP can also affect E-cadherin mRNA splicing. In contrast to the Pnn-dependent positive impact on splicing, splicing reactions performed in the presence of CtBP resulted in a reduction of the ratio of spliced to total E-cadherin mRNA, indicating that CtBP can negatively affect splicing efficiency (Fig. 6A).

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FIG. 6. CtBP can negatively modulate E-cadherin mRNA splicing and can promote CD44 variant exon inclusion. (A) Splicing reactions using the E-cadherin splicing reporter were performed in the presence of the CtBP-Flag vector. CtBP-Flag expression resulted in a decreased ratio of spliced mRNA to total mRNA. Histograms on the right represent the ratio of spliced mRNA to total mRNA obtained from three independent assays. Bars represent standard deviations. (B) RT-PCR analysis of CD44 variant exon inclusion utilizing HeLa or HeLa CtBP-Flag-HA cells. RT reactions were subjected to PCR using upstream primers specific for variant exons 4, 5, 6, 7, and 8 (v4, v5, v6, v7, and v8, respectively) and a downstream primer (c3') that anneals to a constitutive downstream 3' region. Primers that anneal to the constitutive 5' and 3' regions (c5' and c3', respectively) were used to detect a standard CD44 form. (C) PCRs were run on a polyacrylamide gel followed by the quantification of the PCR band intensity (bottom). The degree of variant exon inclusion was calculated as a ratio of the intensity of amplicons of the variant exons to that of the amplicons of the standard CD44 form. The ratio of each variable amplicon (v4, v5, v6, v7, and v8) to a standard CD44 form in case of control HeLa cells was assigned a value of 1. HeLa CtBP-Flag-HA cells displayed increased expression of CD44 variants containing variable exons v4 and v5 but not v6 to v8 compared to control HeLa cells (C). Histograms represent average values of the ratios of each variable exon (v4, v5, v6, v7, and v8) to a standard CD44 form obtained from three independent experiments. Bars reflect standard deviations of data from three independent experiments.

We next examined whether CtBP can impact the splicing pattern of the alternative exons. We reasoned that because CtBP can modulate Pol II phosphorylation and inhibit constitutive splicing, CtBP may also exert an effect on alternative splicing, as those events are functionally linked (11, 42, 43). Therefore, we examined human CD44, a gene regulated through alternative splicing and involved in metastasis. This gene consists of 10 constitutive exons and a cluster of nine variable exons, which confer significant variability to the CD44 splice variants, often resulting in enhanced metastasis (5, 6). Examination of control HeLa and HeLa CtBP-Flag-HA cells for CD44 splicing patterns using primers against variable exons v4, v5, v6, v7, and v8 (Fig. 6B) revealed a CtBP-dependent differential modulation of various CD44 isoforms (Fig. 6C). For example, CtBP expression promoted the inclusion of v4 and v5 exons but not v6, v7, and v8 exons (Fig. 6C). These data demonstrate that CtBP is capable of modulating alternative splicing events. However, the functional impact of this CtBP-dependent alteration of splicing on the metatstatic potential of cells awaits further investigation.

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Our results provide a novel insight into the regulatory cascade involving corepressor CtBP and Pnn, which modulate the expression of a tumor suppressor gene at different control points via basal transcriptional and splicing machineries. These data support the contention that various aspects of gene expression are affected by a number of multifunctional proteins and that the expression level of each protein can have profound consequences on diverse processes and complex interconnections within the gene expression machinery of the living cell.

In the complex set of events which govern the regulation of E-cadherin gene expression, we were able to track functional interactions of the corepressor CtBP and Pnn at the promoter via the repressors ZEB1 and mSin3A, which are known to associate with CtBP, and target E-cadherin (7, 8, 14, 15, 16, 23, 38, 41, 51, 61).

The fact that Pnn, a protein with no known role in transcriptional repression, is recruited to the CtBP-dependent complexes implies that the composition of the repression apparatus may not be limited to proteins involved directly in gene silencing but rather may include proteins that function in mRNA processing and even factors capable of reversing the silencing effect. Indeed, we demonstrate that the presence of Pnn at the E-cadherin promoter correlated with increased histone H4 acetylation, a decrease in histone H3K9 dimethylation, as well as the increased presence of RNA Pol II, which are correlated with transcriptionally active chromatin. These findings provide indirect support for the context-dependent effect of regulatory complexes on a target gene. For example, a protein complex can deliver a silencing or activating effect on the gene promoter, depending on its factor composition. Indeed, this postulate parallels a newly described model showing a cofactor-mediated specificity for histone demethylation exerted by a CtBP-associated factor, LSD1 (49). For instance, LSD1 may act as an activator or repressor depending on the constituents of the LSD1-associated protein complex (59). Therefore, Pnn might be capable of attenuating CtBP-mediated repression by being recruited to the CtBP complex, where CtBP serves as a bridging molecule.

The idea that CtBP and Pnn may directly or indirectly coordinate various aspects of gene expression is substantiated by our findings that Pnn and CtBP have differential effects on the mRNA splicing efficiency. By utilizing a splicing reporter construct driven by the basal promoter of E-cadherin and containing the exon 4-intron-exon 5 cassette, we demonstrated that Pnn expression enhances E-cadherin mRNA splicing efficiency. Unexpectedly, yet perhaps more interestingly, we found that CtBP inhibits the correct splicing of E-cadherin mRNA and promotes the inclusion of alternative exons of the CD44 gene. Furthermore, CtBP inhibited Ser-5 and Ser-2 phosphorylation of the C-terminal domain of RNA Pol II, leading to the depletion of the nuclear pool of initiation-specific and elongation-specific Pol II. We speculate that in addition to its silencing effect through chromatin remodeling, CtBP can affect gene expression through altering Pol II phosphorylation and preventing the assembly of regulators of splicing, such as Pnn, on the elongating Pol II. The fact that Pnn has indeed been described as being part of the Pol II-associated complexes involved in transcriptional initiation and elongation, such as Mediator and CA150 (45, 53), further supports the idea of the CtBP-dependent influence on basal transcriptional machinery.

Therefore, CtBP and Pnn might have differential effects on E-cadherin gene expression through the association with regulatory complexes at the E-cadherin promoter as well as through their effect on splicing, linking promoter-related events and transcription-coupled mRNA processing. These findings resonate recent data describing the promoter-dependent control of splicing decisions exerted by hormone receptor coregulators (3, 24) as well as regulation of splicing by the chromatin-remodeling factor Brm (5, 25).

The conceptual mechanism of transcription-coupled splicing involving CtBP and Pnn can be visualized as efficiency-driven equilibrium, where CtBP modulates gene expression through transcriptional as well as splicing-related protein interactions. In this situation, gene repression can be coordinated through a CtBP-mediated effect at the gene promoters along with its negative influence on the recruitment of the splicing machinery by Pol II. This coupling delivers an efficient "brake" control to the CtBP target genes. It is unclear at this point whether the CtBP-mediated recruitment of the silencing factors to the gene promoters proceeds with a similar or equal rate compared to the dissociation of splicing factors from Pol II. However, it is likely that because CtBP serves as a common interface between these two events, these processes may progress in a compatible manner and are subject to orchestrated regulation by CtBP.

The CtBP-mediated effect on the splicing apparatus carries an important implication with respect to the mechanisms involved in the control of tumor suppressor genes such as E-cadherin and CD44. The fact that CtBP inhibits mRNA processing of a major tumor suppressor gene, E-cadherin, stresses the importance of thorough examinations of tumor-promoting transcriptional repressors that might in fact confer metastatic potential to the transformed cells through both transcriptional inhibition and modulation of mRNA splicing. In addition, by promoting the inclusion of the CD44 alternative exons, CtBP might also influence tumor progression. It is widely accepted that CD44 splice variants heavily influence the metastatic states of numerous tumors (5, 6). Our observations that CtBP influences the inclusion of some of the alternative CD44 exons provide additional evidence of CtBP-mediated posttranscriptional events known to be involved in tumorigenesis. Further detailed analysis of CtBP's impact on the splicing of CD44 and its consequences with respect to cellular metastasis is certainly in order. Importantly, in this context, gene expression profiles of various tumors based on microarray technology alone, which may not provide significant insight into the differential gene expression patterns, might not adequately address the possibility that a significant number of genes may also be regulated through posttranscriptional mRNA processing events. Therefore, approaches such as splice variant-specific microarrays might become increasingly relevant in correlative gene expression profiling, which links transcriptional repressors, such as CtBP, and the tumor-specific potential to metastasize.

Taken together, our data suggest that the differential regulation of tumor suppressor genes, especially under pathological conditions such as cancer, might be heavily influenced by the transcription-associated bridging proteins that are capable of coordinating promoter-specific and mRNA splicing events.

In conclusion, we propose the concept of the functional coupling of processes governing gene expression where CtBP and Pnn serve as an example of unique protein factors that contribute to promoter-related complex interactions as well as impact mRNA processing (Fig. 7). This idea provides an exciting opportunity for pharmacological targeting of various cancers, whereby a carefully designed targeting strategy directed toward a single key regulatory molecule will have a cumulative and hopefully beneficial effect at multiple control points.

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FIG. 7. Diagram depicting a possible mechanism of Pnn and CtBP involvement in multiple steps of regulation of E-cadherin gene expression. (A) CtBP participates in the repression of the E-cadherin gene. (B) Pnn may be recruited to CtBP-associated repressor complexes via CtBP, where Pnn can attenuate CtBP-mediated repression. (C) CtBP and Pnn may also regulate E-cadherin gene expression at the mRNA level, where Pnn might couple transcription and splicing. Importantly, CtBP might modulate the degree of association of Pnn as well as other splicing factors with Pol II through the modulation of Pol II phosphorylation, thus affecting mRNA processing events.

 
We declare that there are no competing financial interests.

We thank A. A. Postigo and D. C. Dean (Washington University) for the ZEB1-myc vector and E. Seto (University of South Florida) for the mSin3A-myc vector.

This work was supported by NIH grant EY07883 to S.P.S.

 
* Corresponding author. Mailing address: Department of Anatomy and Cell Biology, University of Florida College of Medicine, 1600 SW Archer Road, Box 100235, Gainesville, FL 32610. Phone: (352) 392-3569. Fax: (352) 392-3305. E-mail: Sugrue{at}anatomy.med.ufl.edu

Published ahead of print on 17 December 2007.

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Molecular and Cellular Biology, March 2008, p. 1584-1595, Vol. 28, No. 5
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