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Keywords:

  • estrogen receptor;
  • WW-domain binding protein 2;
  • coactivator;
  • transactivation;
  • p300;
  • histone modifications

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The link between breast cancer and estrogen receptor (ER) is well established. The ER is a hormone-inducible transcription factor that, upon binding to its ligand, regulates the expression of a variety of genes mainly involved in cell proliferation and differentiation. Coactivators are proteins recruited by the hormone-activated receptor, which allow or enhance the ER transactivation functions by acting as chromatin remodeling enzymes or adaptors between ER and the transcriptional machinery. Our laboratory has previously identified the WW-domain binding protein-2 (WBP-2) as a bona fide coactivator of ER. However, the molecular mechanism underlying WBP-2 coactivation function was not clear yet. In this study, we explore and identify the mechanism by which WBP-2 acts as coactivator of ER. Our data show that WBP-2 is involved in the regulation of ER target genes, and its expression is required for the proper expression of some ER target genes. To clarify the molecular mechanism by which WBP-2 regulates ER function, we performed chromatin immunoprecipitation assays. We demonstrate here that WBP-2 binds to the ER target gene promoter pS2 promoter and is required for the binding of the phosphorylated form of RNA polymerase II (associated with active transcription/elongation) to the same promoter. Furthermore, we also show that WBP-2 is essential for the recruitment of the histone acetyl transferase p300, an important chromatin modifier enzyme and for histone acetylation at the same target region. Collectively, our data indicate that WBP-2 enhances ER transactivation function at certain genes by facilitating the recruitment and/or the stabilization of a histone modifier enzyme that favors a relaxed chromatin structure, permissive of transcription. © 2012 IUBMB Life, 65(1):76–84, 2013


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Modulation of gene transcription is one of the ways each cell of an organism can control its gene expression and is achieved through the formation of distinct and transitory protein and/or RNA complexes, which bind to specific DNA sequences, allowing for differential messenger RNA (mRNA) synthesis. It is now clear that a plethora of transcription factors, coregulators and a more limited (as of today) number of regulatory noncoding RNAs assist RNA polymerase II (RNA Pol-II) in mRNA transcription initiation, elongation and/or termination (1–4). Transcriptional coactivators, in particular, regulate the expression of target genes by indirect binding to their target gene promoters and other cis-regulatory elements (such as enhancers and locus control regions), allowing or aiding mRNA transcription (5–7). Coactivators can bind transcription factors or RNA Pol-II augmenting their activities, or helping the recruitment of chromatin modifying enzymes, or acting as chromatin modifiers themselves, with the result of creating a more relaxed chromatin environment, permissive of transcription. In such ways, coactivators act as regulators in multiple processes and are able to generate tissue-, cell- and promoter-specific effects (8–11).

Steroid hormone receptors are members of a superfamily of ligand-dependent transcription factors, and estrogen receptor alpha (ERα, form here on referred to as ER) is one of such receptors (12–14). It is particularly relevant as it is strongly implicated in breast cancer development and progression. Specifically, in estrogen-responsive cells, ER has an important role in regulating proliferation and differentiation and inhibition of ER is a major strategy for the treatment of breast cancers, at least for those that are ER positive (15, 16). Upon estrogen binding, ER undergoes conformational changes: it dimerizes, translocates to the nucleus and binds to specific DNA sequences (called estrogen response elements) through its DNA-binding domain, at specific gene promoters. Finally, through its transactivation domain, ER aids the basal transcriptional machinery (RNA Pol-II and general transcription factors) in the transcription of estrogen responsive genes (17). Thismechanism is common to all steroid hormone receptors. Coregulators (coactivators and corepressors) play a key role in the regulation of steroid receptor transactivation functions. As of today, more than 350 different coregulators have been identified; they form transitory and multiprotein complexes with other coregulators and the hormone receptors and are essential for proper steroid hormone receptor functions (18, 19).

WW-domain binding protein 2 (WBP-2) has been identified, through a functional screen of a cDNA library, as a binding partner of Yes kinase-associated protein (YAP) (20, 21). YAP is a transcriptional regulator and a component of the Hippo pathway that, originally studied in Drosophila and conserved also in mammals, is important for regulation of cell growth, proliferation and tumorigenesis (22, 23). The binding between WBP-2 and YAP occurs through interaction between WW-domains (two in YAP) and PPXY-motifs (three in WBP-2) (24). The WW-domain is a small module that forms a binding pocket for the PPXY-motif; the name refers to two signature tryptophan (W) residues 20–22 amino acids apart in most of the WW-domains. The PPXY-motif (P, proline, X, any amino acid, Y, tyrosine) is a short motif located within a proline-rich domain (21, 25, 26). Through a similar interaction, WBP-2 interacts with TAZ (transcriptional co-activator with PDZ-binding motif) a structurally and functionally similar protein to YAP and also a component of the Hippo pathway (27, 28); with WW-domain containing oxidoreductase 1 (WWOX1), a protein that has been identified as a putative tumor suppressor (29–31); and with Nedd4, an ubiquitin-protein ligase (32). WBP-2 can also interact with proteins that do not contain WW-domains, as in the case of Pax8 (Paired box gene 8), a thyroid-specific transcription factor (33), or E6-associated protein (E6-AP), an E3 ubiquitin ligase and a coactivator of ER (34). In this case, the interaction may not require the PPXY motifs of WBP-2. Despite the identity of some of its binding partners, little is known about the in vivo functions of WBP-2. Some studies indicate that WBP-2 is a downstream component of the Hippo pathway (35), or state its role for the oncogenic properties of TAZ (36), but the precise mechanism by which WBP-2 exerts these functions is still unclear. Our laboratory has previously identified WBP-2 as a bona fide coactivator of ER. Luciferase reporter gene assays demonstrated indeed that WBP-2 enhances ER transactivation activity (34). However, the exact mechanism by which WBP-2 enhances ER functions remains unknown. In this study, we investigate the molecular mechanism underlying WBP-2 coactivation functions. We demonstrate the role of WBP-2 in regulating ER target gene expression and highlight the mechanism by which this occurs. Specifically, WBP-2 enhances ER transactivation function at certain gene promoters by facilitating the recruitment/retention of the histone modifier enzyme p300. This, upon histone acetylation, favors a relaxed chromatin structure, permissive of transcription.

Experimental Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cell Culture and Reagents

MCF-7 cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle medium (DMEM) high glucose (Invitrogen, San Diego, CA) with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 100 μg/ml penicillin/streptomycin (Invitrogen), at 37 °C in a humidified 95% air/5% CO2 atmosphere. Cells were proven to be mycoplasma-free by routine testing with Lookout Mycoplasma detection kit (Sigma, St. Louis, MO). Prior to estrogen stimulation, cells were starved for 72 h in phenol-free DMEM high glucose (Invitrogen) with 10% charcoal/dextran-treated fetal bovine serum (Atlanta Biologicals), 100 μg/ml penicillin/streptomycin (Invitrogen). 17β-Estradiol/estrogen (Sigma) was prepared in ethanol and added to the described starvation media for the indicated time at a final concentration of 10−8 M. In this article, the terms hormone and estrogen are used interchangeably.

Plasmid Reverse Transfection

MCF-7 cells were transfected using LipofectamineLTX and Plus reagents (Invitrogen). 4 × 105 cells in complete medium were reverse transfected, in six-well plates, with DNA–Lipofectamine complexes containing 1 μg DNA, 1 μl Plus reagent, 6 μl LipofectamineLTX according to the manufacturer's recommendations. Efficiency of transfection was assessed at every transfection by parallel transfection of a Green Fluorescent Protein (GFP) expressing plasmid. Starvation of the cells was started 16 h post-transfection.

small interfering RNA (siRNA) Reverse Transfection

siRNA was purchased from Dharmacon (Lafayette, CO), as ON-TARGETplus pools: mixtures of four siRNA provided as a single reagent and proven to be both effective and specific in the knockdown. MCF-7 cells were transfected with LipofectamineRNAiMAX reagent (Invitrogen). 6 × 105 cells in complete medium were reverse transfected, in six-well plates, with siRNA–Lipofectamine complexes containing 20 pmol siRNA ON-TARGETplus SMARTpool for hWBP-2 or siRNA ON-TARGETplus nontargeting pool (Dharmacon, cat # L-017572-00-10 and D-00810-10-20, respectively) and 2.5 μl LipofectamineRNAiMAX according to the manufacturer's recommendations. Alternatively, 6 × 106 cells were reverse transfected, in p100 plates, using 100 pmol siRNA ON-TARGETplus SMARTpool for hWBP-2 siRNA ON-TARGETplus nontargeting pool and 12.5 μl LipofectamineRNAiMAX. Starvation of the cells was started 16 h post-transfection.

Total RNA Isolation and Reverse Transcription

Total RNA was isolated from cells grown in six-well plates using the QIAshredder (Qiagen, Valencia, CA), and the RNeasy Mini Kit (Qiagen), following the manufacturer's instructions. 2 μg of total RNA was reverse transcribed using the Maxima First Strand cDNA Synthesis kit (Fermentas, Waltham, MA), according to the manufacturer's protocol but performing the reverse transcription step of the reaction at 55 °C.

Quantitative Polymerase Chain Reaction with cDNA

Each polymerase chain reaction (PCR) was prepared with 25 ng of cDNA, 300 nM forward primer, 300 nM reverse primer, 1× iQ SYBR Green Supermix (Bio-Rad, Hercules, CA)) in a final volume of 10 μl. Reactions were run in triplicates in a LightCycler 480 (Roche, Indianapolis, IN) using the following program: 95 °C, 3 min (4.8 °C/sec); 40 cycles of 95 °C, 10 sec (4.8 °C/sec)—60 °C, 1 min (2.5 °C/sec); melting curve recorded with 95 °C, 1 min (4.8 °C/sec)—55 °C, 1 min (2.5 °C/sec)—95 °C, 0.11 °C/sec (5 acquisitions/sec).

Induction values were calculated with the formula:

  • equation image

according to the Pfaffl method to quantify quantitative PCR (qPCR) results (37) and using 36B4 as the reference gene. Primers used were: 36B4for: 5′-GACAATGGCA GCA TCTAC-3′; 36B4rev: 5′-AAGGTGTAATCCGTCTCC-3′; WBP-2for: 5′-CTGGTC-TGTGCTGGTCTC-3′; WBP-2rev: 5′-AG GGA AGGGAAGGAAGGG-3′; pS2for: 5′-GCGCCCTGGTCCT GGTG TCCAT-3′; pS2rev: 5′-GAAACCACAATTCTGTCTT TC AC-3′; GREB1for: 5′-ATCAGCTGCTCGGACTTGCTG-3′; GR EB1rev: 5′-TGAGCTCCGGTCCTGACAGATG-3′; PRfor: 5′-CC CACAA TACAGCTTCGAGTC-3′; PRrev: 5′-GCGGATTTTA TC AACGA TGCAG-3′.

Cell Lysates Preparation and Western Blot

Cells were washed once with phosphate buffered saline (PBS) and lysed in radio immunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor Cocktail (Sigma), as previously described (38). Cell lysates were separated by SDS/PAGE and transferred to nitrocellulose membrane at 100 V for 2 h or 40 mA overnight (ON), at 4 °C. Membranes were blocked with blocking solution (4% non-fat dry milk, 0.1% Tween-20 in PBS) for 1 hour at room temperature (RT) and incubated with the primary antibody (in 3% albumin fraction V (Sigma), 0.1% Tween-20, 0.02% Na-azide in PBS for 2 h at RT, or ON at 4 °C. Primary antibodies used WBP-2 (N-14) (sc-160905, Santa Cruz Biotechnology, Santa Cruz, CA) (1:500) and β-tubulin (ab522339, Abcam, Cambridge, MA)) (1:2,000). After washing three times with the blocking solution, membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated antibody (anti-goat from Santa Cruz Biotechnology, anti-rabbit from Bio-Rad) (1:4,000) in the blocking solution for 1 h at RT, washed twice with 0.1% Tween-20 in PBS and once with PBS. Protein signals were detected by chemiluminescence, using the SupersignalWest Pico Substrate (Pierce, Rockford, IL) and exposing the membranes to X-ray films. Protein gel images were arranged using Adobe Photoshop 7 and Adobe Illustrator CS softwares.

Chromatin Immunoprecipitation

The buffers used have been previously described (38). Cells grown in p100 plates were fixed with 1% formaldehyde PBS for 10 min, quenched with 125 mM glycine in PBS for 5 min and washed twice in ice-cold PBS for 5 min. Cells were then scraped in ice-cold PBS containing PMSF and protease inhibitor Cocktail, spun and resuspended in lysis buffer with PMSF and protease inhibitor for 20 min rocking, at 4 °C. Nuclear fraction was then collected by centrifuging at 2,655 relative force centrifuge (rcf) at 4 °C and resuspended in 600 μl of shearing buffer with PMSF and protease inhibitor. Chromatin was sheared using a Misonix S-4000 sonicator (Qsonica, Newtown, CT) with a 419 tip, set at 20% amplitude, with 12 pulses of 10 sec each (total energy transferred per sample was ∼650 J, which allows shearing the chromatin into 200–500 base pair fragments). After centrifugation at 20,800 rcf for 12 min at 4 °C, sheared chromatin was used for the IP or stored up to 2 months at −80 °C. 50 μl of the sheared chromatin was kept as input. For the IP, 100 μl of sheared chromatin was diluted 10-fold in buffer Y and precleared with 2 μg of appropriate IgG antibody and 40 μl of salmon sperm DNA/Protein A or G (depending on the antibody isotype) agarose beads (Millipore, Billerica, MA) for 2 h at 4 °C, rocking. Beads were spun at 106 rcf for 1 min at 4 °C, and supernatant was moved to a fresh tube. 2 μg of specific antibody was then used for ON IP at 4 °C, rocking. The day after, salmon sperm DNA/Protein A or G agarose beads were added for 2 h. Beads were washed once in TSEI, once in Tris/Sucrose/Ethylenediaminetetraacetic acid-EDTA (TSE) I, once in TSEIII and twice in Tris/EDTA (TE), 2 min for each wash, rocking. DNA was then eluted in 150 μl of elution buffer, rocking for 20 min, room temperature. Eluted chromatin and input chromatin (50 μl input + 100 μl water) were then reverse crosslinked by adding NaCl to a final concentration of 200 mM and incubating in a water bath, ON at 65 °C. Proteins were removed by bringing samples to a final concentration of 10 mM EDTA and 40 mM Tris pH 6.8 with 1.25 units of Proteinase K for 1.5 h at 45 °C. Finally, DNA was purified using a QIAquick PCR purification kit (Quiagen). The following antibodies were used: WBP2 (ab76680, Abcam), pPol-II (ab5095, Abcam), p300 (sc585, Santa Cruz Biotechnology), acH3K14 (ab52946, Abcam), acH3K9/18 (07-593, Millipore), acH4K8 (07-328, Millipore), acH4K5 (07-327, Millipore).

qPCR for ChIP Samples

Input DNA was diluted five times prior to qPCR. Each reaction was prepared with 5 μl DNA (from input or IPs), 300 nM forward primer, 300 nM reverse primer, 1× iQ SYBR Green Supermix (Bio-Rad) in a final volume of 10 μl. Reactions were run in duplicates or triplicates in a LightCycler 480 (Roche) using the same program described above.

Induction values were calculated with the Plaffl method, normalizing to the inputs and taking into account the nonspecific binding of chromatin to appropriate control IgGs.

Primers used were: pS2ChIPfor: 5′-GGCCATCTCTCACTA TGAATCACTTCTGC-3′; pS2ChIPrev: 5′-GGCAGGCTCTGTTT GCTTAAAGAGCG-3′.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

WBP-2 Regulates ER Target Gene Expression

Previous luciferase reporter gene assays from our lab suggest that WBP-2 acts as a coactivator of ER (34). However, the mechanism(s) by which WBP-2 regulates ER target genes is still unknown. To understand the physiological role of WBP-2, we examined the effects of its downregulation or overexpression on ER-mediated gene transcription using MCF-7 breast cancer cells as a model. This cell line was chosen because it is an excellent model to test the effects of estrogen, due to its abundance of ER and reliance on exogenous estrogen for proliferation (39). To determine if WBP-2 is indeed required for maximal ER activation, we knocked down WBP-2 expression in MCF-7 cells by RNA interference. For this purpose, we transfected cells either with WBP-2-specific siRNA (siWBP-2) or with control siRNA (siScrambled) (see Experimental Procedures for details). After estrogen starvation, important to minimize the basal level of estrogen-related effects in the cells, cells were treated with either vehicle (−H) or hormone (+H). WBP-2 knockdown was confirmed both at mRNA level via qPCR and at protein level via Western blot (Figs. 1A and 1B). To determine the effects of WBP-2 knockdown on ER function, we measured the mRNA expression of well-studied estrogen-regulated genes such as pS2 (40), growth regulation by estrogen in breast cancer 1 (GREB1) (41) and progesterone receptor (PR) (42). Notably, WBP-2 knockdown resulted in reduced mRNA levels of the ER target gene pS2 in the presence of hormone, compared to siScrambled transfected cells (Fig. 1C). Similar results were obtained for the mRNA levels of GREB1 (Fig. 1D). However, knockdown of WBP-2 did not have any effect on the mRNA levels of PR (Fig. 1E). As WBP-2 knockdown affects endogenous pS2 and GREB1 mRNA levels, we decided to test whether, on the contrary, WBP-2 overexpression increases the expression of these ER-regulated genes. MCF-7 cells were transfected with either a control plasmid or a plasmid expressing WBP-2. After estrogen starvation, cells were treated with either vehicle (−H) or hormone (+H), and total mRNA was isolated and analyzed focusing on the genes pS2 and GREB1, together with WBP-2 (to confirm overexpression) (Figs. 2A–2C). WBP-2 overexpression had little effect on the transactivation functions of ER in the absence of hormone. However, WBP-2 overexpression significantly enhanced both pS2 and GREB1 expression suggesting that WBP-2 acts as an ER coactivator for pS2 and GREB1 transcription. These data indicate that WBP-2 is required for the complete biological activity of ER in MCF-7 cells.

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Figure 1. WBP-2 knockdown affects the expression of certain ERα-regulated genes. MCF-7 cells were transfected with control (siScrambled) or siWBP-2 siRNAs, as described in the Experimental Procedures section. After 72 h of estrogen starvation, cells were treated either with vehicle (−H) or estrogen (+H) for 12 hours. mRNA (A, C–E) and total cell lysates (B) were isolated. WBP-2, pS2, GREB1 and PR mRNA levels were analyzed by qPCR (in A, C, D, E, respectively). Results are normalized against 36B4 transcripts. Data are plotted as relative mRNA level, where vehicle-treated control siRNA transfected cells serve as the reference sample and are set as 1. Error bars are the standard deviation (STD) from three independent experiments. Student's t-test was used to calculate significant differences (*P<0.05) between the indicated datasets. (B): Total cell lysates were separated by SDS/PAGE and Western blotted with the indicated antibodies.

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WBP-2 is Recruited to the pS2 Promoter; Its Presence Correlates with Active Transcription

To substantiate the coactivation function of WBP-2 on ER target gene expression, we used ChIP assays in MCF-7 cells and decided to focus on the pS2 gene, which is a well characterized ER target gene. We wanted to investigate the involvement of WBP-2 in the recruitment of different factors to the pS2 promoter. Hence, we performed ChIP experiments in control cells and in cells in which WBP-2 levels had been knocked down by RNA interference. Previously published data show that ER directs the cyclical recruitment of cofactors on promoters (43, 44). To determine the time point at which to conduct our assays, we conducted pilot time course experiments to evaluate the recruitment of RNA polymerase-II phosphorylated at serine 2 (pPol-II) and various transcription factors and cofactors (ER and p300), at the pS2 promoter at different times after hormone stimulation (data not shown). Forty five minutes of hormone stimulation of the cells correspond, in our hands, to the maximal recruitment of the investigated factors. Therefore, we decided to perform all subsequent ChIP experiments at this time point. MCF-7 cells were treated with either vehicle (−H) or hormone (+H), and chromatin associated with WBP-2 was precipitated using a WBP-2-specific antibody. The precipitated genomic DNA was amplified by qPCR using primer sets specific for the pS2 promoter. ChIP analyses demonstrated the recruitment of WBP-2 onto the pS2 promoter in the presence of estrogen (+H). As expected, upon hormone stimulation, knockdown of WBP-2 is associated with reduction in WBP-2 recruitment at the promoter (Fig. 3A). Unexpectedly, in the absence of hormone, knockdown of WBP-2 is not associated with a significant decrease in its occupancy at the pS2 promoter compared to control cells (siScrambled). This phenomenon, reproducible in different experiments, could be due to a not yet identified feedback mechanism that retains more of this coactivator at the promoter when its cellular levels are very low. Alternatively, it is also possible that a small amount of WBP-2, which is constitutively bound to the promoter of ER target genes, is required for cell survival. Studies are on going to address this point.

We found that WBP-2 binds to pS2 promoter upon estrogen treatment. Additionally, knockdown of WBP-2 in MCF-7 cells resulted in reduced transcription of pS2 upon hormone stimulation (Fig. 1C), and the reduction of pS2 expression correlates with reduced recruitment of WBP-2 (Fig. 3A). Therefore, we decided to explore the mechanism by which WBP-2 strengthens the hormone-dependent functions of ER. Toward this end, we examined the effects of WBP-2 knockdown (performed as described above) on the recruitment of RNA polymerase II phosphorylated on Serine 2 (pPol-II) to the ER target gene promoter pS2. While the presence of RNA polymerase II at a gene promoter indicates an active gene that can therefore be transcribed, the promoter occupancy of pPol-II is associated with a state of productive elongation (45, 46). Upon hormone stimulation, knockdown of WBP-2 resulted in reduced occupancy of pPol-II at the pS2 promoter compared to control cells (siScrambled) (Fig. 3B). These data suggest that WBP-2 acts as a coactivator of ER and is associated with active transcription (as measured by occupancy of pPol-II) at some ER target gene promoters.

WBP-2 Affects the Recruitment of the Chromatin-Modifying Enzyme p300 at Target Gene Promoters

Next we investigated a possible cause for decreased transcription at the pS2 promoter in the context of WBP-2 knockdown. WBP-2 might affect the recruitment of chromatin-modifying enzymes, which regulate the accessibility of chromatin and therefore create an environment permissive for active transcription, exemplified by the observed occupancy of pPol-II. To test this possibility, we performed ChIP assays (as described above) and examined the effects of WBP-2 knockdown on the recruitment of some chromatin modifying enzymes. We decided to focus on p300, because this enzyme has been associated with the initial chromatin modifications that happen at the pS2 promoter to allow transcription, compared to other histone acetyl transferases (43). Under WBP-2 knockdown conditions, the hormone-dependent recruitment of p300 to the pS2 promoter was impaired compared to control cells (siScrambled) (Fig. 4). These data suggest that WBP-2 is required for the recruitment of p300 to the ER target gene promoter pS2, shedding important mechanistic insight into WBP-2's coactivation function.

Specificity of Action in WBP-2-Mediated p300 Recruitment at the pS2 Promoter

p300 is a histone acetyl transferase that promotes an “open” chromatin state by acetylating histones at different lysine residues and therefore promotes/enhances transcription (47). Because WBP-2 is associated with p300 recruitment at the pS2 promoter, we sought to investigate whether WBP-2 is also associated with an open chromatin environment by examining the acetylation status of histones at the pS2 promoter. We performed ChIP assays under WBP-2 knockdown conditions and examined the acetylation status of multiple sites (histone lysine residues) that have been proven to be targets of p300 (48). Knockdown of WBP-2 had no significant effect on the acetylation status of lysine (K) 9 or 18 of histone H3 (H3K9, H3K18), or of lysine 5 or 8 of histone H4 (H4K5 and H4K8) (Figs. 5B–5D). However, knockdown of WBP-2 correlated with a reduction in the acetylation of H3K14 (Fig. 5A). This modification is known to be associated with an active gene status for certain genes and it is the primary acetylation site mediated by p300 (48). Collectively, these data suggest that WBP-2 enhances ER transactivation function at some target genes by facilitating the recruitment and/or the retention of a specific histone-modifying enzyme that favors a relaxed chromatin structure, permissive of transcription.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Coregulators (coactivators and corepressors) play a key role in the regulation of steroid receptor transactivation functions (18, 19) and represent yet another mechanism for tight control of gene expression. In this study, we investigated the coactivation functions of a putative ER coactivator: WBP-2. Previous experiments from our laboratory pointed to WBP-2 as a bona fide coactivator of ER (34). Here, we further investigated WBP-2's coactivation functions and dissected the molecular mechanism of its action.

We demonstrate that WBP-2 is important in regulating certain ER target gene expression. In the past we have noted the specificity in WBP-2's coactivation function for different steroid hormone receptors: indeed, in luciferase gene reporter assays, it seems that WBP-2 can modulate the activity of ER and PR but not of androgen receptor or glucocorticoid receptor (34). Now we also demonstrate specificity regarding WBP-2 action on different ER target genes. More specifically, WBP-2 is required for the full expression of some ER target genes, such as pS2 and GREB1, two well-studied estrogen responsive genes (40, 41) (Figs. 1 and 2). Conversely, WBP-2 levels are not important for the expression of PR, another known estrogen responsive gene (42). Therefore, WBP-2 acts as a selective coactivator of some estrogen responsive genes. As of today we do not know the large-scale (genome-wide) extent of specificity for WBP-2-regulated subsets of ER responsive genes, but the subset specificity provides a general rationale for coactivator function. This is true also for most coactivators: for example, E6 associated protein (49) is an ER coactivator, but it is recruited only to a subset of ER responsive promoters (our unpublished data). Such specificity is also exhibited by the steroid receptor coactivator family (SRC) (50), CBP (51) and p300 (47), among many others. In relation to WBP-2, it is also unknown is if different estrogen responsive genes can be differentially regulated in response to specific stimuli in the cell and the identity of these stimuli. Furthermore, it is uncertain if a group of genes exists that are regulated by WBP-2 independently from ER. This is the case for several steroid hormone receptors (50). Therefore, it would not be surprising to identify WBP-2 as a coactivator also of ER independent genes. Future studies clarifying these possibilities could help in understanding how coactivators in general, and WBP-2 specifically, act in generating tissue-, cell-, receptor- and promoter specific effects (19).

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Figure 2. WBP-2 overexpression is associated with increased expression of certain ERα-regulated genes. MCF-7 cells were transfected with a control plasmid or a plasmid overexpressing WBP-2, as described in the Experimental Procedures section. After 72 h of estrogen starvation, cells were treated either with vehicle (−H) or estrogen (+H) for 12 h. mRNA was isolated and WBP-2, pS2 and GREB1 mRNA levels were analyzed by qPCR (A, B and C, respectively). Results are normalized against 36B4 transcripts. Data are plotted as relative mRNA level, where vehicle-treated control siRNA transfected cells serve as the reference sample and are set as 1. Error bars are the STD from three independent experiments. Student's t-test was used to calculate significant differences (*P<0.05) between the indicated datasets.

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Our present work not only demonstrates WBP-2 has a selective role in ER target gene expression but also sheds light on its mechanism of action. WBP-2 is essential for the full activation of estrogen-induced pS2 expression. In agreement with this, we observed a correlation between WBP-2 levels in the cell and occupancy of phosphoRNA pol-II—associated with a state of productive elongation (45, 46)—at the pS2 promoter (Fig. 3B). WBP-2 seems to act as a coactivator by promoting elongation of RNA pol-II-mediated transcription at ER target gene promoters.

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Figure 3. WBP-2 is recruited to the pS2 promoter and is important for RNA Pol-II elongation. MCF-7 cells were transfected with control (siScrambled) or siWBP-2 siRNAs. After 72 h of estrogen starvation, cells were treated either with vehicle (−H) or estrogen (+H) for 45 min. Then, chromatin was prepared and subjected to ChIPs as described in the Experimental Procedures section. Antibodies used for IPs were specific for WBP-2 (A) or pPol-II (B). Immunoprecipitated DNA was analyzed by qPCR. Data reported are a representative of three independent experiments. Data are plotted as relative fold enrichment, where (for a specified protein) vehicle-treated control siRNA transfected cells serve as the reference sample and are set as 1. Data show mean and STD of triplicates measurements within one experiment. Student's t-test was used to calculate significant differences (*P<0.05) between the indicated datasets.

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As mentioned above, WBP-2 can bind, through similar interactions, structurally and functionally related proteins YAP and TAZ and WWOX1. YAP and TAZ are both involved in regulation of cell growth, proliferation and tumorigenesis (27), and YAP is a putative ER coactivator (34); WWOX1, on the contrary, is a putative tumor suppressor (31), and our unpublished data show that it also attenuates the transactivation functions of ER. It is intriguing to speculate that WBP-2 might simultaneously regulate the oncogenic activities of YAP and TAZ and the tumor suppressor functions of WWOX1. This possible regulation of oncogenic signaling is particularly relevant for ER, as ER signaling promotes cell proliferation, especially in the context of breast cancer. Through preferential interaction of WBP-2 with YAP or WWOX1, the cell could indeed be pushed more toward proliferation or quiescence, respectively, depending on specific stimuli at specific times. Studies are ongoing in our laboratory to determine this possibility.

The significance of p300 in ER function has long been established (52). This study highlights, for the first time, the importance of yet another component of the ER coactivator complex: WBP-2. We find that WBP-2 can enhance ER transactivation by facilitating the recruitment of p300, an important histone modifier that, upon histone acetylation, favors a relaxed chromatin structure, which permits transcription (36) (Fig. 4). Another possibility is that WBP-2 helps instead the retention of p300 to some target gene promoters. In either case, WBP-2 appears to act as a scaffolding protein, bringing/keeping p300 to the promoter region of a subset of ER target genes. Ongoing studies in our laboratory are investigating the biochemical details of WBP-2 action on p300, with emphasis in discerning between p300 recruitment or maintenance at some ER-responsive promoters.

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Figure 4. WBP-2 knockdown affects p300 recruitment at the pS2 promoter. MCF-7 cells were transfected with control (siScrambled) or siWBP-2 siRNAs. After 72 h of estrogen starvation, cells were treated either with vehicle (−H) or estrogen (+H) for 45 min. Then, chromatin was prepared and subjected to ChIPs as described in the Experimental Procedures section. The antibody used for IP was specific for p300. Immunoprecipitated DNA was analyzed by qPCR. Data reported are a representative of three independent experiments. Data are plotted as relative fold enrichment, where (for a specified protein) vehicle-treated control siRNA transfected cells serve as the reference sample and are set as 1. Data show mean and STD of triplicates measurements within one experiment. Student's t-test was used to calculate significant differences (*P<0.05) between the indicated datasets.

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Together with a prevalent role as histone acetyl transferase, p300 can also act as a transcription factor acetyl transferase, or as a scaffold molecule, for components of the transcription machinery (53). We observed a link between p300 recruitment and specific histones acetylation markers. However, even in this context, p300 might have some additional function.

p300 can acetylate histones at different residues: histone H3 at lysine 14 and lysine 18, as well as histone H4 at lysine 5 and lysine 8 and histone H2B at lysine 12 and lysine 15 (48). We find that WBP-2 is important for one specific p300-dependent histone modification: acetylation of histone H3 on lysine 14, the primary acetylation site mediated by p300 (Fig. 5). This result supports the idea that the fine-tuning of gene expression is achieved by sequential and combinatorial assembly of transcription factors and coregulators at promoters, which control, by modulation of their activity in relation to the specific context they are in, each and every step of gene expression, allowing for tight control on mRNA production. This step-wise knowledge of gene expression in cancer cells not only contributes to our understanding of the basic mechanisms involved in ER signaling but could also provide new future points of intervention for therapeutic purposes.

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Figure 5. WBP-2 dependent p300 recruitment to chromatin selectively alters H3K14 acetylation status. MCF-7 cells were transfected with control (siScrambled) or siWBP-2 siRNAs. After 72 h of estrogen starvation, cells were treated either with vehicle (−H) or estrogen (+H) for 45 min. Then, chromatin was prepared and subjected to ChIPs as described in the Experimental Procedures section. Antibodies used for IPs were specific for histone H3 acetylated on Lys 14 (acH3K14) (A), or histone H3 acetylated on Lys 9/18 (acH3K9/18) (B), or histone H4 acetylated on Lys 5 (acH4K5) (C) or histone H4 acetylated on Lys 8 (acH4K8) (D). Immunoprecipitated DNA was analyzed by qPCR. Data reported are a representative of three independent experiments. Data are plotted as relative fold enrichment, where (for a specified protein) vehicle-treated control siRNA transfected cells serve as the reference sample and are set as 1. Data show mean and STD of triplicates measurements within one experiment. Student's t-test was used to calculate significant differences (*P<0.05) between the indicated datasets.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors would like to acknowledge Fernando Cruz-Guilloty and Feng Gong for critically reading the manuscript. This research was supported by the 1R01DK079217-01A2 grant from NIH to Zafar Nawaz.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental Procedures
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Fuda, N. J., Ardehali, M. B., and Lis, J. T. ( 2009) Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature 461, 186192.
  • 2
    Pan, Y., Tsai, C. J., Ma, B., and Nussinov, R. ( 2009) How do transcription factors select specific binding sites in the genome? Nat. Struct. Mol. Biol. 16, 11181120.
  • 3
    Pan, Y., Tsai, C. J., Ma, B., and Nussinov, R. ( 2010) Mechanisms of transcription factor selectivity. Trends Genet. 26, 7583.
  • 4
    Guenther, M. G., Levine, S. S., Boyer, L. A., Jaenisch, R., and Young, R. A. ( 2007) A chromatin landmark and transcription initiation at most promoters in human cells. Cell 130, 7788.
  • 5
    Forsberg, E. C., Johnson, K., Zaboikina, T. N., Mosser, E. A., and Bresnick, E. H. ( 1999) Requirement of an E1A-sensitive coactivator for long-range transactivation by the beta-globin locus control region. J. Biol. Chem. 274, 2685026859.
  • 6
    McKenna, N. J., Lanz, R. B., and O'Malley, B. W. ( 1999) Nuclear receptor coregulators: cellular and molecular biology. Endocr. Rev. 20, 321344.
  • 7
    Li, Q., Peterson, K. R., Fang, X., and Stamatoyannopoulos, G. ( 2002) Locus control regions. Blood 100, 30773086.
  • 8
    Naar, A. M., Lemon, B. D., and Tjian, R. ( 2001) Transcriptional coactivator complexes. Annu. Rev. Biochem. 70, 475501.
  • 9
    Gao, X., Loggie, B. W., and Nawaz, Z. ( 2002) The roles of sex steroid receptor coregulators in cancer. Mol. Cancer 1, 7.
  • 10
    Perissi, V., Aggarwal, A., Glass, C. K., Rose, D. W., and Rosenfeld, M. G. ( 2004) A corepressor/coactivator exchange complex required for transcriptional activation by nuclear receptors and other regulated transcription factors. Cell 116, 511526.
  • 11
    Lonard, D. M., and O'Malley B, W. ( 2007) Nuclear receptor coregulators: judges, juries, and executioners of cellular regulation. Mol Cell 27, 691700.
  • 12
    Carson-Jurica, M. A., Schrader, W. T., and O'Malley, B. W. ( 1990) Steroid receptor family: structure and functions. Endocr. Rev. 11, 201220.
  • 13
    Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., et al. ( 1995) The nuclear receptor superfamily: the second decade. Cell 83, 835839.
  • 14
    Novac, N. and Heinzel, T. ( 2004) Nuclear receptors: overview and classification. Curr. Drug Targets Inflamm. Allergy 3, 335346.
  • 15
    Sommer, S. and Fuqua, S. A. ( 2001) Estrogen receptor and breast cancer. Semin. Cancer Biol. 11, 339352.
  • 16
    Bai, Z. and Gust, R. ( 2009) Breast cancer, estrogen receptor and ligands. Arch. Pharm. (Weinheim) 342, 133149.
  • 17
    Dahlman-Wright, K., Cavailles, V., Fuqua, S. A., Jordan, V. C., Katzenellenbogen, J. A., et al. ( 2006) International Union of Pharmacology. LXIV. Estrogen receptors. Pharmacol. Rev. 58, 773781.
  • 18
    McKenna, N. J. and O'Malley, B. W. ( 2002) Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108, 465474.
  • 19
    York, B. and O'Malley, B. W. ( 2010) Steroid receptor coactivator (SRC) family: masters of systems biology. J. Biol. Chem. 285, 3874338750.
  • 20
    Chen, H. I. and Sudol, M. ( 1995) The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. Proc. Natl. Acad. Sci. USA 92, 78197823.
  • 21
    Sudol, M., Chen, H. I., Bougeret, C., Einbond, A., and Bork, P. ( 1995) Characterization of a novel protein-binding module—the WW domain. FEBS Lett. 369, 6771.
  • 22
    Komuro, A., Nagai, M., Navin, N. E., and Sudol, M. ( 2003) WW domain-containing protein YAP associates with ErbB-4 and acts as a co-transcriptional activator for the carboxyl-terminal fragment of ErbB-4 that translocates to the nucleus. J. Biol. Chem. 278, 3333433341.
  • 23
    Overholtzer, M., Zhang, J., Smolen, G. A., Muir, B., Li, W., et al. ( 2006) Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc. Natl. Acad. Sci. USA 103, 1240512410.
  • 24
    McDonald, C. B., McIntosh, S. K., Mikles, D. C., Bhat, V., Deegan, B. J., et al. ( 2011) Biophysical analysis of binding of WW domains of the YAP2 transcriptional regulator to PPXY motifs within WBP1 and WBP2 adaptors. Biochemistry 50, 96169627.
  • 25
    Sudol, M. ( 1996) Structure and function of the WW domain. Prog. Biophys. Mol. Biol. 65, 113132.
  • 26
    Sudol, M. and Hunter, T. ( 2000) NeW wrinkles for an old domain. Cell 103, 10011004.
  • 27
    Wang, K., Degerny, C., Xu, M., and Yang, X. J. ( 2009) YAP, TAZ, and Yorkie: a conserved family of signal-responsive transcriptional coregulators in animal development and human disease. Biochem. Cell Biol. 87, 7791.
  • 28
    Harvey, K. and Tapon, N. ( 2007) The Salvador-Warts-Hippo pathway—an emerging tumour-suppressor network. Nat. Rev. Cancer 7, 182191.
  • 29
    Ludes-Meyers, J. H., Kil, H., Bednarek, A. K., Drake, J., Bedford, M. T., et al. ( 2004) WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins. Oncogene 23, 50495055.
  • 30
    Bednarek, A. K., Laflin, K. J., Daniel, R. L., Liao, Q., Hawkins, K. A., et al. ( 2000) WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. Cancer Res. 60, 21402145.
  • 31
    Del Mare, S., Kurek, K. C., Stein, G. S., Lian, J. B., and Aqeilan, R. I. ( 2011) Role of the WWOX tumor suppressor gene in bone homeostasis and the pathogenesis of osteosarcoma. Am. J. Cancer Res. 1, 585594.
  • 32
    Jolliffe, C. N., Harvey, K. F., Haines, B. P., Parasivam, G., and Kumar, S. ( 2000) Identification of multiple proteins expressed in murine embryos as binding partners for the WW domains of the ubiquitin-protein ligase Nedd4. Biochem. J. 351 ( pt 3), 557565.
  • 33
    Nitsch, R., Di Palma, T., Mascia, A., and Zannini, M. ( 2004) WBP-2, a WW domain binding protein, interacts with the thyroid-specific transcription factor Pax8. Biochem. J. 377, 553560.
  • 34
    Dhananjayan, S. C., Ramamoorthy, S., Khan, O. Y., Ismail, A., Sun, J., et al. ( 2006) WW domain binding protein-2, an E6-associated protein interacting protein, acts as a coactivator of estrogen and progesterone receptors. Mol. Endocrinol. 20, 23432354.
  • 35
    Zhang, X., Milton, C. C., Poon, C. L., Hong, W., and Harvey, K. F. ( 2011) Wbp2 cooperates with Yorkie to drive tissue growth downstream of the Salvador-Warts-Hippo pathway. Cell Death Differ. 18, 13461355.
  • 36
    Chan, S. W., Lim, C. J., Huang, C., Chong, Y. F., Gunaratne, H. J., et al. ( 2011) WW domain-mediated interaction with Wbp2 is important for the oncogenic property of TAZ. Oncogene 30, 600610.
  • 37
    Pfaffl, M. W. ( 2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45.
  • 38
    Catoe, H. W. and Nawaz, Z. ( 2011) E6-AP facilitates efficient transcription at estrogen responsive promoters through recruitment of chromatin modifiers. Steroids 76, 897902.
  • 39
    Lacroix, M. and Leclercq, G. ( 2004) Relevance of breast cancer cell lines as models for breast tumours: an update. Breast Cancer Res. Treat. 83, 249289.
  • 40
    Barkhem, T., Haldosen, L. A., Gustafsson, J. A., and Nilsson, S. ( 2002) pS2 Gene expression in HepG2 cells: complex regulation through crosstalk between the estrogen receptor alpha, an estrogen-responsive element, and the activator protein 1 response element. Mol. Pharmacol. 61, 12731283.
  • 41
    Sun, J., Nawaz, Z., and Slingerland, J. M. ( 2007) Long-range activation of GREB1 by estrogen receptor via three distal consensus estrogen-responsive elements in breast cancer cells. Mol. Endocrinol. 21, 26512662.
  • 42
    Prange-Kiel, J., Rune, G. M., Zwirner, M., Wallwiener, D., and Kiesel, L. ( 2001) Regulation of estrogen receptor alpha and progesterone receptor (isoform A and B) expression in cultured human endometrial cells. Exp. Clin. Endocrinol. Diabetes 109, 231237.
  • 43
    Metivier, R., Penot, G., Hubner, M. R., Reid, G., Brand, H., et al. ( 2003) Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115, 751763.
  • 44
    Reid, G., Hubner, M. R., Metivier, R., Brand, H., Denger, S., et al. ( 2003) Cyclic, proteasome-mediated turnover of unliganded and liganded ERalpha on responsive promoters is an integral feature of estrogen signaling. Mol. Cell. 11, 695707.
  • 45
    Kim, M., Ahn, S. H., Krogan, N. J., Greenblatt, J. F., and Buratowski, S. ( 2004) Transitions in RNA polymerase II elongation complexes at the 3' ends of genes. EMBO J. 23, 354364.
  • 46
    Ahn, S. H., Kim, M., and Buratowski, S. ( 2004) Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing. Mol. Cell 13, 6776.
  • 47
    Chen, J. and Li, Q. ( 2011) Life and death of transcriptional co-activator p 300. Epigenetics 6, 957961.
  • 48
    Schiltz, R. L., Mizzen, C. A., Vassilev, A., Cook, R. G., Allis, C. D., et al. ( 1999) Overlapping but distinct patterns of histone acetylation by the human coactivators p300 and PCAF within nucleosomal substrates. J. Biol. Chem. 274, 11891192.
  • 49
    Nawaz, Z., Lonard, D. M., Smith, C. L., Lev-Lehman, E., Tsai, S. Y., et al. ( 1999) The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily. Mol. Cell. Biol. 19, 11821189.
  • 50
    Leo, C. and Chen, J. D. ( 2000) The SRC family of nuclear receptor coactivators. Gene 245, 111.
  • 51
    Goodman, R. H. and Smolik, S. ( 2000) CBP/p300 in cell growth, transformation, and development. Genes Dev. 14, 15531577.
  • 52
    Kraus, W. L. and Kadonaga, J. T. ( 1998) p300 and estrogen receptor cooperatively activate transcription via differential enhancement of initiation and reinitiation. Genes Dev. 12, 331342.
  • 53
    Chan, H. M. and La Thangue, N. B. ( 2001) p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J. Cell. Sci. 114, 23632373.