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[Cancer Research 65, 9216-9225, October 15, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Regulation of Krüppel-like Factor 6 Tumor Suppressor Activity by Acetylation

Dan Li1, Steven Yea1, Georgia Dolios2, John A. Martignetti2, Goutham Narla1, Rong Wang2, Martin J. Walsh3 and Scott L. Friedman1

1 Division of Liver Diseases, Department of Medicine; and Departments of 2 Human Genetics and 3 Pediatrics, Mount Sinai School of Medicine, New York, New York

Requests for reprints: Scott L. Friedman, Mount Sinai School of Medicine, Box 1123, 1425 Madison Avenue, Room 11-70C, New York, NY 10029-6574. Phone: 212-659-9501; Fax: 212-849-2574; E-mail: Scott.Friedman{at}mssm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Krüppel-like factor 6 (KLF6) is a zinc finger transcription factor and tumor suppressor that is inactivated in a number of human cancers by mutation, allelic loss, and/or promoter methylation. A key mechanism of growth inhibition by wild-type KLF6 is through p53-independent up-regulation of p21WAF1/cip1 (CDKN1A), which is abrogated in several tumor-derived mutants. Here we show by chromatin immunoprecipitation that transactivation of p21WAF1/cip1 by KLF6 occurs through its direct recruitment to the p21WAF1/cip1 promoter and requires acetylation by histone acetyltransferase activity of either cyclic AMP–responsive element binding protein–binding protein or p300/CBP-associated factor. Direct lysine acetylation of KLF6 peptides can be shown by mass spectrometry. A single lysine-to-arginine point mutation (K209R) derived from prostate cancer reduces acetylation of KLF6 and abrogates its capacity to up-regulate endogenous p21WAF1/cip1 and reduce cell proliferation. These data indicate that acetylation may regulate KLF6 function, and its loss in some tumor-derived mutants could contribute to its failure to suppress growth in prostate cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Krüppel-like factor 6 (KLF6) is an ubiquitously expressed zinc finger protein belonging to the KLF family of transcription factors (1, 2). These proteins are DNA-binding transcriptional regulators that serve myriad roles in differentiation and development (3, 4). All KLF members possess a distinct NH2 terminus activation domain and a highly conserved COOH terminus zinc finger DNA-binding domain that interacts with "GC box" or "CACC" DNA motifs in responsive promoters (3, 4). In contrast to the conserved DNA-binding domain among KLFs, the divergent activation domain of each family member accounts for their diverse biological activities.

KLF6 has broad activity in regulating cell growth, tissue injury, and differentiation. Its transcriptional targets include a placental glycoprotein (5), HIV-1 (6), collagen {alpha}1(I) (2), transforming growth factor ß1 (TGFß1), types I and II TGFß receptors (7), nitric oxide synthase (8), and urokinase type plasminogen activator (9). It is also an immediate early gene up-regulated in hepatic stellate cells during acute liver injury (1) and during the differentiation of preadipocytes into adipocytes (10), suggesting a generalized function of KLF6 in different biological contexts.

KLF6 has recently been identified as a tumor suppressor gene that is inactivated in primary prostate (1, 11), colon (12) nasopharyngeal (13), glial (14), and hepatocellular (15) cancers and is down-regulated in lung and prostate cancers (16, 17), Decreased KLF6 mRNA expression correlates with clinical outcome in prostate cancer (17). Somatic loss of heterozygosity (LOH) and DNA mutations result in functional deletion of the KLF6 gene in ~60% of prostate and 45% of colon tumors (1, 11, 12). KLF6 promoter silencing by methylation also has been uncovered in esophageal cancer cell lines (18).

One mechanism by which KLF6 reduces cell proliferation is through up-regulation of p21WAF1/cip1, a key cyclin-dependent kinase (cdk) inhibitor. This induction does not require p53, a well-established transactivator of p21WAF1/cip1, because p21WAF1/cip1 induction by KLF6 is preserved in p53-null cells (1). The molecular requirements for p21WAF1/cip1 up-regulation by KLF6 have not been defined, and information about transcriptional coactivators within KLF6 transcriptional complexes is limited. Heterologous interaction of KLF6 with KLF4 or Sp1 is required for keratin (19) or endoglin gene expression (20), respectively, but potential interacting proteins in that context have not be explored.

Coactivators, specifically histone acetyltransferases (HAT), contribute to the transcriptional activity of other tumor suppressors (e.g., p53), where recruitment of HATs is vital to its function (21). HATs are chromatin-modifying proteins that directly regulate transcription through interaction with transcription factors (22), including p53 (23, 24), pRb (25), and BRCA1 (26), as well as the zinc finger transcription factors Sp1 (27) and EKLF (28, 29). Altered interactions between HATs and transcription factors may contribute to tumorigenesis. For example, disruption of the p300-p53 interaction may underlie the mechanism by which the viral oncoprotein E1A induces cell transformation (23, 24, 30).

In vivo, HATs function as part of large protein complexes that share a highly conserved acetyl-CoA binding motif but have different substrate specificities. Among the best studied HATs, cyclic AMP–responsive element binding protein–binding protein (CBP) and p300/CBP-associated factor (PCAF) modulate gene transcription through the acetylation of specific lysine residues on chromatin, and function as coactivators for a number of transcription factors regulating cell growth and development (31–34). In addition, acetylation of nonhistone proteins has emerged as a novel mechanism of posttranslational modification (35, 36).

Our identification of a KLF6 lysine-to-arginine mutation in primary prostate cancer raised the prospect that mutation of an acetylation substrate site might contribute to the loss of growth suppressive activity through the abrogation of p21WAF1/cip1 transactivation. In the present study, we have explored the role of CBP and PCAF in up-regulating p21WAF1/cip1 gene expression by KLF6. Loss of lysine through mutation impairs KLF6's capacity to transactivate the p21WAF1/cip1promoter or up-regulate endogenous p21WAF1/cip1. These findings suggest that acetylation of KLF6 plays a critical role in its regulation of target gene expression. Given its role as a tumor suppressor, loss of KLF6 acetylation may be associated with tumorigenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression plasmids. PCIneo-KLF6 (human; previously known as "Zf9") expression plasmid was constructed as previously described (1). HA-CBP was a gift from Dr. R.H. Goodman, Vollum Institute, Oregon Health Sciences University, Portland, OR (37). PCI-PCAF plasmid was a gift from Dr. Yoshihiro Nakatani, Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA (38). p21WAF1/cip1 promoter-luciferase construct was a gift from Dr. Toru Ouchi, Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY (39). pRL-TK Vector used as a control for transfection efficiency was from Dual-luciferase Reporter Assay System (Promega, Madison, WI).

Site-directed mutagenesis. A lysine-to-arginine point mutant of KLF6 (pCIneoK209R) was constructed using Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA), as described below. pCIneo-KLF6 (human) was used as the template for the mutagenesis. The primers used for mutagenesis were K209R sense, 5'-CCACTTTAACGGCTGCAGGAGAGTTTACACCAAAAGC-3' and K209R antisense, 5'-GCTTTTGGTGTAAACTCTCCTGCAGCCGTTAAAGTGG-3'. All mutated constructs were sequenced on both strands to verify these mutations and to confirm that no other alterations were introduced.

Cell culture. Prostate cancer 3 (PC3M) cells, 293 cells, and 293T cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in DMEM supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/mL penicillin and 100 units/mL streptomycin, and 2 mmol/L L-glutamine (Life Technologies, Gaithersburg, MD). Treatment with trichostatin A (Sigma-Aldrich, St. Louis, MO) and suberoylaniline hydroxamic acid (Biomol, Plymouth Meeting, PA) was used at a final concentration of 0.5 µmol/L. Cell were recovered 8 to 12 hours following treatment with individual histone deacetylase (HDAC) inhibitors.

Transfection and luciferase reporter assays. Transient transfections were done using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). For analyzing transfected protein using Western blot, PC3M cells cultured in 10-cm plates (Corning, Acton, MA) were transfected with 10 µg pCIneo empty vector, pCIneoKLF6, or the pCIneoK209R mutant; cells were lysed 36 hours later for Western blot analysis. For luciferase assay, PC3 cells were transfected with pCIneo-KLF6 (1 µg), HA-CBP (5 µg), pCl-PCAF (5 µg), respectively or together, as indicated in text, together with the p21WAF1/cip1 promoter-luciferase construct. Five nanograms of pRL-TK plasmid (Promega) were cotransfected in each transfection as a control for transfection efficiency. Forty-eight hours after tranfection, cells were washed thrice with cold PBS and cell lysates prepared using Dual-luciferase reporter assay system (Promega). The luciferase activity in 10 µL lysate was determined using Dual-luciferase reporter assay system and luminometer (Dynex Technologies, Worthing. West Sussex, United Kingdom). Transfection efficiency was normalized by Renilla luciferase activity measured concurrently in the same lysate. Stable cell lines were generated by cotransfecting the appropriate expression constructs (pCIneo empty vector, KLF6 full length, and KLF6 full length with K209R mutation) with a puromycin expressing plasmid in a 10:1 ratio. Transfected cells were selected with 2.5 µg/mL of puromycin, and pooled clones of cells were used in subsequent experiments.

Chromatin immunoprecipitation assays. Chromatin immunoprecipitation assays were done as previously described (40) with minor modifications described here. Briefly, 293 cells (4 x 106) were transfected with pCIneoKLF6 or pSG4 + Sp1 (gift from R. Tijan, University of California at Berkeley and G. Gill, Harvard University, Boston, MA). Thirty-six hours after transfection, cells were cross-linked with 1% formaldehyde for 10 minutes at 37°C followed by cell lysis and sonication of DNA into 200- to 1,000-bp fragments. Proteins cross-linked to DNA were immunoprecipitated with 10 µg anti-Zf9/KLF6 antibody (R-173; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Sp1 antibody (Upstate Biotechnology, Charlottesville, VA) and 60 µL salmon sperm DNA/protein A agarose beads. Antisera against histone H3 (Upstate Biotechnology) was used as an input control for chromatin in each assay as shown. The protein A agarose/antibody/protein complexes were washed extensively and eluted following the manufacturer's instruction. The cross-linking was reversed by heating at 65°C for 4 hours and proteins were digested by proteinase K for 1 hour at 45°C. DNA was recovered by phenol/chloroform extraction and ethanol precipitation in the presence of 10 µg/mL yeast tRNA carrier and used as a template for PCR reactions. Genomic sequence primers encompassing the p21WAF1/cip1 promoter region –150 to –3 bp upstream of transcriptional start site were used to amplify immunoprecipitated DNA as template: –150F, 5'-GCTGGGCAGCCAGGAGCCTG-3'; –3R, 5'-CTGCTCACACCTCAGCTGGC-3'.

Immunoprecipitation and Western blotting. For coimmunoprecipitation assays, 293T cells were transfected with 20 µg of plasmid DNA. Thirty-six hours after transfection, cells were washed twice with cold PBS and lysed on ice for 20 minutes using immunoprecipitation lysis buffer containing 0.5% NP40, 50 mmol/L Tris-HCl (pH 7.4), 120 mmol/L NaCl, and protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Roche, Nutley, NJ). The cell lysate was precipitated for 30 minutes at 14,000 x g at 4°C. The supernatant was immunoprecipitated at 4°C for 1 hour using 4 µg of anti-Zf9/KLF6 antibody (R-173; Santa Cruz Biotechnology), or 4 µg of anti-HA antibody (Mount Sinai Hybridoma Center, New York, NY) with 50 µL of protein-G beads (Pierce, Rockford, IL), or using 50 µL of M2 anti-agarose (Sigma, St. Louis, MO). For coimmunoprecipitation of endogenous KLF6 and CBP, monoclonal anti-KLF6 antibody was cross-linked to agarose beads using ImmunoPure Protein G IgG Plus Orientation Kit (Pierce). NIH 3T3 cells (1 x 108) were lysed and precipitated, as described above. The supernatant was immunoprecipitated using cross-linked anti-KLF6 agarose. The beads were subsequently washed thrice with 800 µL immunoprecipitation wash buffer [0.5% NP40, 50 mmol/L Tris-HCl (pH 7.4), and 500 mmol/L NaCl], solubilized in Laemmli sample buffer (Sigma) containing 5% ß-mercaptoethanol, boiled, and separated by SDS-PAGE followed by Western blotting as described below. As a negative control, immunoprecipitation was done using control rabbit or mouse IgG (Sigma).

For Western blotting, cell extracts were harvested in radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology, standard protocol). Protein samples (30 µg per sample) were separated on SDS-polyacrylamide gel (6% for CBP and 10% for KLF6 and PCAF) and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were blocked in 5% dried milk in 10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and 0.1% Tween 20 (1x TBS-T) for 1 hour at room temperature. The membrane was incubated with primary antibody: anti-Zf9/KLF6 (R-173), 1:2,000 (Santa Cruz Biotechnology); anti-p21WAF1/cip1 (H-164), 1:250 (Santa Cruz Biotechnology); anti-CBP-NT, 1:1,000 (Upstate Biotechnology); anti-PCAF, 1:1,000 (Upstate Biotechnology); anti-acetyl-lysine antibody, 1:1,000 (Cell Signaling, Beverly, MA). Secondary antibody (horseradish peroxidase–conjugated anti-rabbit or anti-mouse IgG (Amersham Biosciences, Piscataway, NJ) was used according to manufacturer's instruction at 1:2,000 dilution followed by enhanced chemiluminescence protocol (Amersham Pharmacia).

In vitro protein acetyltransferase assay. Protein acetyltransferase reactions were done with in vitro translated KLF6 protein and synthesized by using the TNT in vitro transcription and translation system (Promega) either in the absence of radioisotope or in the presence of [14C]-leucine (50 µCi/mmol, Perkin Elmer, Boston, MA). For acetyltransferase reactions, ~1 µg of in vitro translated KLF6 and 100 ng of immunoprecipitated HA or FLAG-tagged proteins of CBP (wild type), CBP (HAT–; kind gifts from Dr. R.H. Goodman), or PCAF (wild type) were incubated with [14C]-acetyl-CoA (55 µCi/mmol, New England Nuclear) for 1 hour at 30°C. After acetyltransferase reactions, KLF6 protein was immunoprecipitated with anti-Zf9/KLF6 antibody (R-173, Santa Cruz Biotechnology). The entire reaction mixture was separated by 10% SDS-PAGE. Gels containing [14C]-labeled proteins were stained and fixed with 10% glacial acetic acid and 40% methanol for 1 hour, soaked in Amplify (Amersham Pharmacia) for 40 minutes, and exposed to X-ray film for autoradiography for ~10 minutes.

Analysis of proliferation. Proliferation was determined by estimating 3H-thymidine incorporation. BPH1 and PC3M cell lines stably expressing the appropriate expression vectors were plated at a density of 50,000 cells per well in 12-well dishes. Twenty-four hours after plating, 1 µCi/mL 3H-thymidine (Amersham Biosciences) was added. After 2 hours, cells were washed four times with ice-cold PBS and fixed in methanol for 30 minutes at 4°C. After methanol removal and cell drying, cells were solubilized in 0.25% sodium hydroxide/0.25% SDS. After neutralization with hydrochloric acid (1 N), disintegrations per minute were estimated by liquid scintillation counting. This process was repeated at 48 and 72 hours.

Tumor samples. The preparation of tumor samples was as previously described (1). Briefly, 5-µm sections stained with H&E were used as an accurate histologic reference for normal and tumor-derived tissue. Microdissection was done on sequential 20-µm sections and DNA subsequently extracted (Ambion paraffin block isolation kit, Austin, TX). DNA was isolated by proteinase K digestion overnight at 37°C incubator followed by heat inactivation at 95°C for 10 minutes. KLF6 sequence analysis of tumor samples was done as previously described (1).

Peptide acetylation assay. Four peptides were synthesized using commercially available resources (Invitrogen) covering the majority of lysine residues within the KLF6 molecule. The peptides' sequences are as follows: (a) h68-87 ILAREKKEESELKISSSPPE, (b) h115-134 SSEELSPTAKFTSDPIGEVL, (c) h204-223 FNGCRKVYTKSSHLKAHQRT, and (d) h248-267 TRHFRKHTGAKPFKCSHCDR. For peptide acetylation assay, 5 ng of peptide were incubated at 30°C for 1.5 hours with 5 ng CBP or PCAF, 10 µL of 1 mmol/L acetyl CoA, 5 µL of 0.1 mol/L sodium butyrate, in the presence or absence of 10 µL 3H-acetyl CoA (0.5 µCi/µL, Amersham Pharmacia), in 1x HAT assay buffer (Upstate Biotechnology). Following the reaction, the mixture was analyzed by scintillation counting to confirm 3H incorporation followed by mass spectrometry (MS) analysis. To assess 3H incorporation, 5 µL of reaction mixture were spotted onto a small square of filter paper followed by washing with 50 mmol/L Na2HPO4 (pH 9.0), then the filter paper was placed into scintillation fluid overnight for counting the next day.

Mass spectrometry analysis. KLF6 peptides and their acetylation products (1 pmol) were isolated and purified using Poros 20 R2 beads (Applied Biosystems, Foster City, CA) and ZipTipC18 pipette tip (Millipore, Bedford, MA) following the manufacturer's protocol. Molecular masses of the synthetic peptides before and after acetylation reaction were accurately measured by matrix-assisted laser desorption ionization-MS (MALDI-MS) using a QSTAR XL hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems). {alpha}-Cyano-4-hydroxy-cinnamic acid was used as matrix for sample preparation. To determine the acetylation site(s), fragment spectra of peaks corresponding to acetylated peptides were collected and analyzed by MALDI tandem MS (MS/MS) experiment using the same mass spectrometer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
KLF6 targets the p21WAF1/cip1 (CDKN1A) locus and is associated with hyperacetylation of histone H3. To determine if KLF6 interacts directly with the endogenous p21WAF1/cip1 (CDKN1A) promoter, we used chromatin immunoprecipitation (Fig. 1). PCR reactions were carried out using primers encompassing –150 to –3 bp upstream of the start site of p21WAF1/cip1 transcription as shown (Fig. 1A), which contains multiple GC boxes predicted by sequence homology to interact with KLF6 and related family members. Chromatin immunoprecipitation analysis confirmed that KLF6 binds to this region of the endogenous p21WAF1/cip1 promoter, establishing p21WAF1/cip1 as a transcriptional target of KLF6 (Fig. 1B). Interestingly, we were unable to show the binding of Sp1 to the same region despite reports to the contrary based on electromobility shift assay (41). To confirm the ability of the Sp1 antibody to recognize Sp1 in the chromatin immunoprecipitation assay, a positive control was used to verify Sp1 on other loci (data not shown). Furthermore, ectopic expression of KLF6 showed increased recruitment of acetylated histone H3 encompassing the region between –150 to –3 of the KLF6 promoter when compared with the control expression vector (Fig. 1C).



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  		Figure 1. KLF6 is directly associated with the p21WAF1/cip1 (CDKN1A) locus and deposition of acetylated histone H3. A, schematic representation of p21WAF1/cip1 promoter. PCR for chromatin immunoprecipitation (ChIP) assay was done using primers (small arrows) positioned at –150 and –3 bp upstream of transcriptional start site of p21WAF1/cip1 promoter. This GC-rich region contains five putative binding sites for KLF6 as well as for Sp1, all of which are between –102 and –30 bp region, as shown in (A). B, chromatin immunoprecipitation assay at p21WAF1/cip1 promoter in 293 cells. Anti-KLF6 antibody ({alpha}KLF6) was used for immunoprecipitation. Anti-histone H3 ({alpha}Histone H3) antibody was used as a positive control. The binding of Sp1 to p21waf1/cip1 promoter is also examined using anti-Sp1 antibody ({alpha}Sp1). C, acetylation of the p21WAF1/cip1 (CDKN1A) locus within the gene promoter was monitored upon transfection with a KLF6 expression vector by chromatin immunoprecipitation analysis. Input levels of chromatin for immunoprecipitation reactions were monitored for total histone H3. HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) at a final concentration of 0.5 µmol/L were used to compare acetylation of histone H3.

 
KLF6 interacts with CBP and PCAF in vivo. Because CBP and PCAF interact with critical cellular proteins, including p53 (21) and E2F1 (42) leading to altered function, we examined whether KLF6 interacts with CBP and/or PCAF using a coimmunoprecipitation assay. As shown in Fig. 2A (top), CBP was associated with KLF6 when whole cell lysate of 293T cells expressing Flag-tagged KLF6 and HA-tagged CBP was immunoprecipitated using anti-Flag antibody and blotted with anti-HA antibody; this result was confirmed by reciprocal coimmunoprecipitation (Fig. 2A). To show the interaction between endogenous KLF6 and CBP, coimmunoprecipitation was done from a lysate of 1 x 108 NIH 3T3 cells using a monoclonal anti-KLF6 antibody cross-linked to protein G agarose. Western blot using anti-CBP antibody revealed that CBP was coimmunoprecipitated with KLF6 (Fig. 2B). The interaction between KLF6 and PCAF was also shown using a similar approach (Fig. 2C).



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  		Figure 2. KLF6 interacts with CBP and PCAF in vivo. A, KLF6 interacts with CBP. 293T cells were cotransfected with FLAG-tagged KLF6 and HA-tagged CBP constructs. Thirty-six hours after transfection, cells were lysed and coimmunoprecipitated followed by SDS-PAGE/Western analysis. Top, cell lysates were immunoprecipitated with anti-FLAG antibody. Anti-HA antibody was used in a Western blot. Bottom, cell lysates were immunoprecipitated with anti-HA antibody. Anti-FLAG antibody was used in Western blot. B, KLF6 interacts with CBP in vivo. Lysate of 1 x 108 NIH 3T3 cells was immunoprecipitated with agarose-linked anti-KLF6 antibody to pull down endogenous KLF6. Following SDS-PAGE, anti-CBP antibody was used in Western blot to detect the endogenous CBP that was coimmunoprecipitated with KLF6. C, KLF6 interacts with PCAF. 293T cells were cotransfected with KLF6 and FLAG-tagged PCAF constructs. Thirty-six hours after transfection, cells were lysed and coimmunoprecipitated followed by SDS-PAGE/Western analysis. Top, cell lysates were immunoprecipitated with anti-FLAG antibody. Anti-KLF6 antibody was used in Western blot. Bottom, cell lysates were immunoprecipitated with anti-KLF6 antibody. Anti-PCAF antibody was used in Western blot.

 
CBP and PCAF synergize with KLF6 in transactivating p21WAF1/cip1. To establish the functional significance of the interaction between KLF6 and CBP and/or PCAF, we explored whether CBP and PCAF potentiated the transactivation of p21WAF1/cip1 by KLF6 (Fig. 3). As shown in Fig. 3A, KLF6 transactivated the p21WAF1/cip1 (CDKN1A) promoter 3-fold, which was further increased to 12-fold in the presence of CBP. When a HDAC inhibitor, trichostatin A was added, the transactivation of p21WAF1/cip1 by KLF6 was further increased, with the maximal transactivation >40-fold achieved in the presence of CBP plus trichostatin A.



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  		Figure 3. CBP and PCAF potentiate p21WAF1/cip1 up-regulation by KLF6, which is further enhanced by trichostatin A (TSA). A, CBP potentiates KLF6 in transactivating the p21WAF1/cip1 promoter. Left columns, PC3 cells were cotransfected with KLF6 and CBP constructs, along with a p21WAF1/cip1 promoter reporter. Right columns, the same cotransfection were done in the presence of an HDAC inhibitor, trichostatin A. B, PCAF potentiates KLF6 in transactivating p21WAF1/cip1 promoter. Left columns, PC3 cells were cotransfected with KLF6 and PCAF constructs, along with a p21WAF1/cip1 promoter reporter. Right columns, the same cotransfection were done in the presence of the HDAC inhibitor, trichostatin A. Transfection efficiencies were normalized using Renilla luciferase assay measured in the same lysate at the same time.

 
In addition, we also examined the synergistic effects of KLF6 and PCAF in driving p21WAF1/cip1 transactivation (Fig. 3B). In the presence of PCAF, the transactivation of p21WAF1/cip1 by KLF6 was enhanced 14-fold. Again, trichostatin A maximally increased the synergistic effects of KLF6 and PCAF. These results suggest the functional involvement of CBP and PCAF in KLF6-mediated p21WAF1/cip1 up-regulation. Considering the roles of CBP and PCAF as acetyltransferases, these results suggested that enhanced acetylation of KLF6 by trichostatin A promoted the transactivation of p21WAF1/cip1 promoter. Previous studies showed that trichostatin A itself enhances transactivation of the p21WAF1/cip1 promoter (41). This is consistent with the results shown in Fig. 3, in which the luciferase activity in empty vector control was also increased in the presence of trichostatin A. It is likely that this effect is due to the enhanced acetylation by trichostatin A on endogenous transcription factors that transactivate the exogenous p21WAF1/cip1 promoter construct.

KLF6 is acetylated by CBP and PCAF, in vitro and in vivo. The data above suggested that KLF6 and either CBP or PCAF functionally interact on the p21WAF1/cip1 promoter and raised the possibility that acetylation of KLF6 by either these two HATs promoted p21WAF1/cip1 transactivation. To test to this hypothesis, we did an in vitro acetylation assay. As shown in Fig. 4A, KLF6 was acetylated in vitro by both CBP and PCAF but not by a CBP mutant lacking the HAT domain. To verify that KLF6 was also acetylated in vivo, we immunoprecipitated KLF6 from 293T cells and blotted with an anti-acetylated lysine antibody (Fig. 4B).



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  		Figure 4. KLF6 is acetylated in vitro and in vivo. A, KLF6 is acetylated in vitro by CBP and PCAF. Equivalent molar amount of CBP (wild type), CBP (HAT–), and PCAF (wild type) were immunoprecipitated from cells expressing HA-tagged CBP, HA-tagged CBP (HAT–), and FLAG-tagged PCAF (wild type). In vitro translated KLF6 was incubated with immunoprecipitation-purified CBP (wild type), CBP (HAT–), and PCAF (wild type), together with [14C]-acetyl CoA. KLF6 was synthesized with [14C]-leucine in vitro and immunoprecipitated as a control. Immunoprecipitated products were resolved on SDS/PAGE followed by autoradiography. B, KLF6 is acetylated in vivo. 293T cells expressing FLAG-tagged KLF6 were immunoprecipitated with anti-FLAG agarose, followed by SDS-PAGE and Western blot analysis. Immunoprecipitated KLF6 was detected by anti-acetylated lysine antibody. Moreover, using lysates from the same cell line, FLAG-tagged KLF6 was immunoprecipitated by anti-acetylated lysine antibody and detected by anti-FLAG antibody using Western blot.

 
Determination of key acetylation sites on KLF6. To determine which lysine residues on KLF6 are acetylated, we used synthetic KLF6 peptide fragments covering the majority of lysine residues of KLF6 (Fig. 5A). Two peptides were located in the transactivation domain (peptides 1 and 2) and the other two were located in the DNA-binding domain (peptides 3 and 4). HAT assay in vitro was done using 3H-labeled acetyl-CoA to identify acetylated residues (Fig. 5B and C). When KLF6 peptides were subjected to acetylation by CBP, a 4.5-fold increase in counts per minute (CPM) in peptide 3 and 2.7-fold increase in CPM in peptide 4 were observed (Fig. 5B). On the other hand, no significant increase in cpm was observed when KLF6 peptides were subjected to acetylation by PCAF (Fig. 5C). These data indicate that lysine residues in the DNA-binding domain of KLF6 are acetylation sites for CBP.



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  		Figure 5. Determination of key acetylation sites on KLF6. A, the location of four synthetic peptides covering the majority of lysine residues on KLF6 molecule (*K73, this residue is only present in the normal human but not murine KLF6 sequence). B and C, peptide acetylation assays by CBP and PCAF. CBP acetylates peptides 3 and 4 encompassing most lysine residues on the DNA-binding domain. PCAF acetylates none of the lysine residues on the four synthetic peptides. Histone H4 peptide fragment was used as a positive control. D, MALDI-MS spectra of peptide 3 before (top) and after CBP acetylation (bottom). Peaks corresponding to protonated molecular ions were labeled with measured monoisotopic protonated molecular masses and denoted with M+H+, M-Ac+H+, M-2Ac+H+, and M-3Ac+H+ for nonacetylated, single, double, and triple acetylations, respectively. E, fragment ion spectra resulted from MS/MS analysis of peptide ions 2361.3, 2403.3, and 2445.3 (top, middle, and bottom). Peaks corresponding to y-series of fragment ions were labeled and aligned with reversed sequence of peptide 3 (NH2-terminal is indicated by -NH2).

 
Molecular mass measurement of the KLF6 peptides by MS also confirmed that molecular mass shifting resulted from the acetylation (42 amu per acetylation). Molecular mass spectra of peptide 3 before and after acetylation by CBP (Fig. 5D) indicated that all of the three lysine residues in peptide 3 could be acetylated, but the majority form was singly acetylated followed by a doubly acetylated form. To identify the acetylation site(s) on peptide 3, molecular ions corresponding to nonacetylated, singly, and doubly acetylated peptide 3 were analyzed by fragment mass analysis using MS/MS (Fig. 5E). To simplify data interpretation, we only labeled those peaks corresponding to y-series peptide fragments and a b-type fragment in the spectra. As shown in the fragment mass spectrum of 2,361.3 (nonacetylated form; Fig. 5E, top), a series of peaks were observed that corresponded to y-ion (y4-y11). Acetylation of any of the three lysine residues was easily identified. In the fragment mass spectrum of 2,403.3 (singly acetylated form; Fig. 5E, middle), we observed two peaks corresponding to y11 ions. The major one (labeled with y11*) indicated the acetylation of Lys213, and the minor one indicated the acetylation of 209, respectively. In the fragment mass spectrum of 2,445.3 (doubly acetylated form; Fig. 5E, bottom), we observed similar y-series peaks as in the singly acetylated peptide 3, which indicated the acetylation of Lys209 and Lys213. Two additional y-ion peaks were also detected (labeled y8* and y9*) in the same spectrum. Because no mass shift was observed for peaks of y6 and y7, we interpreted this observation to have resulted from acetylation of His216, together with the acetylation of Lys213 or Lys209 (most likely 213). Based on the mass spectrometric analysis, the data indicate that the acetylation accessibility of the three lysine residues on KLF6 peptide 3 is in the order of 213, 209, and 218. The histidine residue can also be acetylated by CBP.

A tumor-derived K-to-R mutant has impaired ability to up-regulate p21WAF1/cip1 and suppress growth. We have shown a high frequency of LOH and point mutations of KLF6 in human prostate cancer (1). Importantly, in addition to the point mutants of KLF6 originally identified from prostate cancer (1), several additional lysine mutations were identified in prostate, colon, and hepatocellular cancers (11, 12, 15). Of these, we focused on a single new mutation not previously reported, K209R (Fig. 6A), to explore the potential effect of loss of an acetylation site on KLF6 function. Given that K209 can be acetylated by CBP as confirmed by MS (Fig. 5), a K209R mutation would be predicted to abrogate acetylation at this site. To assess the ability of K209R to transactivate the p21WAF1/cip1 promoter, we cotransfected the mutant with a p21WAF1/cip1 promoter reporter construct into PC3M cells. As shown in Fig. 6B, compared with wild-type KLF6, K209R completely lost the ability to transactivate the p21WAF1/cip1 promoter. To correlate this change with p21WAF1/cip1 expression in vivo, K209R was introduced into PC3 cells and the endogenous p21WAF1/cip1 level was assessed by Western blot. As shown in Fig. 6C, K209R lost the ability to up-regulate endogenous p21WAF1/cip1.



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  		Figure 6. A lysine (K) to arginine (R) mutant (K209R) identified from prostate cancer has impaired ability to up-regulate p21WAF1/cip1 expression. A, a KLF6 mutation (K209R) affecting an acetylation site in primary prostate cancer. Sequencing chromatogram of DNA derived from microdissected primary prostate cancer is shown, along with normal sequence from the surrounding unaffected sequence. B, K209 tumor mutant has reduced ability to transactivate p21WAF1/cip1 promoter. Wild-type KLF6 and K209R were cotransfected with a p21WAF1/cip1 promoter reporter, respectively. Empty expression vector was transfected as a control for baseline luciferase activity. Transfection efficiencies were normalized using Renilla luciferase assay measured in the same lysate at the same time. C, K209R tumor mutant has reduced ability to up-regulate endogenous p21WAF1/cip1. Wild-type KLF6 and K209R were transfected into PC3 cells, respectively. Thirty-six hours after transfection, cells were lysed and loaded onto SDS-PAGE followed by Western blot analysis. Anti-p21WAF1/cip1 antibody was used to detect the expression of endogenous p21WAF1/cip1 protein. The protein expression of transfected KLF6 and mutants constructed was detected by anti-KLF6 antibody. Tubulin was blotted as a control for protein loading. D, impaired growth suppression by K209R mutant in stable prostate cell lines. Both PC3M and BPH cell lines were stably transfected with either pCI-neo empty vector, KLF6 wild type, or K209R mutant as described in Materials and Methods, and cell proliferation was assessed by estimating 3H-thymidine incorporation at 24, 48, and 72 hours after replating equal numbers of cells. The differences were statistically significant, as indicated by Ps. *, P<0.05; ***, P<0.01.

 
Loss of growth suppression by the K209R mutant was also shown in stably transfected prostate cancer cell lines. K209R was stably expressed in both PC3M cells (a metastatic prostate cancer line) and BPH1 cells (a line derived from benign prostate). In contrast to cells expressing wild-type KLF6, the K209R mutant was unable to decrease cell proliferation in either BPH1 or PC3M cell lines (Fig. 6D). Moreover, in PC3M cells but not BPH cells, the K209R mutant actually increased proliferation when compared with cells stably expressing the pCIneo empty vector. These results were validated in two independent sets of stable cell lines for both BPH1 and PC3M cells. Collectively, these data suggest that the acetylation at K209 is necessary for KLF6 to up-regulate p21WAF1/cip1 gene expression, which in turn can inhibit cellular proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
KLF6 has been established as a tumor suppressor gene involved in key intracellular pathways in a number of human cancers (1, 12, 14, 15, 43, 44). Among several target genes transcriptionally regulated by KLF6, p21WAF1/cip1 has been particularly relevant to prostate cancer given p21WAF1/cip1's central role in growth regulation, especially as a mediator of p53-stimulated cell cycle arrest. The acetylation status of the human p21WAF1/cip1 (CDKN1A) locus has been extensively characterized in cellular contexts that are tied to cancer development (45). These activities require specific GC-rich elements (46) in the p21WAF1/cip1 promoter, which are consensus target sequences for KLF6 binding (1). However, little is known about either the biochemical requirements for transcriptional activity of KLF6, or the complexes through which KLF6 regulates target gene activity.

An important goal in studying KLF6 has been to understand the physiologic significance of protein complexes that modify its function in regulating the activity of its target genes. Recently, we showed that KLF6 sequesters cyclin D1 to reduce cyclin D/cdk4 interactions and enhance the phosphorylation of pRb, thereby promoting G1 cell cycle arrest (47). This activity alters the equilibrium of p21WAF1/cip1 levels, with induction and titration onto cdk2 complexes and further growth suppression (47). These findings suggest that covalent modifications may differentially regulate KLF6 activity.

Despite clear evidence that KLF6 transcriptionally activates p21WAF1/cip1 (1, 12), the biochemical mechanisms underlying this observation have not been clarified. Here, we establish that KLF6 activity is associated with increased complexing of acetylated histones within the p21WAF1/cip1 (CDKN1A) locus. Furthermore, we show that KLF6 interacts with the CBP/p300 complex and its acetylation by CBP contributes to normal KLF6 activity. Moreover, MS shows acetylation of a specific lysine residue of KLF6 that is mutated in a primary prostate tumor. Collectively, the findings provide evidence that acetylation of KLF6 is biologically significant, and its dysregulation in human cancer may contribute to loss of KLF6's growth suppressive activity.

Our findings also provide an important functional link between protein acetyltransferases and KLF6 by showing that KLF6 recruits CBP and PCAF to the p21WAF1/cip1 locus. In contrast to the evidence that KLF6 occupies the p21WAF1/cip1 promoter using chromosomal immunoprecipitation, the lack of promoter occupation by Sp1 was unexpected. One potential explanation may be the weak affinity of different antisera used against Sp1 from cross-linked material to detect the protein in these experiments. Alternatively, Sp1 may represent a low-abundance protein involved primarily in stimulated rather than basal expression of p21WAF1/cip1. This possibility is supported by studies showing that p21WAF1/cip1 transactivation by Sp1 is greatly enhanced by TGFß signaling, for example (48). Although our study does not exclude the possible participation of Sp1-like factors in regulating the expression of p21WAF1/cip1 (49), it may indicate a greater role for KLF6 than Sp1 in regulating p21WAF1/cip1 within this specific cellular context. Additionally, Sp1 may use different GC boxes in the p21WAF1/cip1 promoter from those used by KLF6 under our experimental conditions. In other contexts, there may be cooperation between KLF6 and Sp1, as suggested by their transcriptional synergy in regulating the expression of TGFß and of endoglin, a TGFß-binding protein (20). Thus, there may be a precise stoichiometry between KLF6 and Sp1 that is promoter and context specific in regulating their target genes.

The identification of several point mutations of KLF6 in human cancer affecting lysine residues led us to explore the potential role of lysine modification in regulating KLF6 activity. These have included mutations in prostate (K186R; ref. 1), colon (K74R; ref. 12), and hepatocellular carcinoma (K182R; ref. 15). Here we tested the functional activity of an additional prostate cancer–derived mutant of KLF6, K209R, for its capacity to both transactivate the p21waf1/cip1 promoter, and to function as a substrate for protein acetyltransferase activity by CBP. Our findings indicate that acetylation of a specific Lys209 was closely linked with CBP-dependent activation of endogenous p21WAF1/cip1 in vitro. Moreover, its mutation to arginine as seen in prostate cancer abrogated p21WAF1/cip1 transactivation. Interestingly, this lysine is within the peptide consensus sequence for acetylation of p53 by p300/CBP (21, 50, 51), consistent with recent models proposed for acetylation of p53 (21). However, despite ample evidence that acetylation of p53 regulates several of its functions (52), the relative biological significance of p53 acetylation still remains uncertain. Because p53 has been one of the most thoroughly studied molecules that is covalently modified (52), more recent efforts have focused on the interdependence of its different covalent modifications, including phosphorylation, acetylation, methylation, ubiquitination, and sumoylation, and how they affect p53's interaction with other cellular and viral factors, thereby altering protein p53 levels (52–55).

Acetylation of specific lysine residues by different acetyltransferases may lead to divergent functional outcomes. For example, acetylation of pRb at specific lysine residues by p300 has been linked to inhibition of cell cycle progression (25), whereas acetylation of pRb by PCAF of key Lys873/874 near the COOH terminus correlates with cell differentiation (56). These divergent outcomes may also reflect differences between p300 and PCAF in promoting cell differentiation in vivo (57). It seems possible that similar, subtle differences in KLF6 acetylation may also underlie functional differences as well, a possibility that merits further study. Interestingly, rodent and human KLF6 carry the consensus R-K-X-X-T-K sequence at residues 208 to 213, which is as a substrate site for p300/CBP in nuclear factor-{kappa}B (relA)–mediated inflammation and interaction with I{kappa}B{alpha} (58). Thus far, KLF5 and KLF13 are the only KLFs reported to be regulated by acetylation (59, 60).

In summary, our data establish a role of KLF6 in recruiting a specific coactivator complex containing CBP and PCAF to promote transcriptional activation of p21WAF1/cip1 and provide evidence that KLF6 serves as a substrate for CBP HAT activity. Combined with the loss of growth suppressive activity of a tumor-derived lysine mutant of KLF6, these findings point to acetylation as a key modification in regulating its growth- and tumor-suppressive activities.


   Acknowledgments
 
Grant support: Department of Defense grants DAMD17-03-1-0100 (S.L. Friedman), DAMD17-02-1-0720, and DAMD17-03-1-0129 (J.A. Martignetti); NIH grants DK37340 (S.L. Friedman), CA088325 (R. Wang), HL67099, and CA98552 (M.J. Walsh); Bendheim Foundation (S.L. Friedman); and Howard Hughes Medical Student Research Fellowships (S. Yea and G. Narla).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3/29/05. Revised 7/31/05. Accepted 8/ 8/05.


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 Materials and Methods
 Results
 Discussion
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