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MCAF1/AM Is Involved in Sp1-mediated Maintenance of Cancer-associated Telomerase Activity*

  • Lifeng Liu
    Affiliations
    Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811
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  • Ko Ishihara
    Affiliations
    Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811
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  • Takaya Ichimura
    Affiliations
    Department of Pathology and Experimental Medicine, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan
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  • Naoyuki Fujita
    Affiliations
    Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan
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  • Shinjiro Hino
    Affiliations
    Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811
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  • Saori Tomita
    Affiliations
    Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811
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  • Sugiko Watanabe
    Affiliations
    Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811
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  • Noriko Saitoh
    Affiliations
    Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811
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  • Takaaki Ito
    Affiliations
    Department of Pathology and Experimental Medicine, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan
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  • Mitsuyoshi Nakao
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-96-373-6800; Fax: 81-96-373-6804
    Affiliations
    Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811
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  • Author Footnotes
    * This work was supported by a Grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (to M. N.) and a Grant-in-aid for Global COE (Center of Excellence) “Cell Fate Regulation Research and Education Unit,” Kumamoto University. 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.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1–S10.
Open AccessPublished:December 23, 2008DOI:https://doi.org/10.1074/jbc.M807098200
      Telomerase maintains telomere length and is implicated in senescence and immortalization of mammalian cells. Two essential components for this enzyme are telomerase reverse transcriptase (TERT) and the telomerase RNA component (encoded by the TERC gene). These telomerase subunit genes are known to be mainly expressed by specificity protein 1 (Sp1). MBD1-containing chromatin-associated factor 1 (MCAF1), also known as ATFa-associated modulator (AM) and activating transcription factor 7-interacting protein (ATF7IP), mediates gene regulation, although the precise function of MCAF1 remains to be elucidated. Here, we report that MCAF1 is involved in Sp1-dependent maintenance of telomerase activity in cancer cells. Two evolutionarily conserved domains of MCAF1 directly interact with Sp1 and the general transcriptional apparatus. Selective depletion of MCAF1 or Sp1 down-regulates TERT and TERC genes in cultured cells, which results in decreased telomerase activity. The transcriptionally active form of RNA polymerase II and the general transcription factor ERCC3 decreased in the TERT promoter under the loss of MCAF1 or Sp1. Consistently, MCAF1 is found to be frequently overexpressed in naturally occurring cancers that originate in different tissues. Our data suggest that transcriptional function of MCAF1 facilitates telomerase expression by Sp1, which may be a common mechanism in proliferative cancer cells.
      Telomerase is a multiple subunit complex that contains RNA-dependent DNA polymerase that adds telomeric hexamer repeats to the ends of chromosomes (
      • Colgin L.M.
      • Reddel R.R.
      ,
      • Nugent C.I.
      • Lundblad V.
      ). This enzymatic activity is normally absent in most somatic cells, which results in a gradual loss of telomeres through cell division. In contrast, more than 85% of cancerous cells and in vitro immortalized cells gain telomerase activity, which is required for unlimited proliferation (
      • Kim N.W.
      • Piatyszek M.A.
      • Prowse K.R.
      • Harley C.B.
      • West M.D.
      • Ho P.L.
      • Coviello G.M.
      • Wright W.E.
      • Weinrich S.L.
      • Shay J.W.
      ,
      • Kumakura S.
      • Tsutsui T.W.
      • Yagisawa J.
      • Barrett J.C.
      • Tsutsui T.
      ,
      • Nakamura T.M.
      • Morin G.B.
      • Chapman K.B.
      • Weinrich S.L.
      • Andrews W.H.
      • Lingner J.
      • Harley C.B.
      • Cech T.R.
      ,
      • Herbert B.
      • Pitts A.E.
      • Baker S.I.
      • Hamilton S.E.
      • Wright W.E.
      • Shay J.W.
      • Corey D.R.
      ). The reactivation of telomerase activity may contribute to cell transformation by increasing the potential risk of genetic and epigenetic defects (
      • Bellon M.
      • Nicot C.
      ). The telomerase enzyme consists of two essential components, telomerase reverse transcriptase (TERT)
      The abbreviations used are: TERT, telomerase reverse transcriptase; TR, telomerase RNA component; Sp1, specificity protein 1; MBD1, methylated DNA-binding domain protein 1; MCAF1, MBD1-containing chromatin-associated factor 1; AM, ATFa-associated modulator; ATF7IP, activating transcription factor 7-interacting protein; SETBD1, SET domain bifurcated 1; GST, glutathione S-transferase; TF, transcription factor; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; RT, reverse transcriptase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TBP, TATA box-binding protein.
      2The abbreviations used are: TERT, telomerase reverse transcriptase; TR, telomerase RNA component; Sp1, specificity protein 1; MBD1, methylated DNA-binding domain protein 1; MCAF1, MBD1-containing chromatin-associated factor 1; AM, ATFa-associated modulator; ATF7IP, activating transcription factor 7-interacting protein; SETBD1, SET domain bifurcated 1; GST, glutathione S-transferase; TF, transcription factor; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; RT, reverse transcriptase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TBP, TATA box-binding protein.
      (encoded by the TERT gene) and the telomerase RNA component (TR; encoded by the TERC gene), which serves as a template for telomeric DNA synthesis. TR is ubiquitously expressed in most tissues, whereas TERT is expressed only in telomerase-positive cells (
      • Yi X.
      • Tesmer V.M.
      • Savre-Train I.
      • Shay J.W.
      • Wright W.E.
      ,
      • Avilion A.A.
      • Piatyszek M.A.
      • Gupta J.
      • Shay J.W.
      • Bacchetti S.
      • Greider C.W.
      ). Introduction of exogenous TERT can immortalize primary human cells (
      • Bodnar A.G.
      • Ouellette M.
      • Frolkis M.
      • Holt S.E.
      • Chiu C.P.
      • Morin G.B.
      • Harley C.B.
      • Shay J.W.
      • Lichtsteiner S.
      • Wright W.E.
      ,
      • Yang J.
      • Chang E.
      • Cherry A.M.
      • Bangs C.D.
      • Oei Y.
      • Bodnar A.
      • Bronstein A.
      • Chiu C.P.
      • Herron G.S.
      ), and overexpression of both TERT and TR synergistically increases telomerase activity (
      • Cristofari G.
      • Lingner J.
      ). Because the telomerase subunit genes must be appropriately regulated in vivo, abnormal expression of TERT and TERC genes induces cell deregulation.
      The specificity protein 1 (Sp1) plays a crucial role in transcriptional control of the TERT gene (
      • Cong Y.S.
      • Wright W.E.
      • Shay J.W.
      ). There are five GC boxes that are consecutive binding sites of Sp1 in the TERT core promoter (
      • Takakura M.
      • Kyo S.
      • Kanaya T.
      • Hirano H.
      • Takeda J.
      • Yutsudo M.
      • Inoue M.
      ). In addition, Sp1 is known to cooperate with c-Myc to transactivate the TERT gene in a cell type-specific manner (
      • Kyo S.
      • Takakura M.
      • Taira T.
      • Kanaya T.
      • Itoh H.
      • Yutsudo M.
      • Ariga H.
      • Inoue M.
      ). Although Sp1 is a ubiquitous protein that targets both core promoter and enhancers, it is interesting that this protein is highly expressed in various cancers of the stomach, colon and rectum, pancreas, breast, and thyroid (
      • Safe S.
      • Abdelrahim M.
      ). Thus, Sp1-dependent expression of the TERT gene may provide a frequent pathway toward cancer development. However, the precise mechanism of Sp1-dependent control of telomerase expression remains to be elucidated.
      We initially found that methylated DNA-binding domain protein 1 (MBD1) interacts with MBD1-containing chromatin-associated factor 1 (MCAF1) (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ), also known as ATFa-associated modulator (AM) and activating transcription factor 7-interacting protein (ATF7IP) (
      • De Graeve F.
      • Bahr A.
      • Chatton B.
      • Kedinger C.
      ). MCAF1 binds the transcriptional repression domain of MBD1 and further recruits SET domain bifurcated 1 (SETDB1) for gene silencing in methylated DNA regions (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ,
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ). AM/MCAF1 facilitates conversion of dimethyl to trimethyl histone H3 at lysine residue 9 (H3K9) by ets-related gene-associated protein with SET domain, a murine homologue of human SETDB1 (
      • Wang H.
      • An W.
      • Cao R.
      • Xia L.
      • Erdjument-Bromage H.
      • Chatton B.
      • Tempst P.
      • Roeder R.G.
      • Zhang Y.
      ). Thus, MCAF1 is involved in MBD1-dependent transcriptional repression via heterochromatin assembly. Previous observations have suggested that MCAF1 and Sp1 are functionally related (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ,
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ,
      • Gunther M.
      • Laithier M.
      • Brison O.
      ); for instance, MCAF1 enhances Sp1-mediated transcription by Rta protein of Epstein-Barr virus (
      • Chang L.K.
      • Chung J.Y.
      • Hong Y.R.
      • Ichimura T.
      • Nakao M.
      • Liu S.T.
      ). In addition, Sp1 is known to interact with basal transcriptional factors including TFIIB, TFIIE, TBP/TFIID, and further with the transcriptional mediators such as the cofactor required for Sp1 (
      • Wierstra I.
      ). The transcriptional mediators transduce regulatory information between Sp1 and the basal transcription machinery including the core RNA polymerase II (
      • Myers L.C.
      • Kornberg R.D.
      ). It is of great interest to investigate the transcriptional role of MCAF1 and Sp1 in the telomerase subunit genes.
      During our investigations, we have found that MCAF1 is highly expressed in human cancers that originated in many different tissues, suggesting that MCAF1 has an important role in tumorigenesis. Consistently, our data demonstrate that MCAF1 is required for Sp1-dependent maintenance of telomerase activity in cancer cells. Specific knockdown of MCAF1 or Sp1 reduced both TERT and TERC expression, which resulted in a decrease of telomerase activity. The active form of RNA polymerase II and general transcription factor were released from the TERT promoter by the loss of MCAF1 or Sp1. Two evolutionarily conserved domains of MCAF1 were responsible for direct interaction with Sp1 and the general transcriptional apparatus. These data shed light on the transcriptional mediator-like function of MCAF1 that facilitates Sp1-dependent expression of the telomerase subunit genes, which may be a common mechanism in oncogenesis.

      EXPERIMENTAL PROCEDURES

      Cell Culture—HeLa cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's minimum essential medium and Ham's F-12 nutrient medium (Sigma) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and penicillin/streptomycin.
      Antibodies—Anti-MCAF1 antibodies were as described previously (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ). The antibodies utilized were anti-Sp1 (Santa Cruz Biotechnology), anti-β-tubulin (Amersham Biosciences Bioscience), anti-GST (DAKO), anti-RNA polymerase II CTD4H8 (Upstate Biotechnology), anti-His (Qiagen), anti-ERCC3 (Santa Cruz Biotechnology), anti-ERCC2 (Santa Cruz Biotechnology), anti-TFIIEα (Santa Cruz Biotechnology), and anti-TFIIEβ (Santa Cruz Biotechnology). Western blot and immunofluorescence analyses were carried out as described elsewhere (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ,
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ).
      Glutathione S-Transferase (GST) Pull-down Assay—Bacterially expressed GST and GST fusion proteins (1 μg) were immobilized on glutathione-agarose beads, and incubated with HeLa nuclear extract (60 μg) or His-tagged proteins (1 μg) in buffer that contained 0.1% Triton X-100, 50 mm HEPES (pH 7.4), 100 mm NaCl, 5% glycerol, 2 mm dithiothreitol, and protease inhibitors for 1 h at 4 °C. The input indicated 10% of the proteins in the reaction mixture.
      Immunoprecipitation—HeLa nuclear extract (Promega) was diluted 10 times with buffer (40 mm Tris-HCl (pH 7.8), 100 mm KCl, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, and 25% glycerol), and mixed with anti-MCAF1 antibodies, anti-Sp1 antibodies, or control IgG for 1 h at 4 °C, followed by incubation for 1 h after adding 30 μl of protein A/G-agarose beads (Amersham Biosciences). After washing, the bound proteins were detected by Western blot analysis.
      siRNA-mediated Knockdown of Sp1, MCAF1, and TERT—HeLa cells were transfected with siRNAs with Lipofectamine™ RNAiMAX (Invitrogen) and harvested at 60 h after transfection. Twenty-one nucleotide siRNA duplexes were designed to target mRNAs that encoded human Sp1, MCAF1, or TERT. The selected siRNA target sequences were submitted to human genome and EST databases to ensure the target specificities. These sequences are described in supplemental Table S1. The siRNAs for GL3 were as described previously (
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ).
      Chromatin Immunoprecipitation (ChIP) and Quantitative PCR—HeLa cells (1 × 106) were cross-linked with 1% formaldehyde at 37 °C for 10 min. Crude cell lysates were sonicated to generate 200–1000-bp DNA fragments. ChIP was performed with anti-Sp1 or anti-MCAF1 antibodies, or control IgG, according to the manufacturer's protocols (Upstate Biotechnology). PCR amplification of the TERT gene promoter was carried out for 20 (the first PCR) and 26 cycles (the second PCR) under conditions of 20 s at 98 °C, 30 s at 65 °C, and 30 s at 72 °C. Amplification of the TERC gene promoter was performed for 35 cycles under conditions of 20 s at 98 °C, 30 s at 58 °C, and 30 s at 72 °C. Amplification of the ∼8 kb upstream of the TERT gene was performed for 30 cycles under conditions of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. Quantitative PCR was performed on the products of ChIP in triplicate by the SYBR Green method using the Power SYBR Green PCR Master Mix and ABI 7300 Real Time PCR System (Applied Biosystems). Standard curves were generated using serial dilutions of input DNA (10.0, 2.0, 0.4, and 0.08%). The Ct of each reaction was quantified against the standard curve. The primer sets are listed in supplemental Table S2.
      Quantitative RT-PCR—Total RNA was isolated using Isogen (Nippon Gene). For cDNA synthesis, 3 μg of total RNA was reverse-transcribed with Superscript III (Invitrogen) using oligo(dT) primers. Quantitative PCR of the target cDNAs was performed by the SYBR Green method using Power SYBR Green PCR Master Mix (Applied Biosystems). Each experiment was carried out at least three times. The fold relative enrichment was quantified, together with normalization by the β-actin level. The primer sets are listed in supplemental Table S2.
      Measurement of Telomerase Activity—Telomerase activity was measured using the quantitative telomeric repeat amplification protocol (Q-TRAP) assay (
      • Wege H.
      • Chui M.S.
      • Le H.T.
      • Tran J.M.
      • Zern M.A.
      ). Briefly, cell pellets were resuspended in a lysis buffer (10 mm Tris-HCl (pH 7.5), 1 mm MgCl2, 1 mm EGTA, 0.1 mm benzamidine, 5 mm β-mercaptoethanol, 0.5% CHAPS, and 10% glycerol) and incubated for 30 min on ice. After centrifugation at 12,000 × g for 20 min at 4 °C, aliquots of the supernatant were rapidly frozen and stored at -80 °C. Protein concentration of lysates was determined with GeneQuant Pro (Bio-Rad). To prepare a control lysate, telomerase was inactivated by incubating at 85 °C for 10 min. The SYBR Green Q-TRAP assay was conducted with cell lysates (1.0, 0.5, 0.2, and 0.1 μg), 0.1 μg of telomerase primer TS, and 0.1 μg of anchored return primer ACX, in 25 μl with SYBR Green PCR Master Mix (Applied Biosystems). Primer sequences are listed in supplemental Table S2. Using the ABI PRISM 7300 thermal cycler (Applied Biosystems), samples were incubated for 20 min at 25 °C, and amplified in 35 cycles of 30 s at 95 °C and 90 s at 60 °C. Samples, inactivated samples, and lysis buffer were used on all PCR plates. Each sample was analyzed at least in duplicate. Telomerase activity is shown relative to that in control cells.
      Bisulfite Genomic DNA Sequencing—Genomic DNAs were purified and treated with sodium bisulfite using the BisulFast DNA Modification Kit (Toyobo) (
      • Aoto T.
      • Saitoh N.
      • Ichimura T.
      • Niwa H.
      • Nakao M.
      ), followed by PCR amplification with each of the specific primers, which contained no CpG sequences, using LA Taq polymerase (Takara). The amplified region corresponded to nucleotides 3791–4105 of the unmodified human TERT gene sequences (GenBank™ AF097365). PCR was carried out for 32 cycles under conditions of 30 s at 98 °C, 30 s at 56 °C, and 60 s at 72 °C. PCR products were subcloned into pGEM-T easy vector (Promega) and sequenced (at least 15 individual clones). Primers used are shown in supplemental Table S2.
      Immunohistochemistry—Immunohistochemistry was performed with human Tumor Tissue Arrays including stomach, breast, and lung tumors, and various normal tissues (BioChain Institute, Inc. and ISU Abxis Co., Ltd.). The array slides were deparaffinized and then incubated in methanol with 0.3% hydrogen peroxide for 30 min to block the endogeneous peroxidase activity. Thereafter, tissue sections were immersed in 1.0% Block-Ace (Dainippon Sumitomo Pharma Co., Ltd.) in phosphate-buffered saline for 30 min, covered with anti-MCAF1 antibodies, and incubated 1 h at room temperature. Visualization of the immunoreaction was performed using Histofine Simple Stain MAX-PO (Nichirei Bioscience) and 3,3-diaminobenzidine tetrahydrochloride (Dako). The slides were counterstained with hematoxylin and mounted with Malinol (Muto Pure Chemicals).
      Statistical Analysis—Differences between groups were analyzed using Student's t test. p < 0.05 was considered statistically significant.

      RESULTS

      MCAF1 Complexes with Sp1 via the Evolutionally Conserved Domains—The MCAF/AM family of proteins is characterized by two functional domains, Domain 1 and Domain 2, which are evolutionarily conserved from Drosophila to humans (Fig. 1A) (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ,
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ). There is no DNA binding motif found in MCAF1. On the other hand, Sp1 has two transcriptional activation domains and a region that is mainly composed of three zinc finger motifs (
      • Suske G.
      ). Previous observations have suggested that MCAF1 and Sp1 are functionally related (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ,
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ,
      • Gunther M.
      • Laithier M.
      • Brison O.
      ,
      • Chang L.K.
      • Chung J.Y.
      • Hong Y.R.
      • Ichimura T.
      • Nakao M.
      • Liu S.T.
      ). To examine the direct interaction between MCAF1 and Sp1 at the domain levels, we prepared bacterially expressed deletion mutants of MCAF1 and Sp1, which were fused to GST or His-tagged. The representatives included MCAF1 (Δ10, Δ18, and Δ8) (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ) and Sp1 (Δ5, Δ6, and Δ7). GST and GST-fused portions of MCAF1 were immobilized on glutathione-agarose beads and incubated with the HeLa nuclear extract (Fig. 1B, left). Sp1 bound both Domain 1 (Δ18) and Domain 2 (Δ8), but not the amino-terminal region (Δ10) of MCAF1. To further determine the region of Sp1 responsible for interacting with MCAF1, we used GST-fused portions of Sp1 and His-tagged MCAF1 (Fig. 1B, right). GST and GST-fused portions of Sp1 on glutathione-agarose beads were mixed with His-tagged MCAF1 (Δ18 or Δ8). Domain 1 (Δ18) of MCAF1 bound Δ5 and Δ6, whereas Domain 2 (Δ8) bound Δ6 and Δ7 of Sp1. The amino-terminal region of Sp1 that contained activation domains did not bind MCAF1, and the regions of MCAF1 except Domains 1 and 2 did not bind Sp1 (data not shown). To confirm whether MCAF1 formed a complex with Sp1, immunoprecipitation analysis using anti-MCAF1 and anti-Sp1 antibodies were performed in HeLa cells (Fig. 1C). MCAF1 was found in the immunoprecipitates with Sp1. Conversely, Sp1 was present in the MCAF1 immunoprecipitates. These data suggest that the conserved domains of MCAF1 directly bind the carboxyl-terminal region of Sp1, resulting in the formation of an MCAF1-Sp1 complex.
      Figure thumbnail gr1
      FIGURE 1MCAF1 complexes with Sp1 via the evolutionarily conserved domains. A, functional domains of MCAF1 and Sp1. MCAF1 has two evolutionally conserved domains, Domain 1 and Domain 2. Sp1 contains two transcriptional activation domains with serine and threonine-rich regions (S/T), and the three zinc fingers with DNA binding activity. Bacterially expressed portions of MCAF1 and Sp1 are indicated for a pull-down assay. B, direct interaction between MCAF1 and Sp1. Immobilized GST-fused MCAF1 (Δ10, Δ18, and Δ8) was incubated with HeLa nuclear extracts (left). Both Domains 1 (Δ18) and 2 (Δ8) of MCAF1 bound to the carboxyl-terminal region of Sp1 (right). C, complex formation of MCAF1 and Sp1. Endogenous MCAF1 and Sp1 were immunoprecipitated in HeLa cells. Two isoforms of Sp1 (95 and 105 kDa) are normally detected. The full-length of MCAF1 is shown in the presence of the fast-migrating degradation product, which disappeared by the knockdown (). Input indicates 10% of the proteins in the reaction mixture (B and C).
      Formation of MCAF1-Sp1 Complex in Telomerase Subunit Gene Promoters—To investigate the involvement of the MCAF1-Sp1 complex in endogenous gene regulation, ChIP analysis was carried out on HeLa cells. Telomerase-associated TERT and TERC genes are reported to be transactivated by Sp1, through five and four binding sites within the core promoter regions, respectively (Fig. 2A) (
      • Horikawa I.
      • Cable P.L.
      • Afshari C.
      • Barrett J.C.
      ,
      • Zhao J.Q.
      • Hoare S.F.
      • McFarlane R.
      • Muir S.
      • Parkinson E.K.
      • Black D.M.
      • Keith W.N.
      ). After formaldehyde-based cross-linking of protein and DNA, chromatin fragmented by sonication was immunoprecipitated with anti-MCAF1 or anti-Sp1 antibodies, followed by PCR amplification using specific primers for the promoter sequences (Fig. 2B). MCAF1 and Sp1 were found in the TERT and TERC promoter regions. In addition, the ∼8 kb upstream site of the TERT gene was not bound by these proteins. Species-matched IgG was used for immunoprecipitation as a control. In addition, both proteins were also found in the distinct Sp1-dependent BRCA1 gene promoter (supplemental Fig. S1). These data show that MCAF1 and Sp1 coexist in the TERT and TERC gene promoters, which suggests a role for MCAF1 in the Sp1-dependent telomerase expression.
      Figure thumbnail gr2
      FIGURE 2Existence of MCAF1 in telomerase subunit gene promoters in an Sp1-dependent manner. A, schematic representation of human TERT and TERC promoter regions. Both genes had promoter-associated CpG islands that contained multiple Sp1-binding sites (open boxes) and exons (closed boxes). For ChIP analysis, PCR-amplified fragments are shown with gray lines. B, localization of MCAF1 and Sp1 in telomerase subunit gene promoters. ChIP analysis of MCAF1 and Sp1 in the TERT and TERC gene promoters. MCAF1 and Sp1 were cross-linked to DNAs and precipitated with specific antibodies, followed by PCR amplification with specific primers. Genomic DNAs were used as a control (input). C and D, specific siRNA-mediated knockdown of MCAF1 and Sp1. siGL3 was used as control siRNAs. For Western blot analysis, β-tubulin is shown as a control (C). For quantitative RT-PCR, the relative mRNA levels with the use of control siRNAs were normalized to 1 (D). **, p < 0.01. E, Sp1-dependent localization of MCAF1 in the TERT promoter. Control siRNAs and species-matched IgG were used as a control. Values are given as mean ± S.E. from more than three independent experiments. **, p < 0.01. IP, immunoprecipitated.
      To address the functional relationship of MCAF1 and Sp1, we knocked down MCAF1 or Sp1 by using more than two siRNAs specific for each protein. HeLa cells were transfected with these siRNAs and then grown for 60 h. Western blot and quantitative RT-PCR analysis showed that MCAF1 and Sp1 were depleted at both the protein and mRNA levels (Fig. 2, C and D). Sp1 knockdown did not affect the levels of MCAF1, and vice versa. There was no significant effect of control siRNAs. Because the effect of each siRNA was basically similar, we later presented the data with MCAF1–2 and Sp1–2 siRNAs. Under knockdown conditions, we examined the amount of MCAF1 and Sp1 in the TERT promoter, using quantitative ChIP analysis (Fig. 2E). MCAF1 knockdown reduced the known protein, but not Sp1, bound to the gene promoter (gray bars, p < 0.01). In contrast, the loss of Sp1 markedly decreased MCAF1 as well as Sp1 in the TERT promoter (open bars, p < 0.01), compared with the controls. This suggests that MCAF1 is localized to the TERT gene promoter in an Sp1-dependent manner, via complex formation of these proteins.
      MCAF1 and Sp1 Are Required for Maintaining Telomerase Activity—To clarify the role of MCAF1 and Sp1 in transcriptional control of telomerase components, we performed a quantitative RT-PCR for TERT and TERC mRNAs (Fig. 3A). The transcripts of TERT and TERC genes were significantly down-regulated by the knockdown of MCAF1 or Sp1 in HeLa cells (p < 0.01 or p < 0.05). Similar results were obtained in HCT116 cells (supplemental Fig. S2). The data suggest that MCAF1 cooperates with Sp1 to activate transcription from the TERT and TERC genes. We had difficulty detecting the TERT protein, probably because of recognition inefficiency of anti-TERT antibodies; therefore, we assessed the effect of MCAF1 and Sp1 on endogenous telomerase activity, using a Q-TRAP assay (
      • Wege H.
      • Chui M.S.
      • Le H.T.
      • Tran J.M.
      • Zern M.A.
      ). The cell lysates were prepared from HeLa cells under the knockdown of MCAF1 or Sp1, and were serially diluted for the TRAP assay, followed by quantitative PCR using specific primers for telomeric repeats (Fig. 3B). Compared with the controls, telomerase activity markedly decreased in the MCAF1 or Sp1 knockdown cells (p < 0.01 or p < 0.05). These results suggest that the MCAF1-Sp1 complex maintains telomerase activity by inducing TERT and TERC expression.
      Figure thumbnail gr3
      FIGURE 3MCAF1-Sp1 complex is required for telomerase activity and cell proliferation. A, transactivation of the telomerase subunit genes by the MCAF1-Sp1 complex. Quantitative RT-PCR analysis of telomerase subunit genes was performed in MCAF1- or Sp1-depleted cells. The relative mRNA levels with the use of control siRNAs were normalized to 1. B, maintenance of telomerase activity by the MCAF1-Sp1 complex. Quantitative PCR analysis of telomerase activity was performed in MCAF1- or Sp1-depleted cells. Relative telomerase levels with control siRNAs and 1.0 μg of cell lysate were normalized to 1. Values are given as mean ± S.D. from more than three independent experiments (A and B). **, p < 0.01; *, p < 0.05. C and D, maintenance of cell proliferation by the MCAF1-Sp1 complex. The cell numbers were measured at indicated time points after knockdown of MCAF1, Sp1, or TERT. Results were obtained from three independent experiments. Error bars indicate S.D. K.D., knockdown.
      To further evaluate the effect of the MCAF1-Sp1 complex on cellular proliferation, we counted the cell numbers under knockdown of MCAF1 or Sp1 (Fig. 3C). MCAF1- or Sp1-depleted cells showed lower proliferative activity in comparison to the controls. These data were very analogous to the TERT knockdown by using two specific siRNAs (Fig. 3D). As expected, the knockdown of TERT diminished the known mRNAs and the resulting telomerase activity (supplemental Fig. 3). Collectively, these data suggest that the MCAF1-Sp1 complex is required for maintaining telomerase activity and proliferation in the cancer cells examined.
      MCAF1-Sp1 Complex Is Involved in Maintaining the Localization of Phosphorylated RNA Polymerase II and General Transcription Factor in the TERT Promoter—To examine transcriptional control by the MCAF1-Sp1 complex, we analyzed the status of modified histones in the TERT promoter, by a quantitative ChIP analysis using specific antibodies (supplemental Fig. S4). Among the transcriptionally active marks tested, H3 acetylation alone was diminished in the Sp1 knockdown cells, whereas MCAF1 or Sp1 depletion showed no significant changes in H3K4 methylation and H4 acetylation. These results may have been caused by the originally low levels of these modified histones in the TERT promoter. Because MCAF1 and Sp1 are associated with RNA polymerase II and general transcription factors (
      • De Graeve F.
      • Bahr A.
      • Chatton B.
      • Kedinger C.
      ,
      • Wierstra I.
      ), we then tested for the presence of the transcriptionally active form of RNA polymerase II (Pol II (Ser 5-P)) and the excision-repair, complementing defective, in Chinese hamster 3/xeroderma pigmentosum B (ERCC3/XPB), an essential component of TFIIH, in the TERT promoter (Fig. 4, A and B). Knockdown of MCAF1 or Sp1 resulted in significant reduction of both Pol II (Ser 5-P) and ERCC3 in the gene promoter (p < 0.01 or p < 0.05). Thus, the MCAF1-Sp1 complex maintains the localization of phosphorylated RNA polymerase II and ERCC3 in the TERT core promoter.
      Figure thumbnail gr4
      FIGURE 4Sp1-coupled transcriptional mediator-like function of MCAF1. A and B, loading of general transcriptional apparatus to TERT promoter by MCAF1-Sp1 complex. Knockdown of MCAF1 or Sp1 reduced the active form of RNA polymerase II (Pol II (Ser 5P)) and ERCC3 in the TERT promoter. Values are given as mean ± S.D. from more than three independent experiments (A and B). **, p < 0.01; *, p < 0.05. C, complex formation of MCAF1, general transcription factors, and RNA polymerase II. MCAF1 was precipitated from HeLa nuclear extracts with anti-MCAF1 antibodies (left). ERCC3, RNA polymerase II (Pol II), TFIIEα, and TFIIEβ were similarly immunoprecipitated (IP) (right). The immune complexes were probed with the indicated antibodies. D, interaction of general transcription factors with conserved domains of MCAF1. GST-fused MCAF1 (Δ10, Δ18, and Δ8) was incubated with HeLa nuclear extracts. The bound proteins were probed with the indicated antibodies. E, direct interaction between MCAF1 and general transcription factors. GST-ERCC3 and GST-ERCC2 on glutathione-agarose beads were incubated with His-tagged MCAF1 (Δ10 and Δ8). Conversely, immobilized GST-MCAF1 (Δ10, Δ18, and Δ8) was mixed with His-tagged TFIIEα and TFIIEβ. The input indicates 10% of the proteins in the reaction mixture (CE).
      To confirm the fundamental role of MCAF1, we characterized the ability of the protein to bind the general transcription apparatus. MCAF1 in the HeLa nuclear extract was immunoprecipitated with anti-MCAF1 antibodies, and cellular ERCC3 and Pol II were found in the MCAF1 immunoprecipitates (Fig. 4C, left). The MCAF1 immunoprecipitates also contained TFIIEα, TFIIEβ, and ERCC2/XPD (data not shown). We next immunoprecipitated ERCC3, Pol II, TFIIE-α, and TFIIE-β from the nuclear extract with appropriate antibodies, and the presence of MCAF1 was detected in these immunoprecipitates (Fig. 4C, right). Because TFIID is known to interact with Sp1, we examined whether the TATA box-binding protein (TBP), an essential component of TFIID, interacts with MCAF1 (supplemental Fig. S5). Sp1, but not MCAF1, was found in the TBP immunoprecipitates, suggesting that TBP preferentially coexists with Sp1. To further determine the region of MCAF1 that interacted with general transcription factors, GST-MCAF1 deletions were used for a pull-down assay with the nuclear extract (Fig. 4D). Both ERCC3 and ERCC2 were associated with Domain 2 (Δ8) of MCAF1. In contrast, TFIIE-α and TFIIE-β bound both Domain 1 (Δ18) and Domain 2(Δ8) of the protein. To check the direct interaction between MCAF1 and general transcription factors, a pull-down analysis was performed using purified proteins (Fig. 4E). GST-fused ERCC3 and ERCC2 on glutathione-agarose beads were incubated with His-tagged MCAF1 (Δ10 and Δ8). Both ERCC3 and ERCC2 directly bound the Δ8 of MCAF1 but not Δ10, which was consistent with the data in Fig. 4D. Conversely, immobilized GST-MCAF1 (Δ10, Δ18, and Δ8) was mixed with His-tagged TFIIE-α and TFIIE-β. TFIIE-α was found to bind Δ18 alone, whereas TFIIE-β bound both Δ18 and Δ8 of MCAF1. Taken together, these data suggest that two conserved domains of MCAF1 directly bind Sp1 and the general transcription apparatus, like the transcriptional mediators.
      MCAF1-Sp1 Complex May Affect CpG Methylation Status in the TERT Promoter Region—Recent studies have reported that the human TERT promoter region is uniquely methylated in telomerase-positive cell lines (
      • Kim N.W.
      • Piatyszek M.A.
      • Prowse K.R.
      • Harley C.B.
      • West M.D.
      • Ho P.L.
      • Coviello G.M.
      • Wright W.E.
      • Weinrich S.L.
      • Shay J.W.
      ,
      • Devereux T.R.
      • Horikawa I.
      • Anna C.H.
      • Annab L.A.
      • Afshari C.A.
      • Barrett J.C.
      ), and that the unmethylated region just upstream of the transcriptional start site may permit low transcriptional activity of the TERT gene in tumor cells (
      • Yi X.
      • Shay J.W.
      • Wright W.E.
      ,
      • Renaud S.
      • Loukinov D.
      • Abdullaev Z.
      • Guilleret I.
      • Bosman F.T.
      • Lobanenkov V.
      • Benhattar J.
      ). In addition, it has been shown that the repressor protein CCCTC-binding factor (CTCF) binds the unmethylated motifs within the first exon to inhibit TERT transcription, whereas CpG methylation at the binding site inhibits CTCF binding, leading to TERT gene transcription in cancer cells (
      • Renaud S.
      • Loukinov D.
      • Abdullaev Z.
      • Guilleret I.
      • Bosman F.T.
      • Lobanenkov V.
      • Benhattar J.
      ). To test the epigenetic role of the MCAF1-Sp1 complex, we investigated the methylation status at 38 CpG dinucleotides in the promoter and first exon of the TERT gene (Fig. 5), using bisulfite genomic sequencing. Compared with the control cells, the CpG sites 5–9 between the Sp1-binding motifs tended to be selectively methylated in MCAF1 or Sp1 knockdown HeLa cells, in which TERT expression was reduced. In addition, the first exon region, which contained the CTCF binding site, was densely methylated in the controls, whereas it tended to be partially unmethylated at some CpG sites under MCAF1 or Sp1 depletion. Thus, the loss of the MCAF1-Sp1 complex may affect the DNA methylation status in the TERT gene. The molecular mechanism underlying the methylation changes on selective CpG sites under the loss of MCAF1 or Sp1 is unknown, but it may result from transcriptional down-regulation of the TERT gene.
      Figure thumbnail gr5
      FIGURE 5MCAF1-Sp1 complex affects CpG methylation in the TERT promoter region. Methylation status of 38 CpG dinucleotides in the promoter and first exon of the TERT gene was analyzed in HeLa cells, by bisulfite genomic sequencing. CpG dinucleotides are indicated (sites 1–38). MCAF1 or Sp1 knockdown changed the methylation status at the CpGs close to the Sp1- and CTCF-binding sites. Open circle, unmethylated CpG; closed circle, methylated CpG.
      It is reported that MCAF1 cooperates with MBD1 and SETDB1 for gene silencing in methylated DNA regions (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ,
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ). To test the implication of the MCAF1-MBD1-SETDB1 complex in the TERT gene, quantitative ChIP analysis was performed with specific antibodies (supplemental Fig. S6). MBD1 was detected in the TERT promoter, which was not affected by depletion of MCAF1 or Sp1. In contrast, SETDB1 did not appear to be significantly enriched in this promoter, in agreement with the low levels of H3K9 trimethylation, suggesting that the MCAF1-MBD1-SETDB1 complex unlikely affects TERT regulation. In addition, the knockdown of MCAF1 or Sp1 did not significantly change CTCF binding to the target site in the TERT gene.
      MCAF1 Is Overexpressed in Human Cancer Tissues—To clarify the involvement of MCAF1 in cancer development, we finally investigated the expression of MCAF1 in human cancer tissues, using an immunohistochemical analysis (Fig. 6). The specificity of rabbit anti-MCAF1 antibodies used was previously demonstrated (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ,
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ). MCAF1 staining was undetectable or at a low level in non-cancerous tissues of the stomach, breast, and lung (Fig. 6, panels b, f, and j). Using the same experimental conditions, in contrast, MCAF1 was detected at very high levels in the corresponding cancerous tissues. The data represent stomach cancer (panel d), breast cancer (panel h), and lung cancer including adenocarcinoma (panel l), and squamous cell carcinoma (panel n), in which MCAF1 was densely stained in the nuclei of cancer cells. Our studies demonstrated that, among 148 primary human cancers (53 adenocarcinoma of stomach, 52 invasive ductal carcinoma of breast, and 14 adenocarcinoma, and 29 squamous cell carcinoma of lung), MCAF1 was positively detected in more than 70% of the cancer tissues tested (summarized in supplemental Fig. S7).
      Figure thumbnail gr6
      FIGURE 6Overexpression of MCAF1 in human cancer tissues. Serial sections of non-cancerous and cancerous tissues: stomach (A, panels ad), breast (B, panels eh), and lung (C, panels in). Normal tissues show undetectable or very low expression of MCAF1 (panels b, f, and j). In contrast, MCAF1 was highly stained in adenocarcinoma of the stomach (panel d), invasive ductal carcinoma of the breast (panel h), and adenocarcinoma and squamous cell carcinoma of the lung (panels l and n, respectively). Hematoxylin and eosin staining are shown in panels a, c, e, g, i, k, and m; immunostaining for MCAF1 are shown in panels b, d, f, h, j, l, and n. Scale bar, 200 μm. The correlation analyses of MCAF1, Sp1, and telomeres are shown in .
      To investigate the correlation of MCAF1 and Sp1 in human cancer, immunohistochemical analysis was carried out using anti-Sp1 antibodies (supplemental Fig. S8). In agreement with previous studies (
      • Safe S.
      • Abdelrahim M.
      ), Sp1 was positively stained in more than 80% of adenocarcinoma of the stomach examined, whereas this protein was detected at low levels in non-cancerous tissues. Both MCAF1 and Sp1 tended to coexist in these gastric cancer tissues. To further test the relationship between MCAF1, Sp1, and the telomere state, we performed a telomere-FISH with the same tissue samples (supplemental Fig. S9). Multiple telomere signals were detected in adenocarcinoma tissues, rather than in non-cancerous tissues. We observed that high expression of MCAF1 and Sp1 was found in telomere-positive cancer cells, suggesting that telomeres may be well maintained in cancer cells that overexpress MCAF1 and Sp1.
      To clarify the role of MCAF1 and Sp1 in telomerase activities, we assessed the mRNA levels of TERT, TERC, MCAF1, and Sp1 in telomerase positive cancer cell lines, using quantitative RT-PCR analysis (supplemental Fig. S10). The expression levels of these genes were significantly higher than those in control primary fibroblasts (p < 0.01). The association between two relative mRNA levels was analyzed using the Pearson correlation coefficient (r), which varies from a perfect negative correlation (-1) to a perfect positive correlation (+1). A significantly correlated expression was found between TERT and TERC (TERT-TERC; r = 0.93), whereas there was no correlation of TERT-GAPDH or TERC-GAPDH. Moderate correlations were found in TERT-MCAF1 (r = 0.41), TERT-Sp1 (r = 0.33), TERC-MCAF1 (r = 0.30), and TERC-Sp1 (r = 0.48) (supplemental Fig. 10F). Thus, these data may support the transcriptional role of MCAF1 and Sp1 in regulating the telomerase subunit genes. Taken together, these results suggest that enhanced expression of MCAF1 is at least correlated with cancer development in a variety of tissue origins.

      DISCUSSION

      The mechanism responsible for maintaining telomerase activity is crucial for understanding cellular immortalization and cancer development (
      • Kim N.W.
      • Piatyszek M.A.
      • Prowse K.R.
      • Harley C.B.
      • West M.D.
      • Ho P.L.
      • Coviello G.M.
      • Wright W.E.
      • Weinrich S.L.
      • Shay J.W.
      ,
      • Hahn W.C.
      • Counter C.M.
      • Lundberg A.S.
      • Beijersbergen R.L.
      • Brooks M.W.
      • Weinberg R.A.
      ,
      • Shay J.W.
      • Bacchetti S.
      ). Our present study reveals that: (i) MCAF1 and Sp1 maintain expression of the TERT and TERC genes, which results in positive telomerase activity; (ii) Sp1 substantially tethers MCAF1 to the TERT promoter; (iii) MCAF1-Sp1 complex maintains the localization of transcriptionally active RNA polymerase II and ERCC3 in the TERT promoter; (iv) two evolutionarily conserved domains of MCAF1 directly bind Sp1 and the general transcriptional apparatus; and (v) MCAF1 is frequently expressed at high levels in most cancer tissues. Thus, overexpressed MCAF1 mediates Sp1-dependent maintenance of telomerase activity, by facilitating the localization of the general transcriptional apparatus and active form of RNA polymerase II in telomerase subunit genes, which may be a common mechanism in proliferative cancer cells.
      MCAF1 Mediates Sp1-dependent Expression of Telomerase Subunit Genes—The increase of telomerase activity is characteristic of cancer and prevents telomere shortening and senescence of tumor cells, which are critical for oncogenesis (
      • Kim N.W.
      • Piatyszek M.A.
      • Prowse K.R.
      • Harley C.B.
      • West M.D.
      • Ho P.L.
      • Coviello G.M.
      • Wright W.E.
      • Weinrich S.L.
      • Shay J.W.
      ,
      • Shay J.W.
      • Bacchetti S.
      ,
      • Janknecht R.
      ). There are at least two possible mechanisms for telomerase up-regulation in cancer. One is the increased copy number of the TERT and TERC genes (
      • Cao Y.
      • Bryan T.M.
      • Reddel R.R.
      ), which are located at human chromosomes 5p15.33 and 3q26.3, respectively. Chromosomal gains and amplifications that involve 5p (13.2%) and 3q (16.4%) have been detected in solid tumors, using comparative genomic hybridization (
      • Rooney P.H.
      • Murray G.I.
      • Stevenson D.A.
      • Haites N.E.
      • Cassidy J.
      • McLeod H.L.
      ). The other mechanism is increased expression of the telomerase subunit genes. Exogenous expression of both TERT and TR, compared with either alone, markedly augmented telomerase activity. For this reason, it is of interest that Sp1 commonly activates the TERT and TERC genes (
      • Kyo S.
      • Takakura M.
      • Taira T.
      • Kanaya T.
      • Itoh H.
      • Yutsudo M.
      • Ariga H.
      • Inoue M.
      ,
      • Zhao J.Q.
      • Glasspool R.M.
      • Hoare S.F.
      • Bilsland A.
      • Szatmari I.
      • Keith W.N.
      ). Because expression of TERT is essential for positive telomerase activity in cancer cells (
      • Nakamura T.M.
      • Morin G.B.
      • Chapman K.B.
      • Weinrich S.L.
      • Andrews W.H.
      • Lingner J.
      • Harley C.B.
      • Cech T.R.
      ,
      • Kyo S.
      • Inoue M.
      ), we focused on characterizing transcriptional regulation of the TERT gene by Sp1. Although the TERT promoter lacks either a TATA box or TATA-like sequence, it has been demonstrated that TATA-less promoters still utilize TATA factors for transcriptional initiation of these genes (
      • Smale S.T.
      ), and that Sp1 tethers TATA factors that lead to transcriptional initiation of TATA-less promoters (
      • Pugh B.F.
      • Tjian R.
      ). In this study, we showed that MCAF1 and Sp1 are required for maintaining the localization of transcriptionally active RNA polymerase II and general transcriptional factors in the TERT promoter, suggesting the possibility that MCAF1, together with Sp1, may stimulate the conversion of unphosphorylated to phosphorylated forms of RNA polymerase II. MCAF1 did not affect the DNA-binding ability of Sp1, whereas it was recruited to the TERT promoter by Sp1. Thus, MCAF1 synergistically participates in transcriptional activation of the telomerase subunit genes by interacting with Sp1, which is necessary for maintenance of telomerase activity in cancer. These data suggest that up-regulation of MCAF1 and Sp1 may be a fundamental event in telomerase maintenance in cancer.
      Epigenetic states in the TERT promoter region are important for transcriptional regulation, including trimethylated histone H3K4, and acetylated H3 and H4 in transcribed genes (
      • Berger S.L.
      ). In telomerase-positive cancer cells, however, we found that these modified histones were originally at low levels and did not significantly change under MCAF1 or Sp1 knockdown. Nevertheless, acetylated H3 alone tended to decline in Sp1-depleted cells, probably due to histone acetyltransferases involved in Sp1-mediated gene activation (
      • Wang X.
      • Pan L.
      • Feng Y.
      • Wang Y.
      • Han Q.
      • Han L.
      • Han S.
      • Guo J.
      • Huang B.
      • Lu J.
      ). Several studies have reported that promoter methylation is also one of the important mechanisms in the TERT regulation in cancer cells (
      • Renaud S.
      • Loukinov D.
      • Abdullaev Z.
      • Guilleret I.
      • Bosman F.T.
      • Lobanenkov V.
      • Benhattar J.
      ,
      • Guilleret I.
      • Yan P.
      • Grange F.
      • Braunschweig R.
      • Bosman F.T.
      • Benhattar J.
      ). CpG methylation of the TERT promoter region is necessary for transcription by blocking access of repressor CTCF to the binding site within the first exon of the gene. In addition, partially unmethylated CpG sites just upstream of the transcription start site of the TERT gene permit transcription (
      • Yi X.
      • Shay J.W.
      • Wright W.E.
      ,
      • Renaud S.
      • Loukinov D.
      • Abdullaev Z.
      • Guilleret I.
      • Bosman F.T.
      • Lobanenkov V.
      • Benhattar J.
      ). Consistent with these studies, our results showed that depletion of MCAF1 or Sp1 reduced TERT transcription, together with alterations in CpG methylation status, which suggests that the MCAF1-Sp1 complex affects DNA methylation of the TERT gene. Our findings that depletion of MCAF1 or Sp1 reduced telomerase activity and inhibited cell proliferation suggest the active involvement of MCAF1 in both telomerase and proliferative activities via cooperating with Sp1.
      MCAF1 Has a Transcriptional Mediator-like Function Coupled with Sp1—Our previous studies have shown that the MBD1-MCAF1-SETDB1 complex plays an important role in transcriptionally repressive chromatin formation (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ,
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ). In contrast, we showed here that MCAF1 facilitated transcriptional activation by Sp1, via interacting with RNA polymerase II and general transcriptional factors. MCAF1 may function as a positive and negative modulator, depending on the interacting partners and on DNA context. Promoter regions of RNA polymerase II-transcribed genes often possess discrete CpG islands, usually containing several GC-box sequences that are targeted by Sp1 (
      • Suske G.
      ,
      • Antequera F.
      • Bird A.
      ). The transcriptional mediators control gene expression through direct interactions with RNA polymerase II, activators with DNA-binding ability, and the general transcriptional factors loaded onto the core promoter (
      • Myers L.C.
      • Kornberg R.D.
      ,
      • Woychik N.A.
      • Hampsey M.
      ). Our results indicated that MCAF1 interacted with Sp1, RNA polymerase II, and general transcriptional factors, through its two evolutionarily conserved domains. The MCAF1-Sp1 complex recruits the general transcriptional apparatus to the TERT gene, which results in subsequent production of telomerase activity. Thus, MCAF1 facilitates transcription by scaffolding functionally distinct transcriptional regulatory molecules to the TERT gene. This suggests that MCAF1 has a transcriptional mediator-like function to stably couple Sp1 to the general transcription apparatus.
      The MBD1-MCAF1-SETDB1 complex is recruited by CpG methylation for transcriptional repression (
      • Fujita N.
      • Watanabe S.
      • Ichimura T.
      • Ohkuma Y.
      • Chiba T.
      • Saya H.
      • Nakao M.
      ,
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ), whereas the CpG island-associated promoter is activated by the MCAF1-Sp1 complex together with the general transcriptional apparatus. In HeLa cells, the CpG-methylated p53BP2 tumor suppressor gene is inactivated by the involvement of the MBD1-MCAF1-SETDB1 complex (
      • Ichimura T.
      • Watanabe S.
      • Sakamoto Y.
      • Aoto T.
      • Fujita N.
      • Nakao M.
      ), whereas the telomerase subunit genes are transactivated by the MCAF1-Sp1 complex coupled with general transcriptional machinery. These findings suggest that both roles of MCAF1 synergistically contribute to cancer phenotype. Furthermore, the association between MCAF1 and MBD1 is enhanced by SUMO modification of MBD1 (
      • Uchimura Y.
      • Ichimura T.
      • Uwada J.
      • Tachibana T.
      • Sugahara S.
      • Nakao M.
      • Saitoh H.
      ). Some modifications such as phosphorylation, acetylation, and SUMOylation of Sp1 may induce the recruitment of MCAF1, and certain modifications of MCAF1 itself may determine the interacting partners. The results of the present study shed light on the mediator-like function of MCAF1, which contributes to maintaining cancer-associated telomerase activity in an Sp1-dependent manner. Overexpression of MCAF1 may enable Sp1 to enhance the telomerase subunit genes as a common feature in naturally occurring human cancer.
      Overexpression of MCAF1 in Human Cancer—We verified that MCAF1 is overexpressed in cancer that originates from many different tissues, but not in non-cancerous tissues, which suggests a role for MCAF1 in oncogenesis. MCAF1 interacts with Sp1, which regulates expression of many genes involved in cell growth and cell cycle progression, including vascular endothelial growth factor, transforming growth factor-β and its receptor, E2F1 and cyclin D1, and telomerase components (
      • Safe S.
      • Abdelrahim M.
      ,
      • Cairney C.J.
      • Keith W.N.
      ). Our data further demonstrated that up-regulation of MCAF1 participates in transcription activation of the telomerase subunit genes by Sp1. In addition, cancer progression requires resistance to apoptosis and stimulation of angiogenesis (
      • Hanahan D.
      • Weinberg R.A.
      ). Because the promoters of many pro-apoptotic, anti-apoptotic, and angiogenesis genes have been found to contain Sp1-binding sites (
      • Black A.R.
      • Black J.D.
      • Azizkhan-Clifford J.
      ), overexpressed MCAF1 may also affect these genes, which therefore accelerates cancer development. Currently, the mechanism of MCAF1 overexpression in tumor cells is unknown, but it may be one of the common events in human cancer that are required for maintaining proliferative phenotypes.

      Acknowledgments

      We thank LinkGenomics, Inc. (Tokyo, Japan) and members of our laboratory for helpful discussions.

      Supplementary Material

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