LOT1 (PLAGL1/ZAC1), the Candidate Tumor Suppressor Gene at Chromosome 6q24–25, Is Epigenetically Regulated in Cancer
2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês
10.1074/jbc.m210361200
ISSN1083-351X
AutoresAbbas Abdollahi, Debra Pisarcik, David W. Roberts, Jillian K. Weinstein, Paul Cairns, Thomas C. Hamilton,
Tópico(s)Prenatal Screening and Diagnostics
ResumoLOT1 is a zinc-finger nuclear transcription factor, which possesses anti-proliferative effects and is frequently silenced in ovarian and breast cancer cells. TheLOT1 gene is localized at chromosome 6q24–25, a chromosomal region maternally imprinted and linked to growth retardation in several organs and progression of disease states such as transient neonatal diabetes mellitus. Toward understanding the molecular mechanism underlying the loss of LOT1 expression in cancer, we have characterized the genomic structure and analyzed its epigenetic regulation. Genome mapping of LOT1 in comparison with the other splice variants, namely ZAC1 andPLAGL1, revealed that its mRNA (∼4.7 kb; GenBankTM accession number U72621) is potentially spliced using six exons spanning at least 70 kb of the human genome. 5′-RACE (rapid amplification of cDNA ends) data indicate the presence of at least two transcription start sites. We found thatin vitro methylation of the LOT1 promoter causes a significant loss in its ability to drive luciferase transcription. To determine the nature of in vivomethylation of LOT1, we used bisulfite-sequencing strategies on genomic DNA. We show that in the ovarian and breast cancer cell lines and/or tumors the 5′-CpG island of LOT1is a differentially methylated region. In these cell lines the ratio of methylated to unmethylated CpG dinucleotides in this region ranged from 31 to 99% and the ovarian tumors have relatively higher cytosine methylation than normal tissues. Furthermore, we show that trichostatin A, a specific inhibitor of histone deacetylase, relieves transcriptional silencing of LOT1 mRNA in malignantly transformed cells. It appears that, unlike DNA methylation, histone deacetylation does not target the promoter, and rather it is indirect and may be elicited by a mechanism upstream of the LOT1regulatory pathway. Taken together, the data suggest that expression ofLOT1 is under the control of two epigenetic modifications and that, in the absence of loss of heterozygosity, the biallelic (two-hit) or maximal silencing of LOT1 requires both processes. LOT1 is a zinc-finger nuclear transcription factor, which possesses anti-proliferative effects and is frequently silenced in ovarian and breast cancer cells. TheLOT1 gene is localized at chromosome 6q24–25, a chromosomal region maternally imprinted and linked to growth retardation in several organs and progression of disease states such as transient neonatal diabetes mellitus. Toward understanding the molecular mechanism underlying the loss of LOT1 expression in cancer, we have characterized the genomic structure and analyzed its epigenetic regulation. Genome mapping of LOT1 in comparison with the other splice variants, namely ZAC1 andPLAGL1, revealed that its mRNA (∼4.7 kb; GenBankTM accession number U72621) is potentially spliced using six exons spanning at least 70 kb of the human genome. 5′-RACE (rapid amplification of cDNA ends) data indicate the presence of at least two transcription start sites. We found thatin vitro methylation of the LOT1 promoter causes a significant loss in its ability to drive luciferase transcription. To determine the nature of in vivomethylation of LOT1, we used bisulfite-sequencing strategies on genomic DNA. We show that in the ovarian and breast cancer cell lines and/or tumors the 5′-CpG island of LOT1is a differentially methylated region. In these cell lines the ratio of methylated to unmethylated CpG dinucleotides in this region ranged from 31 to 99% and the ovarian tumors have relatively higher cytosine methylation than normal tissues. Furthermore, we show that trichostatin A, a specific inhibitor of histone deacetylase, relieves transcriptional silencing of LOT1 mRNA in malignantly transformed cells. It appears that, unlike DNA methylation, histone deacetylation does not target the promoter, and rather it is indirect and may be elicited by a mechanism upstream of the LOT1regulatory pathway. Taken together, the data suggest that expression ofLOT1 is under the control of two epigenetic modifications and that, in the absence of loss of heterozygosity, the biallelic (two-hit) or maximal silencing of LOT1 requires both processes. LOT1 (lost-on-transformation 1) 1The abbreviations used are: LOT1, lost-on-transformation 1; TSA, trichostatin A; ROSE, rat ovarian surface epithelial; RACE, rapid amplification of cDNA ends; UTR, untranslated region; FACS, fluorescence-activated cell sorter; GFP, green fluorescence protein; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation; TSS, transcription start site; ORF, open reading frame; HDAC, histone deacetylase is a growth suppressor gene (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar, 2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar) localized on chromosome 6 at band q24–25, which is a frequent site for loss of heterozygosity in many solid tumors including ovarian cancer (2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar, 3Colitti C. Rodabaugh K. Welch W. Berkowitz R. Mok S. Oncogene. 1998; 16: 555-559Google Scholar). The mouse ortholog of this gene was independently identified by Spengler et al. (4Spengler D. Villalba M. Hoffman A. Pantaloni C. Houssami S. Bockaert J. Journet L. EMBO J. 1997; 16: 2814-2825Google Scholar) and designated as Zac1, which is highly homologous to the hLOT1 and rLot1 genes. A splice variant of LOT1/ZAC1 was identified by Kas et al. (5Kas K. Voz M. Hensen K. Meyen E. Van de ven W. J. Biol. Chem. 1998; 273: 23026-23032Google Scholar) and named as PLAGL1 based on its homology with PLAG1 protein encoded by the genePLAG1 localized on chromosome 8q12 (6Kas K. Voz M. Roijer E. Astrom A.-K. Meyen E. Stenman G. Van de Ven W. Nat. Genet. 1997; 15: 170-174Google Scholar). The presence of other genes with high homology to LOT1/ZAC1/PLAGL1 indicates that this gene is a member of new family of zinc-finger proteins. Our studies and those of others have shown that LOT1 gene expression is frequently down-regulated in the ovarian and breast carcinoma cells (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar, 2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar, 7Bilanges B. Varrault A. Basyuk E. Rodriguez C. Mazumdar A. Pantaloni C. Bockaert J. Theillet C. Spengler D. Journot L. Oncogene. 1999; 18: 3979-3988Google Scholar). Functional analysis of LOT1 demonstrated that it may play a significant role as a transcription factor modulating growth suppression through mitogenic signaling pathways (8Abdollahi A. Bao R. Hamilton T. Oncogene. 1999; 18: 6477-6487Google Scholar). It is increasingly evident that epigenetic modification of genomic DNA by methylation and/or histone deacetylation plays an important role in transcriptional silencing and loss of gene function of certain tumor suppressor genes. Methylation of DNA normally occurs at cytosine residues within CpG dinucleotides in almost all higher eukaryotic organisms (9Jones P. Baylin S. Nat. Rev. Genet. 2002; 3: 415-428Google Scholar, 10Gardiner-Garden M. Frommer M. J. Mol. Biol. 1987; 196: 261-282Google Scholar, 11Bird A. Nature. 1986; 321: 209-213Google Scholar). This type of modification can suppress gene expression directly by interfering with the binding of transcription factors or indirectly by initiating a complex consisting of methyl-CpG-binding proteins (e.g. MeCP2, MBDs) and histone deacetylases (12Jones P. Veenstra G. Wade P. Vermaak D. Kass S. Landsberger N. Strouboulis J. Wolffe A. Nat. Genet. 1998; 19: 187-191Google Scholar, 13Nan X. Ng H. Johnson C. Laherty C. Turner B. Eisenman R. Bird A. Nature. 1998; : 393Google Scholar, 14Ng H. Zhang Y. Hendrich B. Johnson C. Turner B. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 58-61Google Scholar, 15Wade P. Gegonne A. Jones P. Ballestar E. Aubry F. Wolffe A. Nat. Genet. 1999; 23: 62-66Google Scholar, 16Zhang Y. Ng H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Genes Dev. 1999; 13: 1924-1935Google Scholar). These complexes mediate transcriptional repression through chromatin hypoacetylation, which can be partially reversed by TSA. In human cancers, aberrant methylation of CpG islands silencing the promoters of some genes that function in the suppression of malignant phenotype has been found. For example hypermethylation of CpG islands in different cancer cells has been implicated in the transcriptional inactivation of the Rb, p16, estrogen receptor, INK4B/MTS2, VHL, andH19 genes (17Zhang Y. Shields T. Crenshaw T. Hao Y. Moulton T. Tycko B. Am. J. Hum. Genet. 1993; 53: 113-124Google Scholar, 18Gonzalez-Zulueta M. Bender C. Yang A. Nguyen T. Beart R. Van Tornout J. Jones P. Cancer Res. 1995; 55: 4531-4535Google Scholar, 19Greger V. Passarge E. Hopping W. Messmer E. Horsthemke B. Hum Genet. 1989; 83: 155-158Google Scholar, 20Herman J. Merlo A. Mao L. Lapidus R. Issa J. Davidson N. Sidransky D. Baylin S. Cancer Res. 1995; 55: 4525-4530Google Scholar, 21Herman J. Jen J. Merlo A. Baylin S. Cancer Res. 1996; 56: 722-727Google Scholar, 22Ottaviano Y. Issa J. Parl F. Smith H. Baylin S. Davidson N. Cancer Res. 1994; 54: 2552-2555Google Scholar, 23Herman J. Latif F. Weng Y. Lerman M. Zbar B. Liu S. Samid D. Duan D. Gnarra J. Linehan W. Baylin S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9700-9704Google Scholar, 24Sakai T. Toguchida J. Ohtani N. Yandell D. Rapaport J. Dryja T. Am. J. Hum. Genet. 1991; 48: 880-888Google Scholar). Similarly, CpG island methylation may play a significant role in the regulation of imprinted genes (25Barlow D. Science. 1995; 270: 1610-1613Google Scholar) and genes located on the inactive X chromosome (26Wolfe S. Jolly D. Lunnen K. Friedmann T. Migeon B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2806-2810Google Scholar, 27Li E. Nat. Rev. Genet. 2002; 3: 662-673Google Scholar). Recently, the LOT1/ZAC1 locus at chromosome 6q24–25 was identified as a maternally imprinted region (2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar, 4Spengler D. Villalba M. Hoffman A. Pantaloni C. Houssami S. Bockaert J. Journet L. EMBO J. 1997; 16: 2814-2825Google Scholar,28Gardner R. Mackay D. Mungall A. Polychronakos C. Siebert R. Shield J. Temple I. Robinson D. Hum. Mol. Genet. 2000; 9: 589-596Google Scholar, 29Kamiya M. Judson H. Okazaki Y. Kusakabe M. Muramatsu M. Takada S. Takagi N. Arima T. Wake N. Kamimura K. Satomura K. Hermann R. Bonthron D. Hayashizaki Y. Hum. Mol. Genet. 2000; 9: 453-460Google Scholar, 30Piras G. El Kharroubi A. Kozlov S. Escalante-Alcalde D. Hernandez L. Copeland N. Gilbert D. Jenkins N. Stewart C. Mol. Cell. Biol. 2000; 20: 3308-3315Google Scholar). These studies have established that the paternal duplication or loss of maternal imprinting of the LOT1 locus is associated with transient neonatal diabetes mellitus, an imprinted disease characterized by intrauterine fetal growth retardation and insulin dependence. However, little is known about the molecular mechanisms that down-regulate expression of LOT1 in cancer. In this study, we have characterized the LOT1 gene structure with relation to different splice variants and identified the gene's minimal promoter and a CpG island. Our results indicate thatLOT1 is subject to two epigenetic processes, methylation of CpG islands and histone deacetylation, which may synergistically act to regulate the transcriptional silencing of the gene. The ovarian cells were maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum, glutamine (2 mm), insulin (10 μg/ml), penicillin (100 units/ml), and streptomycin (100 μg/ml) in a humidified 5% CO2 atmosphere at 37 °C (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar). Normal rat ovarian surface epithelial (ROSE) cells were obtained from the ovaries of adult Fisher rats by selective trypsinization (31Godwin A. Testa J. Handel L. Liu Z. Vandeveer L. Tracey P. Hamilton T. J. Natl. Cancer Inst. 1992; 84: 592-601Google Scholar, 32Testa J. Getts L. Salazar H. Liu Z. Handel L. Godwin A. Hamilton T. Cancer Res. 1994; 54: 2778-2784Google Scholar). The ROSE cell line NuTu 26 was obtained as described previously (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar). The breast cancer cells MDA-MB-453 and -468 (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum, glutamine (2 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml). To obtain additional sequence information of the 5′-untranslated region (5′-UTR) of hLOT1 cDNA, which we previously reported (2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar), a normal human fetal brain cDNA library in λZAPII vector (Stratagene, La Jolla, CA) was used. A positive clone, FB2, which hybridized to the hLOT1 probe, gave extended 5′-UTR sequences. Insert sequences of all plasmids were determined by automated ABI PRISM dye terminator cycle sequencing with FS AmpliTaq DNA polymerase and run through an Applied Biosystem 373/377 sequencer (ABI/PerkinElmer Life Sciences). The data were analyzed using FASTA and PILEUP DNA sequence homology searches in the Wisconsin Genetics Computer Group package (Madison, WI). We predicted the LOT1 transcription start site using GeneRacer cDNA Kit (Invitrogen). The cDNA for 5′-RACE was derived from human ovary and generated based on oligo-capping and RNA ligase-mediated RACE methods (33Maruyama K. Sugano S. Gene. 1994; 138: 171-174Google Scholar, 34Shaefer B. Anal. Biochem. 1995; 227: 255-273Google Scholar). The PCR was performed using GeneRacer 5′1 adaptor forward primer (5′-GCACGAGGACACUGA CAUGGACUGA-3′) and gene-specific reverse primer (5′-TTCTAAGTGAGGTACAGATGAGTTTCAGATG-3′). The gene-specific primer was designed from the 3′ end of the LOT1 exon II and at the junction of this exon and exon III. The PCR reaction contained the cDNA, primers, buffer, dNTPs, Me2SO, and Taqpolymerase in a volume of 50 μl. The amplification reaction consisted of denaturation at 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, followed by extending at 72 °C for 7 min. We analyzed the PCR products by cloning into pGEMT vector using the TA cloning kit (Promega, Madison, WI). The clones were sequenced using both T7 and Sp6 primers. All the primers used in this study were synthesized by the Fox Chase Cancer Center DNA synthesis facility. To obtain the human LOT1 promoter, a human placental genomic DNA library (EMBL-3-SP6/T7, Clontech, Palo Alto, CA) was screened using the following fragments in the hLOT1cDNA (GenBank accession number U72621.3) as probes: a) a fragment containing the complete open reading frame; b) a 271-bp fragment upstream of the LOT1 translation start codon; and c) a fragment containing bases −1547 to −567 with respect to the LOT1translation start codon. Phage inserts were sequenced using both the polylinker-specific and LOT1-specific primers. These primers as well as cDNA-specific primers were used in PCR to amplify regions of the phage insert and to gain additional insight into the genomic structure of LOT1. We also screened a human P1 library (Genome Systems, St. Louis, MO), which resulted in identification of a new genomic clone designated as clone 17513. This P1 clone was purified by Qiagen Plasmid Purification Kit (Qiagen Inc., Valencia, CA) and used in subsequent promoter analyses. The forward and reverse primer, p63 (5′-TGGGTGTGAGGAGTGTGGGAAGA-3′) and p69 (5′-GCAGTTTGATTACAGAACACGCG-3′) were used to sequence most of the LOT1 open reading frame using genomic DNA as template. Reporter constructs containing luciferase gene under the control of theLOT1 promoter were created by amplifying and subcloning the 5′-flanking regions of the LOT1 promoter at the nucleotide positions as depicted in Fig. 4, using DNA from the phage or P1 clone 17513 as a template. Primers used to amplify the 5′-flanking region of the LOT1 promoter included: TH1645, 5′-CTAGTGGGGTAGGGATAGCATT-3′; TH1647, 5′-CAGGAGGTAAGTTAGTTTGGCC-3′; and TH1648, 5′-CTGCCCCGTCCGTCCGTCCGT-3′. The primers TH1645 and TH1647 were linked to the XhoI recognition sequence on their 5′ ends, and the primer TH1648 contained a HindIII recognition site at its 5′ end. The plasmids constructed with these PCR products were designated as pGL3–45 and pGL3–47, respectively. Another plasmid designated as PGL3–60 was constructed using a PCR fragment obtained by amplifying human normal ovarian genomic DNA as template with the primers IMP4, 5′-GGCCCGTTGGCGAGGTTAGAGCGC-3′ and IMP5, 5′-ACGGCATCTGCCATTTGTCA-3′. The PCR products were subcloned into pGEMT vector using a TA cloning kit (Promega, Madison, WI), and the insert fragments were purified. The DNA fragments obtained by above procedures were ligated into pGL3-Basic reporter vector (Promega) containing the luciferase gene, which was previously digested with either XhoI alone (pGL3–60) or XhoI andHindIII (pGL3–45, pGL3–47) restriction enzymes. The ligation reactions were used to transform DH5αEscherichia coli competent cells and the clones were confirmed by sequencing their insert. All plasmids used for the transfections were purified using the Qiagen kit. The promoter construct pGL3–45 was in vitro methylated by the bacterial methylase,SssI (New England Biolabs), or mock-treated in the absence of enzyme. The DNA was incubated for 24 h at 37 °C withSssI methylase at 1 unit/μg DNA in 50 mm NaCl, 10 mm Tris-HCl, 10 mm MgCl2, and 1 mm dithiothreitol (pH 7.9) supplemented with 160 μm S-adenosylmethionine. GFP-tagged LOT1 mammalian expression vector was constructed as described previously (8Abdollahi A. Bao R. Hamilton T. Oncogene. 1999; 18: 6477-6487Google Scholar). Briefly, the GFP coding region was amplified using forward (5′-CGCCATGGCTAGCAAGGGCCAG-3′) and reverse (5′-CTTGTACAGCTCGTCCATGCC-3′) primers. The PCR product was ligated with XbaI linker to the LOT1 coding region, and the resulting fragment (GFL) was introduced downstream of the cytomegalovirus promoter into the XhoI site of pcDNA3 plasmid. A2780 cells were cultured in RPMI 1 day before transfection. Transient transfections were performed usingTransIT-LT1 transfection reagent (PanVera, Madison, WI) according to the manufacturer's instructions. Cells (2 × 105/well) were seeded in 6-well plates and transfected with 4 μg/well of pcDNA/GFL vector carrying GFP-LOT1 fusion protein. The cells were then monitored 18 h later and imaged with a Quantix 12-bit cooled charge-coupled device camera (Roper, Inc., Tuscon, AZ). Fluorescent GFP and phase contrast images were generated simultaneously using Isee software (Inovision Corp., Durham, NC) to drive the charge-coupled device, a Ludl filter wheel, and a shutter attached to a Nikon TE300 inverted microscope. The cells were observed at near-physiological conditions using a forced-air incubator, which encompasses the microscope. For FACS analysis the transfected cells were collected by trypsinization, washed, and resuspended in PBS. The cell suspensions or GFP-transfectants were sorted using a Becton Dickinson FACS VintageSE flow cytometer (Becton Dickinson, Inc., San Jose, CA). 10,000 cells were subjected to FACS analysis. Ovarian cancer cells at a density of 4 × 105/well were cultured in 6-well plates in RPMI 1640 medium 1 day prior to transfection with the reporter plasmid as indicated. Transient transfections were performed using TransIT-LT1 polyamine transfection reagent (PanVera, Madison, WI) according to the manufacturer's instructions. Briefly, the cells were washed once with serum-free medium and incubated with the DNA (2 μl)/(6 μl) reagent mixture for 5 h in serum-free medium followed by additional incubation for 17 h (overnight) in fresh medium containing 10% fetal bovine serum. Luciferase enzyme was assayed using the Dual-Luciferase Reporter Assay System (Promega). The cells were washed with 1× PBS, harvested with the lysis buffer, and centrifuged, and 5–20 μl of the cell lysate (supernatant) was added to 100 μl of the luciferase substrate. The luciferase enzyme activity was measured immediately or within 15 s of adding the substrate, luciferin, using the Monolight (R) 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). Total RNA was isolated from the cells by the guanidinium isothiocyanate extraction method (35Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar) or Trizol (Invitrogen) and was separated on 1% agarose gels containing 2.2 m formaldehyde. The RNA was transferred to Nylon membranes (Micron Separations, Inc.) by capillary action and hybridized using a procedure described before (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar, 2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar). The probes for visualization of rat Lot1 and human LOT1 transcripts were the same as described previously (1Abdollahi A. Godwin A. Miller P. Getts L. Schultz D. Taguchi T. Testa J. Hamilton T. Cancer Res. 1997; 57: 2029-2034Google Scholar, 2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar). The probe for β-actin was from Clontech, Inc. (Palo Alto, CA). Western blot analysis was performed with lysates obtained from the cells plated on 6-well dishes in RPMI 1640 containing 10% FBS. After incubation with the transfection reagent supplemented with the appropriate DNAs, whole-cell lysates were prepared using 250 μl/well of M-PER Protein Extraction Reagent (PIERCE, Rockford, IL) containing phenylmethylsulfonyl fluoride and dithiothreitol. Equal volumes of protein (15 μl) from each sample were electrophoresed on SDS-PAGE (10%) followed by transfer to a Hybond nylon membrane (Amersham Life Science, UK) for Western blot analysis. Immunoblots were blocked overnight in 5% nonfat dry milk (w/v) in TBST containing 20 mm Tris-HCl (pH 7.6), 150 mm NaCl, and 0.1% Tween 20. Blots were incubated for 1 h with anti-GFP monoclonal antibody (Clontech, Palo Alto, CA) diluted with 1% nonfat dry milk in TBST. The blots were washed with TBST three times, and incubated for 1 h with the second antibody (anti-mouse IgG horseradish peroxidase, Amersham Biosciences). Anti-glyceraldehyde-3-phosphate dehydrogenase antibody was obtained from NeoMarkers (Fremont, CA). The protein bands were detected by the Western Lightning Chemiluminescence Reagent (PerkinElmer Life Sciences) and exposure to x-ray films (Kodak Co, Rochester, NY). Genomic DNA was isolated by digestion with proteinase K (Invitrogen) followed by phenol/chloroform extraction (36Herrmann B. Frischauf A. Methods Enzymol. 1987; 152: 180-187Google Scholar). After precipitation by the addition of 2 volumes of ethanol and 0.5 volumes of ammonium acetate (7.5 m), the DNA was washed twice with ethanol (80%, v/v), and dissolved in water. Normal and tumor DNA samples were obtained from Biosample Repository Core Facility (Andrew Godwin and Joellen Dangel) and Tumor Bank Facility (Andre Klein-Szanto and Robert Page) at Fox Chase Cancer Center. Genomic DNA (1 μg) was treated with sodium bisulfite and used for methylation-sensitive PCR as previously described (37Frommer M. McDonald L. Millar D. Collis C. Watt F. Grigg G. Molloy P. Paul C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1827-1831Google Scholar, 38Herman J. Graff J. Myohanen S. Nelkin B. Baylin S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93Google Scholar). Briefly, the DNA in a volume of 50 μl was denatured by 0.2 m NaOH and then treated with hydroquinone (Sigma) and sodium bisulfite (Sigma) at 50 °C for 16 h. Bisulfite-modified DNA was purified using Wizard DNA Clean-Up system (Promega). Modification was completed by NaOH (0.3 m) for 5 min at room temperature. The bisulfite-treated DNA was precipitated by glycogen, ammonium acetate, and 3 volumes of ethanol, and the pellets were washed once with 70% ethanol and dissolved in 20 μl of water. The modified DNA (4 μl) was amplified by PCR under the following reaction conditions: 1× buffer, 125 μm dNTPs, 7.5 mmMgCl2, and 1 μm of each primer in a 50-μl reaction. The reaction was carried out with the cycling condition of denaturation at 95 °C for 10 min followed by the addition of 0.5 unit of Gold Taq polymerase (Invitrogen) and 32 cycles of PCR (95 °C, 30 s; 58 °C, 30 s; 72 °C, 30 s) with a final extension of 4 min at 72 °C. Second round of PCR was performed using 2 μl of the first PCR and the same reagents and conditions as above. Primers were designed from the interpolated sequence after bisulfite conversion assuming DNA was either methylated or unmethylated at 14 CpG sites or the imprinting control region (28Gardner R. Mackay D. Mungall A. Polychronakos C. Siebert R. Shield J. Temple I. Robinson D. Hum. Mol. Genet. 2000; 9: 589-596Google Scholar, 29Kamiya M. Judson H. Okazaki Y. Kusakabe M. Muramatsu M. Takada S. Takagi N. Arima T. Wake N. Kamimura K. Satomura K. Hermann R. Bonthron D. Hayashizaki Y. Hum. Mol. Genet. 2000; 9: 453-460Google Scholar). Primers used for first PCR were UM-S1: 5′-GGGGTAGTTGTGTTTATAGTTTAGTA-3′, UM-AS1: 5′-CAAACACCCAAACACCTACCCTA-3′ and M-S1: 5′-GGGGTAGTCGTGTTTATAGTTTAGTA-3′, M-AS1: 5′-CGAACACCCAAACACCTACCCTA-3. Primers used for the second PCR were UM-S2: 5′-ATAGTTTAGTAGTGTGGGGT-3′, UM-AS2: 5′-CCTACCCTACAAAACAACAA-3′, M-S2: 5′-ATAGTTTAGTAGCGCGGGGT-3′, and M-AS2: 5′-CCTACCCTACGAAACGACGA-3. Reaction products were separated by electrophoresis on a 2% gel, stained with ethidium bromide, and photographed. The resulting amplification pools were cloned into the pGEMT vector using the TA cloning kit (Promega, Madison, WI). Four to 10 individual clones per PCR reaction were isolated and sequenced. The clones were sequenced using both T7 and Sp6 primers. The methylation-sensitive restriction digest-PCR was performed on 200 ng of genomic DNA samples. The DNA was digested overnight at 37 °C with 20 units of HpaII or MspI in a total volume of 20 μl. These restriction enzymes both cleave at CCGG sites. However, HpaII will not cut the DNA when the internal C is methylated. The DNA was purified with Wizard column (Promega) and eluted in 50 μl water, and 5 μl of the product was subjected to PCR using the primers IMP4 and IMP5 (1 μm) and reagents as mentioned above. The PCR condition consisted of denaturation at 95 °C for 2 min followed by the addition of 0.5 unit ofTaq polymerase and 29 cycles of PCR (96 °C, 30 s; 60 °C, 30 s; 72 °C, 1 min) with a final extension of 4 min at 72 °C. The reaction contained the same reagents as mentioned above. ChIP assay was performed according to the manufacturer's instructions (Upstate Biotech, Lake Placid, NY). Briefly, 1 × 106 cells in 100-mm dish were directly treated with 1% formaldehyde (Fisher Scientific) to form protein-DNA cross-links. The cells were then collected in ice-cold 1× PBS containing protease inhibitors and subjected to centrifugation at 4 °C. The cell pellets were resuspended in SDS lysis buffer and incubated on ice for 10 min. The samples were sonicated on ice with an Ultrasonics sonicator at setting 10 for 10-s pulses and then microcentrifuged. The supernatant or chromatin solution from each sample was diluted 10× with ChIP dilution buffer, and an aliquot was removed and used as input after reversing cross-link. The diluted chromatin solution was precleared for 30 min with Salmon Sperm DNA/protein A-Agarose-50% slurry and incubated overnight at 4 °C with or without acetyl-histone H4 antibody. The samples were then incubated for 1 h at 4 °C with Salmon Sperm DNA/protein A-Agarose-50% Slurry with rotation and washed with different wash buffers supplied in the kit. The chromatin-antibody complexes were eluted, and cross-links were reversed and treated with proteinase K. The DNA was recovered by phenol/chloroform extractions, precipitated, and used in PCR amplification. The primers used for PCR were IMP3: 5′-GCGAGGAGGGTGTGCCTTTG-3′ and IMP4 as shown above. The amplification conditions for the ChIP assay were the same described above for reactions using IMP4 and IMP5 primer set. As a first step toward analysis of the relationship between theLOT1 gene structure, promoter activity, and the presence of differential splicing and regulation of expression, we began to obtain additional sequence information of the gene's 5′-UTR cDNA region, which we previously reported (2Abdollahi A. Roberts D. Godwin A. Schultz D. Sonoda G. Testa J. Hamilton T. Oncogene. 1997; 14: 1973-1979Google Scholar). A normal human fetal brain cDNA library in the λZAPII vector (Stratagene) was used for screening, and a positive clone, FB2, hybridized to the hLOT1 probe and produced an extension of the 5′-UTR sequence. This newly identified nucleotide sequence of the LOT1 gene was added to the previously published sequence that is stored in GenBankTMwith the accession number U72621. We identified the transcription start site (TSS) of LOT1using 5′-RACE technique. The cDNA for 5′-RACE was derived from human ovary and generated based on oligo-capping and RNA ligase-mediated RACE methods (33Maruyama K. Sugano S. Gene. 1994; 138: 171-174Google Scholar, 34Shaefer B. Anal. Biochem. 1995; 227: 255-273Google Scholar) as described in the GeneRacer cDNA Kit (Invitrogen). We detected a strong PCR band corresponding to about 390 nucleotides in size and weaker bands that are smaller or larger (Fig. 1). Two of t
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