cancer. Comparison of DMH and ChIP-on-chip to detect specific
methylation profile and chromatin structure profile was performed in lung cancer patients and cell lines. In addition, correlation of gene promoter hypermethylation / chromatin structure alterations with clinicopathological parameters (tumor type, stage, age, sex, and smoker) and DNMT1 expression status was analyzed in the lung cancer patients.The present study proposed that such epigenomics analyses can lead to the identification of previously uncharacterized CpG islands associated with gene silencing and shed light on other yet unidentified factors governing aberrant methylation.
Specific aim 2: Validated the cancer related genes and TSGs from
DMH and ChIP-on-chip analysis, and characterization of their
transcriptional important CpG sites in primary non-small cell lung
cancer. Several hypermethylated cancer-related genes and TSGs in
patients were identified using the DMH analysis, such as COL14A1,RASSF1A, and BLU. The present study further used the RT-PCR, MSP,
ChIP-PCR, and bisulfite sequencing to validate their potential role as hypermethylated genes in lung cancer patients and cell lines. In addition, MSO microarray was conducted to identify the CpG sites important for gene expression by comparing the methylation and chromatin structure profiles with the mRNA and protein expressions of the candidate gene.The present study aims to validate gene methylation and chromatin status identified by DMH and ChIP-on-chip assay such as COL14A1 in lung cancer patients. Furthermore, transcriptionally important CpG sites which regulate gene expression will be examined in some candidate genes such as RASSF1A and BLU.
AIM 1
Epigenome-wide detection for hypermethylated CpG islands and chromatin profiling and their
clinical association in lung cancer
Purposes
1. Comparison of chromatin immunoprecipitation-on-chip (ChIP-on-chip) using the acetylated histone H3 antibody and differential methylation hybridization (DMH) data to detect specific chromatin structure profile and methylation profile in lung cancer.
2. Correlation of gene promoter hypermethylation / chromatin structure alterations with clinicopathological parameters (tumor type, stage, age, sex, smoker) and prognosis of the lung cancer patients.
3. Identification of the CpG sites important for gene expression by comparing the methylation and chromatin structure profiles with the mRNA and protein expressions of the candidate gene.
Materials and Methods
Tumor specimens and clinical characterizations of patients
Tissues were collected after obtaining permission from the appropriate institutional review board and informed consents from the recruited patients. The cases include 30 surgically resected NSCLC patients admitted to Veterans General Hospital-Taipei, Taiwan between 1993 and 2004. The histologies of tumor types and stages are determined according to the WHO classification method and TNM system, respectively. There were 15 AD patients and 15 SQ patients.
The patients consisted of 15 early stage patients (stages I and II) and 15 late stage patients (stages III and IV).
Cell lines and culture conditions
Human normal lung cell line IMR90 (obtained from the American Type Culture Collection, ATCC), human NSCLC carcinoma A549 and H1299 (obtained from ATCC), and CL1-1 (obtained from Dr. Pan-Chyr Yang, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan) cell lines were cultured in DMEM medium (GIBCO, Grand Island, N.Y.) containing 10% fetal bovine serum (BIOCHROM AG, Leonorenstr, Berlin) and 1% penicillin-streptomycin (GIBCO), and incubated at 37°C in 5% CO2 atmosphere.
Fractionation of DNA using an MBD column and preparation of a CpG island library
DNA encoding the methyl-CpG binding domain (MBD) from MeCP2 was amplified by PCR and cloned into a bacterial expression vector containing a His-tag. The fusion histidine-MBD protein was attached to a nickel-agarose matrix column. Human DNA digested with
MseI (recognition site TTAA), which cuts bulk DNA into small
fragments but leaves CpG island relatively intact, was loaded onto the MBD protein column. The DNA eluted in high salt fractions contained genomic regions of CpG islands, this DNA was collected for the CpG island DNA and construction of the CpG island library (CGI).Preparation of CpG island array slides
A total of ~8000 CGI clones were individually organized in 96-well culture chambers as master plates. Clones of CGI were picked and placed into 96-well PCR microplates by MULTI-PRINT replicator. The insert from each clone was amplified by PCR. Prior to printing, spotting solution (Genetix, Hampshire, UK) was added to the PCR products for uniform dot morphology. The QArray2 arrayer (Genetix) was used to print PCR product on UltraGAPS coated slides (Corning, NY, USA). Printed slides were crosslinked by UV, post-processed, and stored in a desiccated chamber.
Amplicon generation
DMH method was performed as described previously (Yan et al., 2002). Briefly, DNA (2 μg) was extracted from surgically resected tumors and their matched normal tissues and then digested with MseI, which restricted DNA into small fragment but leaves CpG islands relatively intact. The cleaved ends were ligated with linkers, H-12/H-24 (linkers primer are shown in Table 1), in a buffer containing Fast Link Ligase (Epicentre, Madison, Wisconsin) at room temperature for 1 hour.
After ligation, the subtracted DNA was digested with BstUI and HpaII (New England Biolabs, Beverly, MA) methylation sensitive endonucleases. The restricted DNA was purified in a QIAquick column (Qiagen, Valencia, CA) Linker-PCR reactions were performed with the
BstUI and HpaII-resistant DNA. Reactions were carried out in a
volume of 300μl with digested DNA, 10 μM H-24 primers, 10x PCR buffer, 10 mM dNTP, and Deep Vent (exo-) DNA polymerase (New England Biolabs) on a DNA thermal cycler. PCR reaction condition was 5 min at 72oC, followed by 20 cycles of 1 min at 97oC, 3 min at 72oC, with a final extension of 10 min at 72oC. The amplified product was purified in a QIAquick column. At least 5-6μg of normal or tumor amplicons was used for fluorescence labeling to produce a strong array hybridization image.
Cy dye coupling and array hybridization
Incorporation of aminoallyl dUTP (Sigma, St. Louis, MO) into amplicons was performed using the BioPrime DNA labeling system (Invitrogen, Carlsbad, CA). Cy3 and Cy5 fluorescent dyes (Amersham, Buckinghamshire, UK) were coupled to aminoallyl dUTP-labeled normal and tumor amplicons, respectively. Both the Cy3-coupled normal and Cy5-coupled tumor amplicons were mixed with 20μg of human Cot-1 DNA (Invitrogen) and cohybridized to the CpG island array slide. Hybridization and post-hybridization washing protocols were according to previous study (Yan et al., 2002). Images of fluorescence intensities were generated by scanning array using Axon GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA)
Data analysis
The fluorescence intensities of each spots were quantified by GenePix Pro 6.0 microarray analysis software (Molecular Devices) and the ratio was then determined as the intensity of the tumor sample vs.
the normal sample (Cy5/Cy3). Raw intensity data of the ratio of the 30 arrays were normalized by LOWESS (limmaGUI package) (Wettenhall
et al., 2004). After normalization, each array intensity data was ranked
and selected for the top 200 spots, which were clustered and intersected with patients’ data by tumor type and tumor stage.
Immunohistochemistry (IHC) assay
Paraffin blocks of tumors were dissected into 5-m slices and then processed using standard deparaffinization and rehydration techniques.
Polyclonal antibody for anti-DNMT1 (A22659, aa 177-550; 1:200; Asia Hepato Gene Co., Kaohsiung, Taiwan) was used as the primary antibodies to detect DNMT1 protein expression. The primary antibody was incubated for 4 oC overnight, then biotinylated secondary antibody for 30 min, streptavidin-horseradish peroxidase for 15 min (DAKO LSAB kit K0675, Dako, Glostrup, Denmark), 3,3’-diaminobenzidine tetrahydrochloride as the chromagen. The evaluation of the immunohistochemistry was conducted blindly without prior knowledge of the clinical and pathologic characteristics of the cases. DNMT1 was graded overexpression when tumor cell staining are >60%. The surrounding normal stroma and epithelial cells served as an internal positive control for each slide.
Cell lines chromatin immunoprecipitation (ChIP)-on-chip assay
Cells grown on a 100 x 20-mm culture dish to approximately 80–90% confluency were cross-linked with 1% formaldehyde for 10 min at 37oC and stopped by the addition of glycine to a final concentration of 0.125 M. Lysates were sonicated by BioruptorTM system (Diagenode, Liège, Belgium) to shear DNA to lengths between 200 and 800 bp. Subsequent steps were performed with the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY, USA) according to the manufacturer’s instructions. Immunoprecipitation for 16 h at 4oC was performed using anti-acetylated histone H3 (1:400; Upstate
Biotechnology) in IMR90 normal lung cell line and 3 lung cancer cell lines (A549, H1299, and CL1-5). After de-crosslinking and purification of DNA, the immunoprecipitated and whole-cell extract DNA were amplified and labeled by ligation-mediated PCR with Cy5 and Cy3 fluorescent dyes, respectively. Both pools of labeled DNA were hybridized to the Agilent human CpG island 244k microarray chip (Agilent Technologies, Foster City, CA). Images of fluorescence intensities were generated by scanning array using GenePix 4000B scanner (Axon Instruments), and then data were extracted and normalized ChIP signals using Agilent Feature Extraction program.
Results
Epigenomic detection of hypermethylated CpG islands and their clinical association in lung cancer patients
To search for the genomic regions containing high frequency of hypermethylation, which may possess cancer related genes or novel candidate TSGs, we established an epigenomic hypermethylation analysis using the microarray-based strategy called DMH (Figure 1a) and used it to identify the regions of hypermethylation in 30 lung cancer patients. Patients included 15 AD patients and 15 SQ patients consisting of 15 early stage patients (stages I and II) and 15 late stage patients (stages III and IV) (Table 2). Several cancer subtype- and stage-specific hypermethylated genes successfully identified by DMH are listed in
Table 3 and Table 4. In addition, we had identified several
hypermethylated TSGs in patients with overexpression of DNMT1 using the DMH and IHC analysis. They are listed in Table 5.Chromatin status was involved in cancer related genes expressions in lung cancer cell lines
To investigate whether compact chromatin structure in addition to promoter hypermethylation of cancer related loci caused the loss of their gene expression, IMR90 normal lung cell line and three lung cancer cell lines (A549, H1299, and CL1-1) were analyzed by ChIP-on-chip
(Figure 1b). ChIP was performed using antibody against acetylated
histone H3, which recognizes loose chromatin structure. The immnoprecipitated DNA was then hybridized to the array containing oligonucleotide probes designed for genes’ promoters. The value of each probe was calculated as the ratio between ChIP-pulled down DNA and total input DNA from the same cell line. The higher the ratio, the looserthe chromatin structure is. For example, among the gene analyzed, a group of 10 oligonucleotide probes was designed to test chromatin status of COL14A1 gene within the CpG island (Figure 2a). We found that the CpG island of COL14A1 gene showed looser chromatin structure in IMR90 normal cell line than did all cancer cell lines (Figure
2b).
Discussion
Down-regulation of cancer-related genes and TSGs by hypermethylation or chromatin alteration of promoter is one of the important events involved in tumor development. In this study, we have established DMH microarray for epigenome-wide hypermethylation analysis to identify the regions of hypermethylation and ChIP-on-chip analysis to identify the regions of condensed or open chromatin in 30 lung cancer patients and several lung cell lines. Some cancer subtype- and stage-specific hypermethylated genes are shown in Table 3 and
Table 4. These genes may serve as biomarkers for early detection (for
example, those hypermethylated genes in early-staged patients) or prognosis prediction (for example, those hypermethylated genes only in late-staged patients). In addition, using an antibody to acetylated histone H3 lysine 9, which indicates an open chromatin structure, several chromosomal regions containing potential TSGs such as COL14A1 andRASSF1 genes were revealed.
DMH has a growing number of scanning methods for detecting hypermethylated genomic regions in cancer (Hatada et al., 1991;
Ushijima et al., 1997; Gonzalgo et al., 1997). This approach has at least three unique features. First, DNA is more stabilization than mRNA and protein in experiment process, so that DNA biomarkers may be better used for clinical-detecting of cancers. Second, the high-density of DMH array-based technology has been used in differential screenings of thousands of cDNA sequences up- or down-regulated in cancer cells.
Therefore, this DNA array chip could be used for dual analysis of DNA methylation and gene expression in cancer cells to provide an effective tool to elucidate the mechanisms of aberrant DNA methylation in cancer
(Shi et al., 2002). Third, the genomic fragments were derived from a library specifically constructed to contain highly enriched CpG island sequences (Cross et al., 1994). Therefore, DMH may lead to the identification of not only known expressed sequences but also novel TSGs down-regulated via methylation in cancer. For these reasons, DMH is useful for a genome-wide screening of methylation in cancer and can be converted into a high-throughput analysis by implementing the aforementioned microarray technologies.
The multiple covalent modifications on the histone tail create specific epigenetic patterns that switch genes between their transcriptionally active and inactive stages (Moggs et al., 2004; Zhang and Reinberg, 2001). HDACs, HATs, HMTs and DNMTs play crucial roles in the epigenetic regulation of gene expression involved in carcinogenesis (Geiman et al., 2004; Sun et al., 1997). Early evidences suggest that histone methylation may even be an important determinant of DNA methylation patterns (Jackson et al., 2002; Tamaru and Selker, 2001). In previous studies, alterations in DNA methylation associated with gene expression have been shown to be intimately associated with changes in chromatin structure (Jones and Baylin, 2002). Thus, epigenetic gene regulation involves a complex interplay between DNA methylation and chromatin modification.
Finally, we have shown that DMH and ChIP-on-chip methods are useful in identifying epigenetic alteration in cancer and have potential applications for cancer diagnostic and prognostic biomarkers. These biomarkers will be validated in a larger cohort with prospective study design. In addition, functional and mechanistic studies for the genes identified will be performed to reveal their roles in lung tumorigenesisis.
AIM II
Validated the cancer related genes and TSGs from DMH
and ChIP-on-chip analysis, and characterization of their
transcriptional important CpG sites in primary non-small
cell lung cancer
Purposes
1. Verification of the status of COL14A1, RASSF1A, and BLU identified from DMH and ChIP-on-chip analysis by comparing the alterations of methylation, mRNA, and protein expression data in NSCLC patients.
2. Identification of the methylated CpG sites of RASSF1A and BLU which are important to transcriptional regulation.
3. Investigation whether there was a regional effect on the gene expression and promoter methylation status of the juxtaposed
RASSF1A and BLU genes. If not, then there may be an insulator
between RASSF1A and BLU genes.Materials and methods
Cell lines and culture conditions
Human normal lung cell line IMR90 and MRC5 (obtained from the American Type Culture Collection, ATCC) and human lung cancer cell lines A549 and H1299 (obtained from ATCC), and CL1-0, CL1-1, CL1-3 and CL1-5 (obtained from Dr. Pan-Chyr Yang, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan) were cultured in DMEM (IMR90, MRC5, A549, H1299, CL1-0, CL1-1, CL1-3, and CL1-5) medium (GIBCO, Grand Island, N.Y.) containing 10% fetal bovine serum (FBS) (BIOCHROM AG, Leonorenstr, Berlin) and 1% penicillin-streptomycin (GIBCO) and incubated at 37°C in 5%
CO2 atmosphere.
Clinical samples preparation and DNA/RNA extraction
Tissues were collected after obtaining appropriate institutional review board permission and informed consent from the recruited patients. Surgically resected tumor tissue and corresponding normal tissue were collected from 76 patients diagnosed with primary non-small cell lung cancer admitted to Veterans General Hospital, Taipei. Of these patients, 55 had adenocarcinomas (AD), 18 had squamous cell carcinomas (SQ), and 3 had large cell carcinomas (LC). Histological classification was determined according to the WHO classification and the tumor-node-metastasis system. Information on the age, sex, and smoking history of the patients was obtained from hospital records. The genomic DNA was prepared using proteinase K digestion and phenol-chloroform extraction. Total RNA was prepared from tumors and normal lung tissues using TRIzol reagent (Invitrogen, Carlsbad, CA).
cDNA was synthesized using SuperScript reverse transcriptase
(Invitrogen).
Semiquantitative multiplex reverse transcription-PCR (RT-PCR) assay
cDNA was synthesized from the isolated RNA by reverse transcription. COL14A1, RASSF1A, and BLU mRNA expression was assayed in a multiplex RT-PCR analysis using the GAPDH gene as an internal control. The primer nucleotide sequences are shown in the Table1. PCR products were separated in 1.5% agarose gels and visualized
under UV illumination. To quantify the relative levels of mRNA expression in the multiplex RT-PCR assay, the value of the GAPDH in each reaction was used as the baseline gene expression of that sample and relative value was calculated for the COL14A1, RASSF1A, and BLU genes for each tumor and matched normal samples. Tumor cells that exhibited justified mRNA expression below that of normal cells were considered to have an abnormal pattern.Bisulfite modification and methylation-specific oligonucleotide (MSO) microarray
Tumor DNA from 32 patients (23 were AD, 8 were SQ, and 1 was LC) was analyzed by MSO microarray. To prepare the methylation-positive DNA, DNA was treated with M. SssI methyltransferase (New England Biolabs, Beverly, MA) that methylates all cytosine residues of CpG dinucleotides in the genome. The test samples and SssI-treated DNA samples were treated with sodium bisulfite as previously described (Gitan et al., 2002). Preparation of MSO arrays was carried out essentially as described previously (Yan et
al., 2004). The oligonucleotides and PCR primers for RASSF1A
methylation assay were described in the previous study (Yan et al., 2003). Twenty sets of paired oligonucleotides were designed to include promoter and first exon of BLU CpG sites to be interrogated (Figure 3a).For DNA target preparation, bisulfite-treated DNA was amplified from two regions (P1 as promoter region; E1 as exon 1 region) located in the
BLU CpG island. Primers used for PCR to amplify bisulfite DNA are
shown in the Table 1. To control the accuracy and reproducibility of the MSO probes, a series of MSO hybridization were performed with mixed samples containing 100, 66, 33, and 0% SssI-treated methylation-positive DNA (Yan et al., 2004). Standardization curves generated for these optimal probe sets were subsequently used to interrogate RASSF1A and BLU methylation in the test tumor DNA samples (data not shown). Slides hybridization and wash were described in the previous study (Gitan et al., 2002; Yan et al., 2004). The microarray slides were scanned with a GenePix 4000A scanner (Axon Instruments). MSO data were normalized according to a global ratio in each microarray image, and the intensity ratio of M/(M+U) (M:methylated probe; U: unmethylated probe) for each probe set was then derived.
Methylation-specific PCR (MSP) assay
MSP assay takes advantage of the fact that unmethylated cytosines are efficiently converted to uracil after sodium bisulfite treatment, whereas methylated cytosines remain unchanged. EZ DNA Methylation-Gold™ Kit (Zymo Research, Orange, CA) was used for bisulfite conversion of DNA. After treatment, COL14A1 MSP analyses were conducted using the primers which are shown in the Table 1. In addition, the methylation status in the E2F1 binding sites of the
RASSF1A and BLU promoters were determined by chemical treatment
with sodium bisulfite and MSP analysis. MSP analyses were conducted using the primers for the –144 to –38 bp (MSP1) and -6 to +94 bp (MSP2) regions in RASSF1A promoter and exon1 and for -156 to +1 bp region in BLU promoter. Location of regions analyzed by MSP is shownin Figure 3a and the primers used are listed in Table 1. Positive control samples of unmethylated IMR90 and MRC5 normal lung cell DNA and
SssI methyltransferase treated methylated DNA were also included for
each set of PCR.Bisulfite DNA sequencing analysis
The methylation status of the upstream and downstream CTCF regions was examined by sequencing analysis after bisulfate modification. Bisulfite sequencing primers were designed for CpG sites -60 to +14 (corresponding to nucleotide numbers from -2380 to +161 bp) in which the first CpG site was defined as +1 corresponding to
RASSF1A transcriptional start site (Figure 4b). Bisulfite-modified DNA
was amplified using three pairs of primers: BS-P1, BS-P2, and BS-P3 shown in the Table 1. The products were isolated and sub-cloned into the pCR2.1-TOPO vector (Invitrogen), and five individual clones per each sample were sequenced.Immunohistochemistry (IHC) assay
Paraffin blocks of tumors were dissected into 5-m slices and then processed using standard deparaffinization and rehydration techniques.
Polyclonal antibody for anti-COL14A1 (1:200; Abnova, Taipei, Taiwan), monoclonal antibody for anti-RASSF1A (1:500; eBioscience, San Diego, CA), and polyclonal antibody for anti-BLU (1:100; Abcam, Cambridge, UK) were used as the primary antibodies to detect COL14A1, RASSF1A and BLU protein expression, respectively. The evaluation of the immunohistochemistry was conducted blindly without prior knowledge of the clinical and pathologic characteristics of the cases.
COL14A1, RASSF1A and BLU were graded low or negative expression when tumor cell staining are <50%, <20% and <40%, respectively. The
surrounding normal stroma and epithelial cells served as an internal positive control for each slide. Normal lung tissue slide from some patients were included to evaluate the IHC results.
Electrophoretic mobility shift assay (EMSA)
The E2F1 binding sites were identified using the transcription factor search program PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/
promo/promoinit.cgi?dirDB=TF_8.3) (Messeguer et al., 2002). The EMSA was performed using Raji nuclear extracts (Active Motif, Carlsbad, CA) and biotin end-labeled double-stranded DNA probes prepared by annealing complementary oligonucleotides. The probes of E2F1 binding sites for RASSF1A gene were as follows: probe 1, -169 to -138 bp; and probe 2, -7 to +25 bp. The probe sequences are shown in the Table 1. Probes were labeled in a reaction using terminal deoxynucleotide transferase (Promega, Madison, WI) and biotin-14-dCTP (Invitrogen). The binding reaction was performed using the LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, IL) and conducted according to the manufacturer’s protocol. Binding mixtures were loaded on a 6% polyacrylamide gel, and gels were run at 100 V for 1 hour at room temperature. Following electrophoresis, the DNA-protein complexes were transferred onto nylon positively charged membranes and detected using chemiluminescence reagent (Pierce).
Chromatin immunoprecipitation (ChIP) and target promoter ChIP-PCR
Cells grown on a 100 x 20-mm culture dish to approximately 80–90% confluency were cross-linked with 1% formaldehyde for 10 min at 37oC and stopped by the addition of glycine to a final concentration of 0.125 M. Lysates were sonicated using BioruptorTM system (Diagenode, Liège, Belgium) to shear DNA to lengths between 200 and 800 bp. Subsequent steps were performed with the ChIP assaykit (Upstate Biotechnology, Lake Placid, NY, USA) according to the manufacturer’s instructions. ChIP was performed using anti-acetylated histone H3 (Lys 9) (1:400; Upstate Biotechnology), anti-trimethylted histone H3 (Lys 27) (1:400; Upstate Biotechnology), anti-E2F1 (1:500;
Active Motif), and anti-CTCF (1:500; Upstate Biotechnology) antibodies for 16 h at 4oC. De-crosslinks and purification of pulled down DNA were performed with the ChIP assay kit (Upstate Biotechnology).
PCR analysis using the primer pairs for RASSF1A, BLU, and
GAPDH promoters as well as CTCF binding domain and regions next to
CTCF binding domain are shown in Table 1.Luciferase reporter gene analysis
Promoter sequences of RASSF1A were cloned by PCR from IMR90
Promoter sequences of RASSF1A were cloned by PCR from IMR90