Supplementary Materials and Methods
Patient samples and RNA isolation from tissues
In each patient case, cancer tissues and surrounding tissues that were free of
diseases, as judged by histological examination, were isolated and used for the
case-matched studies. As the minimum criteria for the usefulness of our studies, we
only chose tumour tissues in which tumour cells occupied a major component (>80%)
of the tumour sample. Information regarding major clinical parameters, including
gender, age, histological tumour subtype, tumour stage, lymphatic invasion, and
distant metastasis, was obtained via standard diagnostic procedures or pathological
examinations. The analysed patients were diagnosed with mucinous colon cancer,
defined as tumours with more than 50% of the tumour volume comprising mucin.
Total RNA was isolated using TRIzol reagent (Invitrogen, CA) from snap-frozen
tissues. The quality of RNA extracted from each specimen was evaluated by the RNA
integrity number (RIN) using an Agilent 2100 Bioanalyzer (Agilent Technologies).
All RNA preparations had RINs over 7.
Reverse transcription reaction and quantitative real-time PCR
First-strand cDNA synthesis was carried out with 500 ng of total RNA from each
sample with a First Strand cDNA Synthesis Kit (Invitrogen), according to the
manufacturer’s instructions. Real-time PCR was performed using cDNA as a template
and a standard SYBR-Green PCR kit on the StepOne Plus system (Applied
Biosystems, Foster City, CA). Primer sequences are listed in Supplementary Table S1.
Relative mRNA expression was normalized to GAPDH mRNA expression (for
1
real-time RT-PCR) or to the total input (for ChIP experiments) and calculated
according to the 2-ΔΔCT method, as appropriate (1) .
Bisulfite sequence analysis
Genomic DNA from tissues and cells were extracted using the Axygen genomic DNA
purification kit (Axygen Biotechnology, Hangzhou, China). Genomic DNA (500 ng)
was bisulfite-converted using the EZ DNA Methylation-Gold kit (Zymo Research,
Orange, CA), following the manufacturer’s protocol. Modified genomic DNA was
then amplified by PCR with primers specific to the respective genomic region. The
PCR amplicons were gel-purified and cloned into pMD-18T vectors (TaKaRa). At
least 6 clones were randomly selected and individually sequenced on an ABI3730xl
DNA Analyzer to ascertain the methylation patterns of each locus. PCR clones with
less than 98% C to T conversion efficiency outside CpG sites were excluded from
further analysis. The percentage of methylation was calculated as the number of
methylated cytosines divided by the total number of cytosines in all of the amplicons
analysed.
Quantitative real-time methylation-specific PCR
Genomic DNA from both tumoral and normal tissues was treated with sodium
bisulfite to modify unmethylated cytosines to uracil (2) . After the DNA conversion,
an aliquot of 2 l was amplified by quantitative real-time PCR using a primer set
specific to the methylated/unmethylated sequence (3) . Appropriate MSP primers were
designed using MethPrimer (University of California, San Francisco). Primer
sequences are listed in Supplementary Table S1. Serial dilutions of methylated or
2
unmethylated control genomic DNAs (Zymo Research, CA, USA) were used to
construct standard curves. To correct for differences in both quality and quantity
between samples, GAPDH was used as an internal control.
The bisulfite reaction and MSP for all samples were repeated at least one time to
confirm the methylation status. Representative PCR products were separated in a 2%
agarose gel, stained with ethidium bromide and visualized under UV illumination.
The MI in each sample was calculated using the following equation:
MI=M/(M+U)100. MI≥0.5 was considered as highly methylatedGAD1. MI<0.5
was considered as lowly methylated GAD1.
Chromatin conformation capture (3C)
We employed the 3C procedure (4) with minor modifications. Briefly, HUVEC,
THLE-3 and SW480 cell extracts were prepared from 107 cells by crosslinking with
1% formaldehyde for 10 minutes at 4°C, followed by quenching with 0.125 M glycine.
Cell lysates were prepared in 10 mM Tris (pH 8.0), 10 mM NaCl and 0.2% NP40,
including proteinase inhibitors. Nuclei were re-suspended in NEB buffer 3 and were
lysed by adding SDS to a final concentration of 0.3% and incubating at 37°C for 1
hour. Triton X-100 was added to a final concentration of 1.8%, followed by
incubation at 37°C for 1 hour. Subsequently, crosslinked nuclear extracts were
digested with 800 U of EcoRI (New England Biolabs) for 16 hours, followed by
restriction enzyme inactivation using SDS at a final concentration of 1.5% and
incubation at 65°C for 20 minutes. Fifteen percent of the crosslinked and digested
extracts was diluted in a total volume of 4 ml and ligated at 16°C for 16 hours using
3
40,000 U of T4 ligase (New England Biolabs) and the appropriate buffer. Religated
products were digested with proteinase K at 65°C for 16 hours, followed by
phenol/chloroform extraction and ethanol precipitation. Religation was tested by PCR
using the primers indicated in Supplementary Table S1.
Bioinformatics and database analysis
To analysis the methylation status of GAD1, we used our previous data on DNA
methylomes from three sets of normal colon, non-CIMP and CIMP colon cancer
samples. These data were obtained from the NCBI Sequence Read Archive (SRA)
(http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi)
under
the
accession
number
SRA029584. RefSeq gene promoters were defined as the regions 1000 bp upstream
and 500 bp downstream of an annotated RefSeq transcription start site. CpG islands
were defined by the CpG island annotation from the reference human genome (NCBI
build 36.1, UCSC Hg18). CTCF binding sites were compiled from all available CTCF
ChIP-Seq data sets deposited by the Broad Institute on the UCSC Genome Browser
(5) .
The methylation status was obtained from the ENCODE Hudson Alpha Methyl-seq
database. The CTCF consensus binding sites (score) were analysed using the in silico
CTCFBS prediction tool (http://insulatordb.uthsc.edu/help.php#tool) (6) .
4
Supplementary Figure Legends
Fig. S1. Summary of GAD1 expression in cancer vs. normal tissues. These data
were obtained from Oncomine, a cancer microarray database and integrated
data-mining platform (www.oncomine.com) (7) . (A) The data in the greenbox
indicated the mRNA expression profile between cancer and normal tissues in different
cancer types. We used stringent selecting thresholds: 1) GAD1 is among the top 10%
differentially expressed genes between cancer and normal tissues; 2) the fold change
of cancer/normal tissue expression is >1.5; 3) the P value is <0.0001. Red indicates
that GAD1 expression is increased in cancer. Blue indicates that GAD1 expression is
decreased in cancer. These data indicated that GAD1 is over-expressed in most
cancers, except in some brain and kidney cancers. (B) Representative data for GAD1
over-expression in colon adenoma tissues. The expression profile is obtained from the
Oncomine database. Raw microarray data are available at Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20916).
(C)
A
representative data for GAD1 down-regulation glioblastoma (8). The expression
profile
is
obtained
from
the
Oncomine
database.
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7696).
Fig. S2 (A) Representative genomic bisulfite sequencing of genomic regions
proximal to the GAD1 promoter in paired colon tumour vs. normal tissue
(patient #C27). The diagram depicts the genomic organization of the human GAD1
locus proximal to the promoter. CpG islands are shown in green. The regions of
bisulfite sequencing are shown in the red line under the CpG islands. Each circle in
5
the bisulfite sequencing data represents a CpG dinucleotide. Black circles, methylated
cytosines; white circles, unmethylated cytosines.(B) GAD1 mRNA levels are
consistent with the DNA methylation status. GAD1 expression was determined
using real time RT-PCR analysis. Error bars indicate the standard deviation.
Fig.S3 (A) Linear plots of quantitative real-time RT-PCR results for GAD1
expression in liver cancer cells. Case-matched samples are represented as
independently connected normal (N) and tumour (T) point pairs. The relative
expression of 1 represents an arbitrarily set value to encompass all variations. Each
point is the average of at least 3 independent quantitative RT-PCR results. (B) The
correlation of GAD1 expression and the extent of methylation in liver cancer. Tumour
cases were classified into two categories, low methylation (MI<0.5) and high
methylation (MI>0.5). GAD1 expression is equivalent to the mRNA fold change for
paired tumour/normal samples. Each box represents the range of T/Nfold changes.
The ends of the boxes represent the 25th and 75thpercentiles, the bars indicate the
10th and 90th percentiles, and theline represents the median. Significant differences
were calculated using the Wilcoxonrank sum test. *, p<0.05.
Fig. S4 Methylation status at the GAD1 locus and GAD1 mRNA levels in HUVECs
and H1-hESC, HCT116, Hela-S3 and K562 cell lines from ENCODE data. The
methylation status was determined by using Methyl 450K Bead Arrays from
ENCODE/HAIB. Orange = methylated (score >= 600); purple = partially methylated
(200 < score < 600); bright blue =unmethylated (0 < score <= 200). The transcription
levels shown were assayed by high-throughput sequencing of polyadenylated RNA
6
(RNA-Seq). The signals are scaled and are present at each of the detected GAD1 exons.
In each cell line, signals from two replicates are shown (http://genome.ucsc.edu/).
Fig. S5 Demethylation by 5-aza-dC treatment decreases GAD1 transcription in
colon and live cancer cells.
The HCT116, SW480, and SMMC7721 and HUVEC cells were treated with 5-aza-dC
for 72 h. (A) methylation status of CpG island 3b were determined by genomic
bisulfite sequencing. Each circle in the bisulfite sequencing data represents a CpG
dinucleotide. Black circles, methylated cytosines; white circles, unmethylated
cytosines. (B) CDKN2A and CA4 mRNA levels were determined by quantitative
RT-PCR. Error bars indicate standard deviations. (C) The diagram depicts the basic
features of the GAD1 locus at the 5’ end, along with the approximate locations of the
primer sets used to analyse the chromatin that immunoprecipitated with the H3K4me3
and H3K27me3 antibodies. The graph depicts the percentages of input chromatin
recovered in the immunoprecipitation for each primer set. The data represent the mean
of two independent replicates.
Fig. S6 CTCF protein and mRNA levels in the HUVECs after transfection with
shRNA-CTCF lentiviruses were detected by Western blot and quantitative RT-PCR.
Fig. S7 Expression of GAD1 correlated well with mucinous colon cancer. Data
were obtained from the TCGA database (http://tcga-data.nci.nih.gov/tcga/) and
compiled by using Oncomine (www.oncomine.com). A total of 215 colorectal
adenocarcinoma and 22 paired normal colorectal tissue samples were analysed.
Fig. S8 GAD1 protein (A) and mRNA (B) levels in SW480 cells after transfection
7
with shRNA-GAD1 lentivirus were detected by Western blot and quantitative
RT-PCR.
Fig.S9 DNA methylation status at the GAD1 locus in 54 cancer or non-cancer
cells. These data were captured in the UCSC genome browser from the methylation
data determined by Methyl 450K Bead Arrays from ENCODE/HAIB. Orange =
methylated (score >= 600), purple = partially methylated (200 < score < 600), bright
blue = unmethylated (0 < score <= 200). The green rectangles indicate the CTCF
binding sites (CTCF-BS2 and CTCF-BS3) located within CpG islands 3a (CGI-3a)
and 5 (CGI-5).
Fig.S10 DNA methylation status at the GAD1 locus in CNS or non-CNS cells.
These data were captured in the UCSC genome browser from the methylation data
determined by Methyl 450K Bead Arrays from ENCODE/HAIB and by Reduced
Representation Bisulfite Seq from ENCODE/Hudson Alpha. For methyl 450k data,
Orange = methylated (score >= 600), purple = partially methylated (200 < score <
600), bright blue = unmethylated (0 < score <= 200). For bisulfite seq data, red=
100% of molecules sequenced are methylated; yellow= 50% of molecules sequenced
are methylated; green= 0% of molecules sequenced are methylated. NH-A: normal
astrocytes; BC_Brain: brain, donor H11058N, age 66, Asian, normal; PFSK-1:
neuroectodermal cell line derived from a cerebral brain tumor; SK-N-SH:
neuroblastoma, cancer; BE2_C: a clone of the SK-N-BE neuroblastoma cell line (see
ATCC CRL-2271), cancer.
8
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