Homeobox gene methylation in lung cancer studied
by genome-wide analysis with a microarray-based
methylated CpG island recovery assay
Tibor Rauch*, Zunde Wang*, Xinmin Zhang†, Xueyan Zhong*, Xiwei Wu‡, Sean K. Lau§, Kemp H. Kernstine¶,
Arthur D. Riggs*?, and Gerd P. Pfeifer*?
*Division of Biology,‡Division of Information Sciences,§Division of Pathology, and¶Division of Surgery, Beckman Research Institute of the City
of Hope, Duarte, CA 91010; and†NimbleGen Systems, Inc., Madison, WI 53711
Contributed by Arthur D. Riggs, February 5, 2007 (sent for review December 11, 2006)
De novo methylation of CpG islands is a common phenomenon in
human cancer, but the mechanisms of cancer-associated DNA
methylation are not known. We have used tiling arrays in combi-
nation with the methylated CpG island recovery assay to investi-
gate methylation of CpG islands genome-wide and at high reso-
lution. We find that all four HOX gene clusters on chromosomes 2,
7, 12, and 17 are preferential targets for DNA methylation in cancer
cell lines and in early-stage lung cancer. CpG islands associated
with many other homeobox genes, such as SIX, LHX, PAX, DLX, and
Engrailed, were highly methylated as well. Altogether, more than
half (104 of 192) of all CpG island-associated homeobox genes in
the lung cancer cell line A549 were methylated. Analysis of paralo-
gous HOX genes showed that not all paralogues undergo cancer-
associated methylation simultaneously. The HOXA cluster was
analyzed in greater detail. Comparison with ENCODE-derived data
shows that lack of methylation at CpG-rich sequences correlates
with presence of the active chromatin mark, histone H3 lysine-4
methylation in the HOXA region. Methylation analysis of HOXA
genes in primary squamous cell carcinomas of the lung led to the
identification of the HOXA7- and HOXA9-associated CpG islands as
frequent methylation targets in stage 1 tumors. Homeobox genes
are potentially useful as DNA methylation markers for early diag-
nosis of the disease. The finding of widespread methylation of
homeobox genes lends support to the hypothesis that a substan-
DNA methylation ? HOX genes ? chromatin ? Polycomb
2). De novo methylation of CpG islands that overlap with promoter
regions is commonly associated with gene silencing and is a
frequent event that accompanies tumorigenesis (3–9). Cancer-
specific hypermethylation of genes that suppress uncontrolled cell
proliferation or promote genome stability is a key step in tumor
Homeobox genes encode a transcription factor family that plays
decisive roles in embryogenesis and differentiation of adult cells
(10). Homeobox proteins are classified into one family on the basis
of their evolutionary conserved helix–loop–helix DNA-binding
motif, called the homeodomain. Apart from the homeodomain,
family members share only limited conservation outside of the
DNA-binding motif. Most of the family members are scattered
throughout the genome, but a subgroup of the homeobox genes,
HOX genes, are organized into clusters. HOX genes were originally
identified in Drosophila as factors involved in homeotic transfor-
mations (11). During mammalian evolution, the ancient HOX
cluster underwent duplications that were followed by gene losses
that finally led to the emergence of the 39 present HOX genes
organized into four clusters (10). HOX proteins are essential
switches of developmental stage- and cell-specific gene regulation,
NA methylation at CpG dinucleotides is an important epige-
netic modification carried out by DNA methyltransferases (1,
and in this way they are key determinants of cell identity and
potential targets during tumorigenesis.
We recently developed a DNA methylation detection technique,
the methylated CpG island recovery assay (MIRA) (12). This
technique is based on the high affinity of a complex of the MBD2b
and MBD3L1 proteins for CpG-methylated DNA (13) and is
compatible with microarray-based methodology (14). We previ-
cancer cell line (14). In the present study, we combined the MIRA
technique with tiling arrays to obtain genome-wide CpG island
coverage as well as high-resolution data for DNA methylation
data indicate that multiple CpG islands within HOX clusters and
near other homeobox genes are frequent methylation targets in
lung cancer. An in-depth analysis of the HOXA cluster revealed
DNA methylation markers for stage 1 lung cancer.
Methylation of the HOXA Cluster. To explore the use of MIRA-
assisted tiling platforms for genome-wide DNA methylation anal-
ysis, we first used NimbleGen’s ENCODE tiling arrays. The EN-
CODE array is designed to identify functional elements in ?1% of
the human genome. On these arrays, the neighboring tiling oligo-
nucleotides (50 bp long) overlap with each other at 12-bp-long
sequences, and in this way 30 Mb, including the HOXA chromo-
somal region, is fully covered. First, we analyzed methylation
patterns by using DNA obtained from the lymphoblastoid cell line
GM06990, which is one of the cell lines used in the ENCODE
project. We compared the MIRA-enriched fraction with the input
fraction. Samples were prepared as described previously (14) by
using MseI digestion, linker ligation, amplification, and labeling of
input and MIRA-enriched DNA. Upon examination of the EN-
CODE regions, we observed that the highest level of methylation
was found on chromosome 7 in a region including the HOXA gene
cluster. Three independent MIRA reactions with GM06990 DNA
were conducted. Fig. 1 Upper shows the high reproducibility of this
approach, which is aided by the overlap features of the tiling array.
We found that several CpG islands within the HOXA cluster were
strongly methylated in GM06990 cells. Confirmation of the meth-
Author contributions: K.H.K., A.D.R., and G.P.P. designed research; T.R., Z.W., X. Zhang, X.
Zhong, and S.K.L. performed research; X. Zhang contributed new reagents/analytic tools;
T.R., Z.W., X.W., A.D.R., and G.P.P. analyzed data; and T.R., A.D.R., and G.P.P. wrote the
The authors declare no conflict of interest.
Abbreviations: COBRA, combined bisulfite restriction analysis; MIRA, methylated CpG
island recovery assay; NHBE cells, normal human bronchial epithelial cells.
?To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or gpfeifer@
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0701059104 PNAS ?
March 27, 2007 ?
vol. 104 ?
no. 13 ?
(COBRA) methylation analysis (Fig. 1 Lower) and by bisulfite
sequencing [Fig. 2 and supporting information (SI) Fig. 5].
The HOXA cluster contains 12 genes (11 HOX genes and EVX1)
and is contained in a 155-kb-long genomic region. Most of the
HOXA promoters are embedded in one of the 39 CpG islands (SI
Table 1) residing in the locus. The high CpG island density makes
the cluster an ideal target for testing the interdependence of
neighboring DNA methylation patterns, including, for example,
theories of long-range epigenetic silencing (15). According to this
hypothesis, genes located in the same neighborhood are coordi-
analysis does show that most CpG islands were methylated within
the HOXA cluster (Fig. 1). However, not all CpG islands were
methylated. Highly methylated and poorly or nonmethylated CpG
islands could be next to each other within the cluster (Figs. 1 and
2; SI Tables 1 and 2). To scrutinize this theory in more detail, we
focused on two neighboring CpG islands that reside just 4 kb apart
in the middle of the HOXA cluster. COBRAs (Fig. 1) and bisulfite
sequencing data (Fig. 2) confirmed that the methylation status of
these two neighboring CpG islands is opposite. Hypermethylation
of the HOXA5 promoter suggests the formation of inactive chro-
promoter should correlate with an active chromatin configuration.
These expectations were confirmed by comparison with chromatin
immunoprecipitation (ChIP)-on-chip experiments previously per-
formed with the GM06990 cell line with anti-histone H3K4me3
antibody (from the ENCODE database) and by tiling expression
array data for the two exons of HOXA5 and HOXA6 (from the
ENCODE database) (Fig. 2). The active gene-associated histone
methylation mark was not detected at the methylated HOXA5
promoter. The region upstream of the HOXA6 gene, in particular
CpG island 16, showed a strong signal for trimethylated K4 on
histone H3 that defines an active chromatin state. Using 5? RACE,
island 16 and containing parts of the two HOXA6 exons (data not
CpG island 15 (Fig. 2).
Cancer Cells. Cancer-associated DNA methylation changes com-
monly affect CpG islands that are often associated with gene
promoters and undergo hypermethylation in tumors. We next used
in a tiling fashion 21 Mb of the human genome. Combining MIRA
with Agilent arrays, we compared the methylation status of CpG
islands genome-wide between the lung cancer cell line A549 and
normal human bronchial epithelial (NHBE) cells. Although the
that each of them is the target of extensive de novo methylation in
lung cancer cells (Fig. 3 and SI Table 2). Fig. 3 shows that besides
ylation hot spots showing ?10-fold enrichment by MIRA in the
lung cancer cell line A549. Strikingly, many of them define other
homeobox genes, for instance the engrailed homologues EN1 and
EN2, on chromosomes 2 and 7, respectively, and the SIX3/2 gene
and TBX20) also were preferred chromosomal methylation sites
(Fig. 3). In addition to those, several paired-box homologues (PAX
genes 1–9, except PAX1 and PAX4) and many other homeobox
genes were methylated in this tumor cell line (SI Table 3). The
occurrence of apparent methylation hot spots as displayed along
the entire length of a chromosome is most often related to the
simultaneous methylation of several CpG islands within a chromo-
somal segment. This observation is illustrated for the HOX gene
clusters (Figs. 1 and 2 and SI Tables 1 and 2) but also can be seen
for other chromosomal methylation hot spots. As an example, we
illustrate the methylation of several CpG islands within or near the
cell line was used for three independent MIRA reactions by using the amplification and labeling procedure as described in Materials and Methods. (Upper) The
signal ratio of MIRA-enriched versus input DNA is plotted along the chromosome. The green boxes indicate the 39 CpG islands (see SI Table 1). The numbering
for the regions indicated to confirm the methylation data obtained by MIRAs. Some of the BstUI cleavage fragments are too small to be visible on the gels. ?,
to control digestion with no BstUI; ?, BstUI-digested samples.
www.pnas.org?cgi?doi?10.1073?pnas.0701059104Rauch et al.
EN1 and TBX20 genes in SI Fig. 6. In all, 104 of 194 (54%) of the
line A549 were methylated (SI Tables 2 and 3). We used rather
stringent criteria for making this assignment, and the percentage of
the elimination of low signal intensity spots, for instance caused by
large spacing of adjacent restriction sites and low amplification
efficiency. Also, homeobox genes without CpG islands are not
represented on the array, and some may be methylated already in
normal cells. Taken together, these findings suggest that the inac-
tivation of clusters of CpG islands near homeobox genes is a
common event in this lung cancer cell line.
Methylation Analysis of HOX Gene Paralogues. HOX proteins be-
longing to a given paralogous group can be functionally similar and
methylation profile of the individual genes that constitute a paralo-
gous group, we analyzed the Agilent CpG island arrays in more
detail. These high-resolution tiling arrays harbor most of the 39
HOX gene promoters. We determined the hypermethylation pro-
file of each HOX gene promoter for all of the HOX gene clusters
in the lung cancer cell line A549 versus NHBE cells (SI Table 2).
The data show that many CpG islands in the HOX clusters were
hypermethylated in A549 lung cancer cells relative to NHBE cells.
We focused our attention on the HOX6 and HOX7 paralogues. We
in this experiment, the MIRA-enriched DNA and the input DNA were processed without amplification and were directly labeled and hybridized to the array to
make these data methodologically comparable with the ChIP data. ChIP with anti-histone H3K4m3 antibody was performed, and the DNA was hybridized onto
Sanger ENCODE3.1.1 DNA microarrays (data were obtained from the ENCODE database). The pink shading indicates the promoter-associated CpG islands. (B)
are from the ENCODE database. (Lower) Bisulfite sequencing verification of the DNA methylation status of the indicated CpG island regions.
DNA and histone methylation profile at HOXA cluster genes in GM06990 cells. (A) DNA and histone methylation profile analysis of the entire HOXA
Rauch et al. PNAS ?
March 27, 2007 ?
vol. 104 ?
no. 13 ?
in NHBE cells, but both promoters were subject to dense DNA
methylation in the tumor cell line (SI Fig. 7). Although HOXC6
belongs to the same paralogous group, there is no CpG island near
its promoter region, and it is not represented on the array. The two
HOX7 paralogous genes (HOXA7 and HOXB7) are methylation-
free in normal cells, but in the lung cancer cell line one of them
(HOXB7) kept the methylation-free status, whereas the other one
(HOXA7) became hypermethylated (SI Fig. 7). Noncoordinate
methylation was seen for other paralogues as well (SI Table 2),
indicating that DNA methylation in tumors does not necessarily
simultaneously affect all members of a paralogous group.
Methylation of HOXA Genes in Primary Lung Tumors. To investigate
whether methylation of HOX cluster genes is present in human
primary lung tumors, we first analyzed pairs of normal lung tissue
and adjacent stage 1 adenocarcinoma or squamous cell carcinoma
(SI Figs. 8 and 9). We found that all four HOX clusters, as well as
several other homeobox genes, were hypermethylated in the early-
HOXB and HOXC clusters were methylated to a lesser extent.
We then analyzed HOXA cluster genes in four stage 1 lung
by COBRA methylation assays (Fig. 4). In pilot experiments, we
found that these early-stage tumor samples were not methylated at
the promoters of the following well known lung cancer DNA
methylation marker genes: RASSF1A (19), PAX5? (20), DLEC1
(14), and RAR?2 (21). The p16 (22) and TCF21 (23) promoters
showed partial methylation (data not shown). We next systemati-
cally checked the methylation status of the CpG islands in the
HOXA cluster. The HOXA genes near the 3? end of the cluster
samples, and the methylation levels were moderately or strongly
elevated in the matching tumor pairs (Fig. 4), suggesting the
possibility that the preexisting low levels of methylation made these
samples (Fig. 4). Distinct from the 3? located genes, DNA meth-
ylation can only weakly be detected at the HOXA6 promoter by
COBRAs in normal lung cells. Upstream of the HOXA6 gene, the
other promoters are not methylated in normal lung samples, but
some of them are cancer-specifically methylated in tumor samples.
The HOXA7 and HOXA9 promoters show high levels of DNA
methylation in stage 1 squamous cell carcinomas.
series of primary lung tumors, we analyzed the methylation status
of the CpG islands associated with the HOXA7 and HOXA9 genes
in primary lung squamous cell carcinomas (SI Fig. 10). No meth-
ylation of HOXA7 or HOXA9 was detected in any of the 18 normal
lung tissues. However, the HOXA7 CpG island was methylated in
in 8 of 10 (80%) of the stage 1 tumors.
To characterize the DNA methylation status of the human HOX
gene clusters we combined the MIRA method with region-specific
genomic and genome-wide CpG island tiling arrays. This technol-
ogy proved to be highly reproducible, and methylation differences
could generally be confirmed by standard bisulfite-based methyl-
ation assays. HOX gene clusters containing multiple CpG islands
were preferential methylation targets in lung cancer cells and
A549 lung cancer cells and from NHBE cells were mixed and hybridized onto Agilent CpG island arrays. The ratio (fold difference) plotted indicates individual
probe signals (blue diamonds) for hypermethylated CpG islands in A549 cells (A549/NHBE signal ratio). The numbers indicate selected areas of densely
hypermethylated CpG island clusters for the genes indicated at the bottom.
DNA methylation profiles of chromosomes 2, 7, 12, and 17 that carry the HOXA, HOXB, HOXC, and HOXD gene clusters. MIRA-enriched fractions from
www.pnas.org?cgi?doi?10.1073?pnas.0701059104 Rauch et al.
HOX clusters were methylated, we found that nonmethylated and
highly methylated CpG islands can still be next to each other in the
gene-rich HOX clusters (Figs. 1 and 2 and SI Tables 1 and 2). For
example, the HOXA5 and HOXA6 promoters are located just 4 kb
apart and are embedded in CpG islands. We found that the
methylation status of the two promoters is the opposite in the
lymphoblastoid cell line GM06990 (Figs. 1 and 2). These findings
on an island-by-island basis within the HOX clusters rather than by
a long-range effect encompassing all CpG islands within a specific
chromosomal domain as reported for the 2q.14.2 chromosomal
trimethylated K4 antibody (ENCODE database) negatively corre-
lated with the MIRA-assisted microarray data. DNA hypermeth-
ylation and histone H3 K4 trimethylation were mutually exclusive
along the HOXA cluster (Fig. 2).
The human HOX genes are organized in four clusters, and based
on homeobox sequence similarities, the individual HOX genes are
classified into 13 paralogous groups. Our data suggest that para-
logues do not always undergo simultaneous methylation in tumor
Many homeobox genes are aberrantly expressed in a variety of
hematological malignancies and solid tumors, most commonly
However, many of these genes are not expressed in adult tissues,
including the lung (24, 26). In some instances, the lack of their
expression may promote de novo methylation during malignant
progression. Several homeobox genes are methylated in tumors of
various histological origins. For example, methylation of HOXB13
occurs in 30% of renal cell carcinomas (27), and methylation of
(28). The HOXA5 promoter region was methylated in 16 of 20
p53-negative breast tumor specimens (29). While this manuscript
cancer was reported (30). Our data indicate that dense methylation
of the four HOX gene clusters and many other homeobox genes
located on different chromosomes occurs in lung cancer.
DNA-binding motif and can bind to similar cis elements in in vitro
experiments (31). However, by interacting with other transcription
factors they can diversify their binding targets. Interacting partners
for HOX proteins can be other homeodomain-containing factors
(32–35), and they synergistically govern the expression of down-
are also frequent targets of hypermethylation (Fig. 3 and SI Table
3). Hypermethylation of clustered and nonclustered homeobox
However, it remains to be determined whether methylation-
induced silencing of homeobox genes contributes to tumorigenesis
directly or is merely associated with it.
The widespread and pervasive nature of homeobox gene meth-
ylation in lung cancer (104 of 194 genes analyzed in A549 cells)
suggests a common mechanistic pathway that promotes de novo
methylation of these genes during tumorigenesis. Polycomb com-
plexes are involved in silencing of homeobox genes (36–38). A
recent genome-wide analysis of the localization of Polycomb com-
ponents including SUZ12, which is required for the histone meth-
yltransferase activity and silencing function of the EED–EZH2
complex, has identified a large fraction of the targets as homeobox
histological types, including precancerous tissues (42, 43). The
Polycomb component EZH2 associates with DNA methyltrans-
ferase activity and can promote DNA hypermethylation (44–46),
and a speculative mechanism can be proposed that links Polycomb
silencing with tumor-associated DNA methylation. This mechanis-
tic link has received support from recent studies finding good
concordance between Polycomb occupancy of genes in noncancer-
hypermethylation events (47–49). Many of the Polycomb targets
are homeobox genes and other key developmental regulatory
genes. They overlap with those genes we find hypermethylated in
lung cancer, lending strong support to the Polycomb connection.
Alternatively, a regional breakdown in activating factors that
maintain active chromatin domains at homeobox genes, such as
MLL1, a member of the mammalian trithorax group, and H3K4
methyltransferase, may ultimately lead to a shift in histone modi-
complete understanding of the range and patterns of CpG island
methylation within tumors and premalignant lesions will be an
important step toward understanding of the mechanistic pathways
leading to hypermethylation of genes during tumorigenesis.
Finally, the extent and tumor specificity of homeobox gene
methylation provide a potentially useful source of DNA methyl-
ation markers for detecting early stage disease (8, 53–56). With
relevance to lung cancer, diagnostic tests of the population at risk
analysis could supplement high-resolution imaging technologies to
of early-stage tumors. In particular, methylation of HOXA9 in a
high percentage of early-stage squamous cell carcinomas may be
one such promising marker.
Materials and Methods
MIRA. MIRA was done essentially as described previously (14) with
minor modifications. Briefly, purified GST-tagged MBD2b protein
was eluted from a glutathione–Sepharose CL-4B matrix (Amer-
sham Biosciences, Piscataway, NJ) with elution buffer (50 mM
Tris?HCl, pH 8.5/150 mM NaCl/20 mM glutathione/0.1% Triton
X-100) for 4 h at 4°C. The eluted GST-MBD2b fraction was
dialyzed against PBS for 5 h and then overnight against 50 mM
Hepes, pH 7.4/150 mM NaCl/5 mM 2-mercaptoethanol/50% (vol/
vol) glycerol. GST-tagged MBD2b protein was kept at ?20°C.
1 squamous cell carcinoma samples. (A) Methylation differences between
squamous cell carcinomas and matching normal pairs (pairs 1–4) were de-
digestion with no BstUI; ?, BstUI-digested samples. (B) Summary of the
methylation status of promoters in the HOXA gene cluster.
Rauch et al. PNAS ?
March 27, 2007 ?
vol. 104 ?
no. 13 ?
His-tagged MBD3L1 was prepared as described previously (14). Download full-text
Genomic DNA was fragmented by MseI digestion, and linker
ligation was done as described earlier (14). The linker-ligated
fraction was incubated with GST-MBD2b and His-MBD3L1 pro-
teins overnight as described previously (14). MagneGST beads (2.5
?l) (Promega, Madison, WI) preblocked with JM110 bacterial
DNA, were added to the binding reaction and incubated at 4°C for
Tris?HCl, pH 7.5/700 mM NaCl/1 mM EDTA/3 mM MgCl2/0.1%
Triton X-100), and the methylated CpG-enriched fraction was
eluted by using Qiaquick PCR purification kits (Qiagen, Valencia,
CA). Eluted fractions or MseI-digested and linker-ligated input
fractions were PCR-amplified as described previously (14).
Sample Labeling and Hybridization to NimbleGen and Agilent Arrays.
The labeling of dsDNA, microarray hybridization, and scanning
were performed by the NimbleGen Service Laboratory (Madison,
which contains ?385,000 50-mer oligonucleotides and covers the
ENCODE regions at 38-bp spacing, was used. Data were extracted
from scanned images by using NimbleScan 2.3 extraction software
(NimbleGen Systems, Inc.).
Human CpG island microarrays, which contain 237,000 oligo-
nucleotide probes covering 27,800 CpG islands, were purchased
from Agilent Technologies (Santa Clara, CA). Genomic DNA was
then subjected to linker ligation and MIRA as described above.
Two micrograms each of the amplicons from MIRA-enriched
Cy5-dCTP (tumor) or Cy3-dCTP (control) in 87.5-?l reactions
(both Cy3- and cy5-dCTP were obtained from GE Healthcare,
microarray hybridization was performed according to the Agilent
ChIP-on-chip protocol (version 9.0). The hybridized arrays were
scanned on an Axon 4000B microarray scanner (Molecular De-
vices, Sunnyvale CA), and the images were analyzed with Axon
GenePix software version 5.1. Image and data analysis were done
as described previously (14).
DNA Methylation Analysis Using COBRA and Bisulfite Sequencing.
and matching normal tissues removed with surgery were obtained
from the frozen tumor bank of the City of Hope National Medical
Center (Duarte, CA). The COBRAs were done according to the
method of Xiong and Laird (58) using digestion with BstUI
(5?-CGCG). DNA was treated and purified with an EpiTect
Bisulfite kit (Qiagen). PCR primers for amplification of specific
targets in bisulfite-treated DNA are listed in SI Table 4. For
sequence analysis, the PCR products obtained after bisulfite con-
version were cloned into the pDrive PCR cloning vector (Qiagen),
and five individual clones were sequenced.
We thank Steven Bates for assistance with cell culture. This work was
supported by National Institutes of Health Grant CA104967 (to G.P.P.).
1. Riggs AD (1975) Cytogenet Cell Genet 14:9–25.
2. Holliday R, Pugh JE (1975) Science 187:226–232.
3. Baylin SB, Ohm JE (2006) Nat Rev Cancer 6:107–116.
4. Jones PA, Baylin SB (2002) Nat Rev Genet 3:415–428.
5. Costello JF, Fruhwald MC, Smiraglia DJ, Rush LJ, Robertson GP, Gao X, Wright
FA, Feramisco JD, Peltomaki P, Lang JC, et al. (2000) Nat Genet 24:132–138.
6. Esteller M, Corn PG, Baylin SB, Herman JG (2001) Cancer Res 61:3225–3229.
7. Issa JP (2004) Nat Rev Cancer 4:988–993.
8. Ushijima T (2005) Nat Rev Cancer 5:223–231.
9. Egger G, Liang G, Aparicio A, Jones PA (2004) Nature 429:457–463.
10. Garcia-Fernandez J (2005) Nat Rev Genet 6:881–892.
11. Lewis EB (1978) Nature 276:565–570.
12. Rauch T, Pfeifer GP (2005) Lab Invest 85:1172–1180.
13. Jiang CL, Jin SG, Pfeifer GP (2004) J Biol Chem 279:52456–52464.
14. Rauch T, Li H, Wu X, Pfeifer GP (2006) Cancer Res 66:7939–7947.
15. Frigola J, Song J, Stirzaker C, Hinshelwood RA, Peinado MA, Clark SJ (2006) Nat
16. Tvrdik P, Capecchi MR (2006) Dev Cell 11:239–250.
17. Greer JM, Puetz J, Thomas KR, Capecchi MR (2000) Nature 403:661–665.
18. Wellik DM, Hawkes PJ, Capecchi MR (2002) Genes Dev 16:1423–1432.
20. Palmisano WA, Crume KP, Grimes MJ, Winters SA, Toyota M, Esteller M, Joste N,
Baylin SB, Belinsky SA (2003) Cancer Res 63:4620–4625.
21. Grote HJ, Schmiemann V, Geddert H, Rohr UP, Kappes R, Gabbert HE, Bocking
A (2005) Int J Cancer 116:720–725.
22. Bearzatto A, Conte D, Frattini M, Zaffaroni N, Andriani F, Balestra D, Tavecchio
L, Daidone MG, Sozzi G (2002) Clin Cancer Res 8:3782–3787.
23. Smith LT, Lin M, Brena RM, Lang JC, Schuller DE, Otterson GA, Morrison CD,
Smiraglia DJ, Plass C (2006) Proc Natl Acad Sci USA 103:982–987.
24. Grier DG, Thompson A, Kwasniewska A, McGonigle GJ, Halliday HL, Lappin TR
(2005) J Pathol 205:154–171.
25. Samuel S, Naora H (2005) Eur J Cancer 41:2428–2437.
(2001) Am J Pathol 158:955–966.
27. Okuda H, Toyota M, Ishida W, Furihata M, Tsuchiya M, Kamada M, Tokino T,
Shuin T (2006) Oncogene 25:1733–1742.
28. Shiraishi M, Sekiguchi A, Oates AJ, Terry MJ, Miyamoto Y (2002) Oncogene
29. Raman V, Martensen SA, Reisman D, Evron E, Odenwald WF, Jaffee E, Marks J,
Sukumar S (2000) Nature 405:974–978.
Kim C, Futscher BW (2006) Cancer Res 66:10664–10670.
31. Catron KM, Iler N, Abate C (1993) Mol Cell Biol 13:2354–2365.
32. Williams TM, Williams ME, Innis JW (2005) Dev Biol 277:457–471.
33. Liu Z, Shi W, Ji X, Sun C, Jee WS, Wu Y, Mao Z, Nagy TR, Li Q, Cao X (2004)
J Biol Chem 279:11313–11319.
35. Shanmugam K, Green NC, Rambaldi I, Saragovi HU, Featherstone MS (1999) Mol
Cell Biol 19:7577–7588.
36. Cao R, Zhang Y (2004) Mol Cell 15:57–67.
37. Kim SY, Paylor SW, Magnuson T, Schumacher A (2006) Development (Cambridge,
38. Erhardt S, Su IH, Schneider R, Barton S, Bannister AJ, Perez-Burgos L, Jenuwein T,
Kouzarides T, Tarakhovsky A, Surani MA (2003) Development (Cambridge, UK)
39. Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, Levine SS,
Wernig M, Tajonar A, Ray MK, et al. (2006) Nature 441:349–353.
40. Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K (2006) Genes Dev
41. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B,
Johnstone SE, Cole MF, Isono K, et al. (2006) Cell 125:301–313.
42. Ding L, Erdmann C, Chinnaiyan AM, Merajver SD, Kleer CG (2006) Cancer Res
43. Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda
MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP, et al. (2002) Nature 419:624–629.
A, Bernard D, Vanderwinden JM, et al. (2006) Nature 439:871–874.
45. Hernandez-Munoz I, Taghavi P, Kuijl C, Neefjes J, van Lohuizen M (2005) Mol Cell
46. Reynolds PA, Sigaroudinia M, Zardo G, Wilson MB, Benton GM, Miller CJ, Hong
C, Fridlyand J, Costello JF, Tlsty TD (2006) J Biol Chem 281:24790–24802.
47. Ohm JE, McGarvey KM, Yu X, Cheng L, Schuebel KE, Cope L, Mohammad HP,
Chen W, Daniel VC, et al. (2007) Nat Genet 39:237–242.
48. Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, Zimmerman J, Eden
E, Yakhini Z, Ben-Shushan E, Reubinoff BE, et al. (2007) Nat Genet 39:232–236.
49. Widschwendter M, Fiegl H, Egle D, Mueller-Holzner E, Spizzo G, Marth C, Weisen-
berger DJ, Campan M, Young J, Jacobs I, Laird PW (2007) Nat Genet 39:157–158.
RA (2005) Proc Natl Acad Sci USA 102:8603–8608.
51. Popovic R, Zeleznik-Le NJ (2005) J Cell Biochem 95:234–242.
52. Terranova R, Agherbi H, Boned A, Meresse S, Djabali M (2006) Proc Natl Acad Sci
53. Belinsky SA (2004) Nat Rev Cancer 4:707–717.
54. Laird PW (2003) Nat Rev Cancer 3:253–266.
55. Ahrendt SA, Chow JT, Xu LH, Yang SC, Eisenberger CF, Esteller M, Herman JG,
Wu L, Decker PA, Jen J, Sidransky D (1999) J Natl Cancer Inst 91:332–339.
56. Belinsky SA, Nikula KJ, Palmisano WA, Michels R, Saccomanno G, Gabrielson E,
Baylin SB, Herman JG (1998) Proc Natl Acad Sci USA 95:11891–11896.
57. Selzer RR, Richmond TA, Pofahl NJ, Green RD, Eis PS, Nair P, Brothman AR,
Stallings RL (2005) Genes Chromosomes Cancer 44:305–319.
58. Xiong Z, Laird PW (1997) Nucleic Acids Res 25:2532–2534.
www.pnas.org?cgi?doi?10.1073?pnas.0701059104 Rauch et al.