Identification of a key role of widespread epigenetic drift in Barrett’s esophagus and esophageal adenocarcinoma
© The Author(s). 2017
Received: 25 May 2017
Accepted: 24 September 2017
Published: 16 October 2017
Recent studies have identified age-related changes in DNA methylation patterns in normal and cancer tissues in a process that is called epigenetic drift. However, the evolving patterns, functional consequences, and dynamics of epigenetic drift during carcinogenesis remain largely unexplored. Here we analyze the evolution of epigenetic drift patterns during progression from normal squamous esophagus tissue to Barrett’s esophagus (BE) to esophageal adenocarcinoma (EAC) using 173 tissue samples from 100 (nonfamilial) BE patients, along with publically available datasets including The Cancer Genome Atlas (TCGA).
Our analysis reveals extensive methylomic drift between normal squamous esophagus and BE tissues in nonprogressed BE patients, with differential drift affecting 4024 (24%) of 16,984 normally hypomethylated cytosine-guanine dinucleotides (CpGs) occurring in CpG islands. The majority (63%) of islands that include drift CpGs are associated with gene promoter regions. Island CpGs that drift have stronger pairwise correlations than static islands, reflecting collective drift consistent with processive DNA methylation maintenance. Individual BE tissues are extremely heterogeneous in their distribution of methylomic drift and encompass unimodal low-drift to bimodal high-drift patterns, reflective of differences in BE tissue age. Further analysis of longitudinally collected biopsy samples from 20 BE patients confirm the time-dependent evolution of these drift patterns. Drift patterns in EAC are similar to those in BE, but frequently exhibit enhanced bimodality and advanced mode drift. To better understand the observed drift patterns, we developed a multicellular stochastic model at the CpG island level. Importantly, we find that nonlinear feedback in the model between mean island methylation and CpG methylation rates is able to explain the widely heterogeneous collective drift patterns. Using matched gene expression and DNA methylation data in EAC from TCGA and other publically available data, we also find that advanced methylomic drift is correlated with significant transcriptional repression of ~ 200 genes in important regulatory and developmental pathways, including several checkpoint and tumor suppressor-like genes.
Taken together, our findings suggest that epigenetic drift evolution acts to significantly reduce the expression of developmental genes that may alter tissue characteristics and improve functional adaptation during BE to EAC progression.
Research into the connection between aging and cancer is being fueled by advances in molecular profiling of age-related processes such as chronic inflammation, accumulation of somatic DNA/mtDNA mutations, and epigenetic changes in tissues in which cancers arise . Two distinct, albeit not entirely independent, concepts have emerged recently that relate changes in DNA methylation to biological tissue age. The first is based on the discovery (of sets) of CpG dinucleotides (CpGs) in the genome that are subject to age-dependent, possibly complex changes in methylation levels that, in aggregate, correlate strongly with chronological age [2–4]. We refer to these types of CpGs as clock-CpGs. A second and simpler concept is based on the observation of gradual age-related changes in methylation levels at specific CpG sites or CpG-rich regions, a process commonly referred to as epigenetic or methylomic drift [5–11]. For example, some CpG islands show very low methylation levels early in life but are known to become gradually methylated over time as a result of sporadic de novo methylation events during DNA replication. We identify these as drift CpGs. It is worth pointing out that data supporting these concepts come mainly from cross-sectional studies that include individuals of different age. In contrast, individual-level (longitudinal) drift, unless studied directly in select individuals over time as we have collected for this study, is typically inferred from population drift.
In a recent study, we used a combination of cross-sectional and longitudinally collected biopsy samples to identify a set of highly correlated CpGs in premalignant Barrett’s esophagus (BE) tissue that undergo differential epigenetic drift relative to normal squamous (NS) tissue . This study arrived at a set of 67 drift CpGs that show significant age-related methylation differences between NS and BE and was used as an epigenetic clock to estimate the time of BE onset. Unlike previous epigenetic clocks that were constructed to predict the age of an individual, the BE tissue-specific clock model was designed to infer unknown tissue ages. Because BE is essentially asymptomatic, it is usually not known how long a patient has lived with BE. However, the time a patient has lived with BE may be considered a risk factor since older BE tissue has had more time for cancer to evolve compared to younger BE tissue which is more likely to be free of neoplastic changes [13, 14]. Although the earlier study was the first to develop an epigenetic clock for BE tissue age, we did not evaluate the full scope of epigenetic drift occurring in BE following its formation, nor its dynamics or functional consequences at the genomic level.
The aims of this study are to characterize individual heterogeneity and genome-wide patterns of age-related epigenetic drift in tissue samples from BE and EAC patients, to develop a mechanistic understanding of methylation dynamics during tissue aging, including the degree of cooperativity and possible presence of nonlinear feedback within CpG islands during the temporal evolution of epigenetic drift, and to explore the impact of advanced drift on gene expression in EACs for which we have both gene expression and methylation data.
To accomplish the aims of this study, it was necessary to analyze methylation drift patterns from an extensive collection of NS, BE, and EAC biopsy tissue samples, including 173 tissue samples from 100 nonprogressed and progressed (nonfamilial) BE patients, along with methylation and gene expression data in 87 EAC from The Cancer Genome Atlas (TCGA)  and in 47 EAC + 4 BE samples previously analyzed by Krause et al. .
Using data from the HM450 methylation arrays (see “Methods”), we aimed to better characterize the full extent of epigenetic drift occurring in BE and the temporal dynamics of epigenetic drift and to explore the impact of advanced drift on gene expression in EAC for which we have both gene expression and methylation data. Methylation levels are measured either in terms of a β value (methylation fraction) or M value (logit2(β)) as indicated in the text.
Genomic scope of drift
The majority (63%) of islands that include drift CpGs are associated with gene promoter regions, i.e., they involve a transcription start site (TSS200 or TSS1500), while only 11% of islands that undergo drift overlap with the gene body, compared to 73% (TSS-associated) and 10% (body-associated) CpG islands on the HM450 array, respectively. In contrast, the relative abundance of intergenic CpG islands is significantly higher among CpG islands that undergo drift compared to the fraction of intergenic CpG islands found on the array (24 vs. 16%, see Fig. 1).
Pairwise correlations between island-associated CpGs
Figure 4 also shows that the pairwise correlations decay with genomic distance and, for drift CpGs, extend further into the island shelves than static CpGs. Islands that show tissue age-related drift are also significantly larger (in terms of genomic length) than static islands. The mean sizes of the static vs. drift CpG islands were 0.9 vs 1.1 kb, respectively (p = 2 × 10−10, two-sided t test).
Bimodal nature of epigenetic drift in BE and EAC
Advanced drift is associated with low tumor stage
Using tumor stage information from the TCGA, we found a statistically significant association (p value = 0.024; Fisher’s exact test) of low tumor stage (AJCC stage I) vs advanced stage (AJCC stage III and higher) with the type of drift pattern (group H vs group L + I). Specifically, low-stage tumors are more prevalent in group H compared with group L + I among the 74 TCGA EAC for which tumor stage information was available (odds ratio 6.0 (1.1–63.3)).
Differential gene expression by drift group
To see whether gene expression patterns (at the mRNA level) differed between EAC samples that showed minor (unimodal low) drift and samples that showed advanced methylomic drift on a gene by gene basis, we matched 1240 drift CpG islands (out of 1317 CpG islands with 5 or more drift CpGs per island) with one or more (overlapping) genes to evaluate the relationship between gene expression and island-level methylation for the TCGA and the Krause et al. data sets. Specifically, we identified differentially expressed genes for which expression differed significantly between low- and high-drift samples by setting a threshold of β = 0.2 to delineate the two groups and using a two-sided Mann-Whitney-Wilcoxon test (q < 0.01) on normalized gene expression data.
In total, we identified 200 genes that were significantly underexpressed in the advanced drift group while only 10 genes were significantly overexpressed (Additional file 2: Table S1). Independently, we found 35 genes that were significantly repressed and none that were significantly overexpressed among 51 (47 EAC + 4 BE) samples provided by Krause et al. . Importantly, several genes (20/35) that were found repressed in the smaller study by Krause et al. were also found repressed in TCGA (Additional file 2: Table S1). In particular, the gene most significantly repressed in TCGA (q = 5 × 10−9) was also ranked most significantly repressed in the data provided by Krause et al., CHFR (checkpoint with forkhead and ring finger domains), a mitotic stress checkpoint gene with tumor suppressive function that has been identified in a wide range of cancers [20, 21], and most recently as a significantly silenced gene in a large clustering analysis of esophageal adenocarcinoma . This striking asymmetry between gene expression changes and methylomic drift is consistent with parallel findings that CpG promoter hypermethylation in cancers often is correlated with gene-silencing . A Gene Ontology (GO)-based over-representation analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) shows a highly significant greater than threefold enrichment of sequence-specific DNA binding transcription factor activity (p = 2 × 10−9, Additional file 2: Table S2). The most prominent group identified by this analysis is a family of repressive Krueppel-associated box (KRAB) domain zinc finger (ZNF) transcription factors (greater than sixfold, p = 1.3 × 10−15, Additional file 2: Table S2). KRAB-mediated transcriptional repression involves the binding of the KRAB domain to co-repressors potentially resulting in heterochromatin formation and silencing of endogenous retroviruses [23, 24].
Evidence for a threshold effect leading to bimodal drift
We note that these findings are surprisingly consistent with the unimodal-to-bimodal epigenetic drift predictions made by Sontag et al.  who proposed a mathematical model that included a nonlinear relationship between de novo methylation and the ambient level of methylation present in a region of CpGs. To demonstrate that such a model results in unimodal-to-bimodal drift transitions over time, we explicitly simulated sporadic de novo methylation on an island of 50–100 CpGs, independently in 1000 cells, mimicking crudely the cell population in the tissue samples. Each CpG was assumed to be in a binary state (0/1 of being (un)methylated), and the states of the CpGs initially (at time t = 0) were sampled from a binomial distribution with probability 0.06 which equals the mean methylation level in our NS tissue samples. The CpG states were then propagated stochastically with a rate (probability per time step) of becoming methylated that increases 100-fold from a background of 10−4 to 10−2 when the mean level of methylation on the island crosses a threshold of β = 0.2.
Without a mathematical exploration of this Markov model, but straightforward in silico experimentation with the baseline distribution of methylation rates (specifically, a gamma distribution with mean 10−4 and variance 4 × 10−8) and threshold value, our simulations show that this simple model generates methylation density trajectories that typically bifurcate and strikingly resemble the observed drift signatures in our samples. Figure 5b shows a typical density trajectory for a region of 50 CpGs, an arrayed population of 1000 cells, every 100 time steps, for a total duration of 3000 time steps. Although our model differs in functional form from the model described in Sontag et al., it shares important features, including a suppression of de novo methylation at low levels and a nonlinear acceleration as the ambient (regional) level of methylation increases. In contrast, models that do not include this ambient methylation feedback on the local (site-specific) rate of methylation do not, in general, lead to bifurcations in the main (initial) mode of the evolving drift pattern, but still exhibit a weak bimodality as shown in Additional file 1: Figure S2. Additional file 1: Figure S3 further illustrates the stochastic nonlinear behavior of our model via simulated time course trajectories of the mean methylation levels for 10 CpG islands that share an identical drift rate distribution across their CpGs.
Here we take a closer look at how differential epigenetic drift is organized in BE-associated genomes, and its scope and association with gene expression, motivating further investigation of its role in neoplastic progression in BE. To do so, we first surveyed the array-based DNA methylome for significant correlations with the mean drift measured by 67 drift CpGs previously identified by our group to estimate BE dwell time, i.e., the time a patient has lived with BE [12, 13]. Following this study, we targeted CpGs that are hypomethylated in NS tissue but are subject to differential drift in BE tissue caused by accelerated age-related de novo methylation. While NS tissue may not be the tissue of origin for BE, the similarity of methylation levels at drift-associated CpGs between NS and other normal tissues, such as fundus (see Additional file 1: Figure S1), justifies the use of NS as a normal reference tissue to identify differential drift in BE. Our previous study did not reveal the full extent of this differential drift due to highly restrictive pre-filtering.
Our genome-wide “drift survey” revealed that, at the island level, > 24% of CpG islands undergo methylomic drift and are predominantly promoter-associated (i.e., overlap transcription start sites (TSS)). To investigate whether epigenetic drift occurs on isolated CpG sites or is a nonlocal phenomenon at the CpG island level, we evaluated correlations of methylation between pairs of CpGs (across BE samples) using all island-associated CpG probes (available on the HM450 platform) as a function of genomic distance between the probes (Fig. 4). Our results confirm the prevailing view that CpG islands essentially exert epigenetic control by their collective methylation state rather than through specific CpG sites [25, 26]. Importantly, we found evidence that drift does not evolve uniformly in BE and EAC but appears to be governed by a nonlinear, threshold-like stochastic methylation process which depends nonlocally on the methylation status of other island CpGs. Simulations using a stochastic model, which reflects these dynamics at the island level, show characteristic transitions from unimodal to bimodal drift similar to what we observe in our data. Although other models may provide similar fits to the observed drift distributions, this model has its origins in earlier work aimed at understanding the stable, somatic inheritance of methylation imprints  and predicts epigenetic drift as a series of sporadic de novo methylation events at the island level. Our nonlinear feedback model for methylomic drift suggests that the various drift distributions we see in our tissue samples may simply be attributed to tissue age itself (i.e., at what points in time the tissue samples were obtained during the dynamic process of methylomic evolution). Furthermore, analyses of consecutive biopsies in the same patient separated by several years further confirmed that epigenetic drift, as defined in this study, involves the sporadic departure from normal (hypomethylated) levels to higher levels as the tissue ages. Taken together, these findings suggest that epigenetic drift in BE advances non-uniformly by departing from unimodal (low-drift) distributions of methylation and gradually bifurcating into bimodal distributions over time. Similar unimodal and bimodal methylation distributions are observed in EAC samples although the bimodality appears more pronounced in EAC.
To investigate potential functional consequences of epigenetic drift, we compared gene expression in BE and EAC samples showing no (or low) drift to gene expression in samples that show definite drift β > 0.2. This comparison revealed statistically significant differences in gene expression between the two sample groups that are predominantly repressive involving several checkpoint and tumor suppressor-like genes, in particular CHFR (checkpoint with forkhead and ring finger domains), a mitotic stress checkpoint gene that has been observed to undergo promoter-associated hypermethylation in colon, gastric, and esophageal cancers and is associated with chromosomal instability [27, 28]. Submitting the 200 differentially repressed genes in the TCGA EAC samples to a statistical over-representation test (Additional file 2: Table S2) further revealed an unexpected high number of KRAB domain zinc finger genes (greater than sixfold enrichment using DAVID) that are subject to epigenetic drift and transcriptional repression possibly compromising their KAP1(TRIM28)-mediated repressive function. This finding is intriguing because KRAB domain ZNF also target endogenous retroviruses and transposable elements.
Finally, comparison of island-level drift with gene expression in NS and BE tissue samples from the Krause study  revealed that the majority of genomic loci undergoing epigenetic drift in BE are transcriptionally silent, consistent with the notion of neutral (clock-like) drift. However, the majority of differentially expressed genes associated with CpG islands that exhibit advanced drift are repressed in EAC when methylation levels increase beyond a threshold of approximately 20%. These findings support the hypothesis that neoplasia, such as dysplastic BE and EAC, may develop in response to epigenetically driven selective pressure exerted on gene expression as methylation levels (on CpG islands associated with gene promoters) advance via random drift beyond a critical, repressive threshold.
Our results are consistent with the hypothesis that epigenetic drift heralds the onset of (epi)genomic instability via bifurcations (as seen in Fig. 5) that associate with the transcriptional repression of important regulatory genes [29–32]. Thus, under this hypothesis, epigenetic drift not only defines tissue aging (i.e., provides a molecular clock) but also “throttles” the expression and function of developmental genes forcing transitions in tissue characteristics that better cope with the erosive and damaging milieu in BE. Further studies of whether changes in methylomic drift simply reflect transcriptional changes during neoplastic progression or induce such changes are therefore of critical importance to better understand mechanisms that drive age-related cancer evolution.
Formalin fixed paraffin-embedded (FFPE) tissue slides and cores were obtained from Case Western Reserve University/University Hospitals of Cleveland (Cleveland, OH) and the Cleveland Clinic (CC) following protocols approved by the Institutional Review Board of each institution. For the cross-sectional analysis, we used HM450 methylation array data from 52 NS, 64 nondysplastic BE, and 24 EAC samples through the Barrett’s Esophagus Translational Research Network (BETRNet) . For the longitudinal drift analysis, we utilized 33 additional tissue samples from two studies with 10 patients each (CC and CW). Each patient had two biopsies separated by at least 3–4 years (40 samples total). Of these, seven samples were included in the cross-sectional analysis. See Additional file 2: Table S4 for relevant clinical information on the patient samples used in this study.
Tissue sample preparation and DNA extraction were performed as described previously . The quality of DNA extracted from FFPE samples were determined with Illumina HD FFPE QC assay (Illumina, San Diego, USA) following the manufactures’ instructions. Two hundred fifty nanograms of DNA samples that passed the QC assay were bisulfite converted using the EZ DNA Methylation Kit (Zymo Research, Irvine, USA). DNA restoration was performed using the Illumina HD FFPE Restoration Kit (Illumina, San Diego, USA) according to the manufacturer’s instructions. Intermediate DNA purifications were performed using the Zymo DNA Clean and Concentrator-5 Kit (Zymo Research, Irvine, USA). The BETRNet DNA samples were run on Illumina HumanMethylation450 BeadChip (HM450) arrays following the manufacturer’s instructions (Illumina Inc.) at the Fred Hutch Genomics Core facility. Data were then accessed as raw two-color channel intensities in idat format and pre-processed. Arrays were normalized using two functions implemented in the minfi (v1.18.6) R module, including an initial background intensity correction identical to the correction implemented in Illumina’s Genome Studio software, followed by subset quantile within-array normalization (SWAN) to harmonize data across assay design types [35, 36]. Probes showing mean detection p value > 0.05 were filtered out. Furthermore, we checked for the presence of previously identified cross-reactive CpGs in our drift CpG sets. Our drift CpG sets are uniformly under-enriched for cross-reactive probes, and we found that the presence of cross-reactive probes did not affect the integrity of our findings.
Methylation and gene expression datasets
Matched methylation (HM450 platform) and gene expression (Illumina HumanHT-12V4.0 expression BeadChip platform) data collected for 4 BE and 47 EAC, and 17 normal esophagus tissue samples published by  were accessed via the Gene Expression Omnibus (GEO) online repository (Series GSE72874). Methylation and expression BeadChip array data were obtained as normalized and filtered intensity counts or β values and prepared as described in .
Additional validation data, including HM450 array methylation and Illumina HiSeq 2000 RNA Sequencing data from the Version 2 analysis pipeline, were obtained for samples provided by the Cancer Genome Atlas (TCGA) via the NCI Genomic Data Commons  and the Firehose resource hosted by the Broad Institute (http://gdac.broadinstitute.org/). HM450 array data were obtained as raw two-color channel intensity readings, which were subjected to the same pre-processing pipeline as the BETRNet cohort data, described above. RNAseq expression sequencing data was obtained as level 3 RNAseq by expectation maximization (RSEM)-normalized and pre-processed intensity counts . A nonparametric Mann-Whitney-Wilcoxon (MWW) U test was applied to gene-specific count data to detect differential gene expression between low methylation samples (β < 0.2) and advanced methylation samples (β ≥ 0.2).
Quantification of drift
The methylation state of a CpG dinucleotide on a specific chromosome is essentially a binary variable; the cytosine is either methylated or unmethylated. However, DNA methylation arrays (such as the Illumina HM450 beadchip) provide only aggregate measurement across thousands of cellular epigenomes in a given tissue sample and therefore can only provide population fractions (i.e., β values) of methylated probes expressed as the ratio β = M/(M + U), with M and U representing the number of methylated and unmethylated probes in the sample, respectively.
Statistical analysis and visualization
All data pre-processing and the majority of statistical testing was performed in R programming language with base R graphics and analysis functions (v3.3.0). The minfi (v1.18.6) and GEOquery (v2.38.4) Bioconductor modules were used to access, pre-process, normalize, and analyze both methylation and gene expression array data, respectively [35, 39, 40].
Data prepared for this study are available online at the GEO website (Series Number: GSE104707). Scripts for study analyses and visualizations are available at https://github.com/gluebeck/Scope-of-methylomic-drift-in-BE .
We thank Prof Trevor Graham (Barts Cancer Institute, London, UK) and Dr. Hamid Bolouri (Fred Hutch) for their helpful comments. This study was supported NIH grant U01CA182940 (EGL, WDH, WMG, SKM, KC); NIH grants P50CA150964, U54CA163060 and P30CA43703 (WMG, JEW, AC); NIH grants UO1CA152756, P30CA015704, and U01CA086402 (SKM, YM, WMG); the DeGregorio Family and Price Family Foundation (WMG, EGL); and Barts Charity, London (KC).
PNT, DTP, AC, JEW, and WMG contributed the biospecimens and materials for this study, DNA processing and HM450 arrays were performd by MY and WMG, bioinformatic analyses were performed by SKM, WDH and EGL. KC and EGL conceived this study and collaborated with WDH and WMG in the analyses of the results. All authors read and approved the final manuscript.
The authors declare that they have no competing interest.
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