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Two distinct classes of 5hmC enrichment profiles are observed at active genes in normal
human colon

We first set out to identify genes marked by 5hmC in colon by hmeDIP-seq in order
to ultimately follow their methylation fate in cancer. Initial hmeDIP-seq on five
DNA samples from normal mucosa of affected patients showed 5hmC enrichment at promoters,
absent at the transcription start site (TSS), abundant within the body of genes and
underrepresented within intergenic regions (Figure 1a and b).

Figure 1. 5hmC promoter profiles and their association with active genes in normal colon. (a) hmeDIP-seq profile for all genes around the TSS in normal colon tissue (n?=?5). (b) Quantification of 5hmC enrichments in genomic features. (c) Two distinct promoter profiles were identified. Left panel: high 5hmC within a promoter
window (-1 kb to +0.5 kb) with a ‘narrow’ promoter profile. Right panel: high 5hmC
within gene bodies (from the TSS to the TTS) with a ‘broad’ promoter profile. Below
are examples of each type of profile. (d) 5hmC and CpG content in the promoter. High, intermediate and low CpG content (HCP,
ICP and LCP, respectively). Inset numbers represent the number of promoters for each
category (LCP numbers not shown). (e) 5hmC content at promoter CpG islands. The levels represent an average of the population
for each promoter type, thus individual loci may not necessarily display the full
profile. Additional file 2 shows further examples. (f) Expression levels (log2 microarray intensity) of genes associated with 5hmC promoter
profiles (P values were obtained by a Wilcox test).

From the profiling of 5hmC content across genes we identified two types of enrichments
at gene promoters (Figure 1c). A ‘narrow’ type was observed after ranking 5hmC read content inside a window of
-1 kb to +0.5 kb of the TSS and a ‘broad’ type after ranking by 5hmC read content
in the gene body (from TSS to the TTS). We identified 2,156 unique ‘narrow’ and 2,199
unique ‘broad’ promoters (listed in Additional file 1).

The ‘narrow’ and ‘broad’ profiles were distinct in terms of promoter CpG content (Figure 1d) and in distribution of 5hmC around promoter CpG islands (Figure 1e). Promoters with the ‘narrow’ profile were enriched for intermediate CpG content
promoters (ICP) whereas the ‘broad’ promoters where mostly high in CpG content (Figure 1d). Both promoter types showed that 5hmC is enriched within the shores of promoter
CpG islands, more so within the upstream shore, and a higher overall content of 5hmC
for the ‘narrow’ type (Figure 1e). Note, however, that the enrichment of 5hmC in the downstream shore of ACTN2 is lower than that for AGAP1. The levels measured over the islands represent an average of the population for
each type of promoter, and thus individual loci may not necessarily display the full
enrichment profile across the associated promoter CpG island. Additional file 2 shows further examples to illustrate this. Interestingly, comparison of the 5hmC
profiles with Illumina expression array data from four normal cases showed that ‘narrow’
promoter genes are less active than the ‘broad’ type (Figure 1f), in accordance with previous correlations made for higher 5hmC content at promoters
and reduced gene activity in mouse and human ES cells 41],42]. Biological processes also typified the 5hmC promoters; gene ontology categories
indicative of gut function were enriched for the ‘narrow’ type whereas cell differentiation
and development where enriched for the ‘broad’ type (Additional file 2).

Together these data show that the content and distribution of 5hmC within promoters
and gene bodies correlates with gene activities involved in normal gut epithelial
function and differentiation.

5hmC enrichment is similar to 5mC enrichment at genic regions

Next we examined DNA methylation content with respect to the 5hmC profiles by comparing
our hmeDIP-seq data to published meDIP-seq data for normal colon tissue 43] (Additional file 3). We generated heatmaps for 5hmC and 5mC enrichment profiles from -3 kb to +20 kb
around the TSS (Additional file 3a). Ten clusters were generated based on the distribution of 5hmC and 5mC within this
window. Overall we found that where 5hmC-specific enrichment is observed, the enrichment
profiles are similar for 5mC (Additional file 3a). The exception was cluster 2 where there was more DNA methylation near the TSS
than 5 hmC. Further comparison of 5hmC and 5mC profiles closer to the TSS (-3 kb to
+3 kb) of all loci suggest that the differences in enrichment patterns for 5hmC and
5mC occur near the TSS and upstream promoter region (Additional file 3b). This suggests that several gene promoters may have DNA methylation without 5 hmC.

The heatmaps also identified the ‘narrow’ promoters as typified by clusters 3 and
8 whereas the ‘broad’ promoters fell within clusters 5, 6, 7 and 9 (Additional file
3c). With the exception of clusters 2 and 3 that showed an enrichment for LCP promoters,
most of the 5hmC/5mC clusters fell with promoters of an intermediate or high CpG content
(Additional file 3e).

We then compared the meDIP-seq methylation clusters to the methylation levels assessed
by the Infinium27k arrays in 17 normal samples from our patient cohort (Additional
file 3e). For the loci plotted in the heatmap the maximal distance of the Infinium probes
to the TSS is 1499 bp. The highest methylation levels for these probes were around
the promoters grouped within clusters 1 and 2, which correspond to the meDIP-seq data
where the highest methylation enrichment was observed (Additional file 3a and e). Similarly clusters 4 to 9 which all reported low amounts of DNA methylation
around the TSS by meDIP-seq also had lower levels of DNA methylation at the corresponding
Infinium probes (Additional file 3a and e).

Thus in our normal colon tissues, the Infinium arrays concur with meDIP-seq enrichment
patterns proximal to the TSS of genes.

Reduced levels of 5hmC in colon tumours do not correlate with changes in TET transcript
levels

Having established profiles for 5hmC and 5mC in normal colon we next analysed their
behaviour in neoplasia. Our colon cancer cohort is composed of 47 normal tissues,
36 adenomas and 31 adenocarcinomas (Additional file 1). We confirmed that 5hmC and 5mC are globally reduced during colon cancer progression
using liquid chromatography mass spectrometry (LCMS) and immunofluorescence (IF) (Figure 2a and b). The IF also shows that 5hmC is concentrated in the differentiated colon
epithelium and is low in the base of the crypts and tumours consistent with previous
reports 21]. Importantly, we observed TET1, TET2 and TET3 were consistently transcribed in normal and tumour tissue and that the absolute levels
of TET1 were low relative to TET2 and TET3 by Sybr-Green qRT-PCR (Figure 2c). Further analysis of TET expression in normal-tumour matched cases by Taqman qRT-PCR showed no correlation
with the changes in global levels of 5hmC (Additional file 4). Moreover, mining of recently published data sets 31],44] indicates that TETs are present in normal crypt and differentiated epithelium and tumours.

Figure 2. Reduced 5hmC in tumours without global changes inTETs transcripts. (a) Global content of 5hmC and 5mC in normal (N), adenoma (Ad) and adenocarcinoma (T)
DNA by mass spectrometry (P values were obtained by a Wilcox test). (b) Representative images from a colon cancer tissue microarray immunofluorescence. Arrows
indicate the epithelium, arrowheads the stroma. (c) Absolute levels of TETs (standard curve method) in selected cases from our colon cancer cohort. Orange vertical
bands represent the median. Negative values indicate TETs transcripts are less abundant than B2M transcripts. There was no significant change in levels across tissues but considerable
variation within tissues.

Mutation at the Fe2 and a-KG binding pockets could account for a lack of TET activity
30] but these were specifically excluded in our sample set through targeted exonic sequencing
(Additional file 5a and Additional methods). We identified non-synonymous mutations elsewhere in the
catalytic domains of TETs but their presence did not correlate with the changes in global 5hmC levels (Additional
file 5b). Reduction of 5hmC in tumours may also be due to inhibition of TETs by metabolites
that accumulate through mutation of IDH1/2, Fumarate hydratase (FH) or Succinate dehydrogenase
(SDH) 39],45]. In our study IDH1/2 mutations were excluded in a subset of samples (not shown) and
recent larger studies have shown IDH1/2, FH or SDH mutation is rare or absent in colon
cancer 31],40].

We do not have TET protein data associated with our sample set and therefore we cannot
exclude that the global reduction in 5hmC could be due to post-transcriptional events
with an impact on variations in the stability or activity of TETs. However, the detection
of mRNA at levels similar to the normal tissues suggests that the reduced levels of
5hmC that we uncover in all our colon tumours is unlikely to be due to an absence
or mutation of TETs or an inhibition by currently recognised onco-metabolites.

5hmC is reduced across the genome of tumours with a small effect on gene transcription

We profiled 5hmC in four matching adenocarcinomas. The hmeDIP-seq read content in
tumours showed an overall similar distribution to the normal tissue but with markedly
reduced 5hmC levels across the genome as assessed by 5hmC content within repetitive
elements (Additional file 6) and within genes (Additional file 7a and b). The reduced level of 5hmC in tumours compared to normal was confirmed at
selected loci by a glycosylase-restriction enzyme sensitive assay (gluc-MS-qPCR –
Additional file 7c) indicating that genes continue to be marked by a reduced amount of 5hmC in tumours.

Illumina expression array data generated from four normals and 14 tumours showed a
small but statistically significant reduction in gene activity for genes with ‘broad’
5hmC promoters (Additional file 7d). Thus, although 5hmC associates with active gene transcription, the reduction of
5hmC in tumours were accompanied by very small expression level changes. These results
indicate that genes that acquire 5hmC in normal colon are transcriptionally active
in tumours and suggest that low levels of 5hmC do not hinder transcription.

Loci marked by 5hmC in normal have an innate resistance to DNA hypermethylation in
cancer

To ascertain whether promoters normally marked by 5hmC undergo DNA methylation changes
in colon cancer, we assessed DNA methylation in 17 tumours matched to the normal tissues
using Infinium methylation arrays. The Infinium27k arrays are a robust platform for
quantitative measurement of the DNA methylation status of 27,578 CpG sites located
at the promoter regions of 14,495 protein-coding genes 43],46]. Infinium technology is based on bisulfite conversion that does not distinguish between
5mC and 5hmC. However, 5hmC only makes up a small percentage of modified cytosines
in normal colon and an even smaller percentage in colon cancer tissue. Based on the
median levels of 5hmC detected by LCMS (Figure 2), only about 2.4% of 5mC reported in the Infinium data is likely to be undistinguishable
from 5hmC in normal cells, and about 0.7% in tumours.

Methylation changes in our patient cohort showed both gain and loss of promoter DNA
methylation (Figure 3a). To refine our analysis of 5hmC content to changes in DNA methylation at the promoters
assessed by the Infinium platform, we counted the hmeDIP-seq reads from normals in
200 bp windows around the Infinium probes (Figure 3b). After ranking by read content we identified the top 3,000 5hmC enriched loci (5hmC-high)
as well as 3,000 loci where 5hmC was low or undetected (5hmC-low). Interestingly,
by this measurement of read counts around the Infinium probes, we observed that promoters
with high 5hmC in normal are either resistant to methylation change or are prone to
methylation loss (79% loss vs. 21% gain from 676 probes with significant change out
of 3,000) and that 5hmC marked promoters more frequently associate with a range of
intermediate levels of methylation in normal (Figure 3c left panel and d). 5hmC low promoters more frequently associated with low levels
of methylation in normal and showed an increased propensity to methylation gain, albeit
methylation loss was also observed (56% gain vs. 44% loss from 379 probes with significant
change out of 3,000) (Figure 3c right panel and d). We also find that the methylation-prone genes that lack 5hmC
in normal have a low level of expression in the normal tissue (Figure 3d, right panel), in agreement with a recent report where propensity to methylation
gain in tumours is frequent at promoters of genes with low expression in the normal
tissue 47].

Figure 3. Promoters marked by 5hmC in normal colon resist DNA methylation gain in tumours. (a) DNA methylation changes in adenocarcinoma (n?=?17) relative to matched normal tissues
(n?=?17) (Infinium arrays). Each dot represents a CpG (grey dots are changes with
P 0.01). (b) 5hmC read content measured in windows around the Infinium probes (black bars). CpG
island (CpGi) as orange bar. (c) Overlay of 5hmC high or 5hmC low promoters on the methylation states. (d) Left panel: 5hmC content around the Infinium probes of promoters with a significant
change in methylation. High 5hmC promoters are prone to loss of DNA methylation in
tumours whereas low 5hmC promoters are prone to methylation gain in tumours (limma
geneSetTest). Middle panel: 5hmC content in normal and levels of DNA methylation in
normal to show that methylation gain or loss occurs across a range of methylation
levels in normal (P values from a Wilcox test). Right panel: 5hmC content in normal and expression levels
in normal. DNA methylation prone genes (5hmC low) have low expression in the normal
tissue (P values from a Wilcox test). (e) Heatmap comparing 5hmC and 5mC levels in normal to the 5mC changes in tumours at
selected loci.

Importantly, the reciprocal pattern of high/low 5hmC in normal with loss/gain of methylation
in adenocarcinoma was already present at the adenoma stages (Additional file 8a and b) and observed at CpG islands and island shores (Additional file 8c). This reciprocal pattern was also present at previously identified colon cancer-specific
small regions of differential DNA methylation (sDMRs) 8] (Additional file 8d) and clearly observed and verified in a number of colon cancer relevant gene promoters
(Figure 3e and Additional file 9).

Together these results indicate that gene promoters marked with 5hmC in normal rarely
become hypermethylated when 5hmC is reduced in tumours. Indeed these promoters have
a tendency to lose DNA methylation in cancer. We also identified 117 promoters where
5hmC was still detected in adenocarcinomas, albeit at very low levels, and found that
these where three times more likely to have lost methylation rather than gain (27%
vs. 8.5%, respectively) (Additional file 10). These results may suggest that DNA demethylation at a subset of proximal promoters
could be mediated via hydroxymethylation and/or that the presence of 5hmC helps to
repel DNA methylating complexes as previously suggested 48],49].

There is strong evidence from cell labelling experiments that colon cancer can originate
from the stem cell/progenitor compartment 50]. Our data, and that of others 21], showing that global 5hmC levels are low in the stem cell compartment and in cancer
tissues may suggest that 5hmC is not lost in colon cancer. Rather, 5hmC may not accumulate
due to an aberrant progenitor-like proliferative state. One explanation for why the
loci that would accumulate 5hmC upon terminal differentiation are seemingly more resistant
to gain of DNA methylation in cancer, in contrast with loci that do not accumulate
5hmC, could be that the TETs in cancer cells are bound to their target promoters to
prevent de novo DNA methylation.

TET2 marks promoters in cancer cells that resist DNA methylation gain in primary tumours
but is not required to maintain a demethylated state

In order to examine whether TETs are bound to DNA in cancer cells we turned to the
colorectal cancer cell line HCT116. This cell line shows low global levels of 5hmC
and TET2 and TET3 transcript levels comparable to that observed in normal and adenocarcinoma tissue
(Additional file 11a to c). Despite the extremely low global content of 5hmC in these cells, lower than
that seen in the primary tissues, TET2 and TET3 proteins can be detected in the nuclear
fraction (Additional file 11d) albeit a sizeable amount of TET2 is present in the cytoplasm (Additional file 11d and e). A similar subcellular distribution of TET2 is observed in normal colon crypts
and tumours by immunohistochemistry (Additional file 12).

Chromatin immunoprecipitation sequencing (ChIP-seq) revealed that TET2 preferentially
binds to gene promoters within 1 kb of the TSS (Figure 4a and b). Overall 3,144 promoters were identified as TET2 targets (Additional file
1) of which the large majority were CpG island-containing promoters of the HCP type
(Figure 4c and d). CpG islands bound by TET2 were largely unmethylated as measured by Infinium450k
arrays (from GSE29290) and CpG island shores showed lower methylation levels at the
TET2 bound sites relative to those not bound by TET2 (Figure 4e). We validated a number of loci identified in the TET2 ChIP-seq by ChIP-qPCR (Additional
file 13). Interestingly, presence of TET2 associated with active genes measured by expression
arrays (GSE36133) or evidenced by a considerable overlap with RNA Pol2 binding sites
(ENCODE Pol2 ChIP-seq) (Figure 4f and g).

Figure 4. TET2 binds promoters of active genes in cancer cells. (a) Example of TET2 binding profile in HCT116 colorectal cancer cells. (b) TET2 binds close to TSSs and (c, d) primarily at CpG islands within HCP promoters. (e) TET2 bound islands are largely unmethylated and (f, g) associate with active genes.

If the TETs bind to DNA and protect against hypermethylation in tumours, then it would
be expected that promoters susceptible to DNA methylation gain in colon tumours would
form a distinct group with a minimal overlap with TET target promoters. We therefore
examined whether loci that gained DNA methylation in our primary tumours (1,597 probes
for 1,077 promoters) were likely TET2 target promoters (4,201 probes for 3,144 promoters).
This analysis showed less than 1% overlap between loci that gain DNA methylation in
tumours and the TET2 bound promoters (Figure 5a). These results could suggest that TET2 might be part of a mechanism that protects
promoters from de novo DNA methylation. To examine this we depleted TET2 in HCT116 cells by stable transfection
of shRNAs (Figure 5b and c). In one instance we used shRNA against TET2 alone (TET2C) and in the other
shRNA against TET2 and TET3 (TET2?+?3 where TET3 mRNA was not affected and therefore
treat this sample as a TET2 only knockdown) (Figure 5c). LCMS after TET2 depletion showed a marked reduction in the global level of 5hmC
(Figure 5d), confirming TET2 oxygenase activity in HCT116, without changes in global levels
of 5mC (Figure 5d) but this could be due to the small contribution of promoter methylation to the
methylome. Infinium arrays identified several loci with changes in DNA methylation
(Figure 5e) that were for the most part low in magnitude (median of change was 10.4%; not shown).
Similar changes in levels of DNA methylation were recently observed after TET1 depletion
in differentiated cells 51]. However in our study, methylation levels at TET2 bound CpG islands were largely
unaffected after TET2 depletion (less than 1%, Figure 5e), suggesting that these promoters do not require high levels of TET2 to maintain
the methylation free state and are intrinsically resistant to methylation changes.

Figure 5. Pervasive maintenance of a methylation-free state at TET2 bound promoters. (a) DNA methylation gain in primary tumours was remarkably scarce at the TET2 bound promoters
identified in HCT116 cells (P 0.0001, binomial test). (b) Western blot for TET2 and beta TUBULIN from whole cell extracts of HCT116 cells stably
transfected with a non-targeting shRNA control (shCtrl.) or with shRNA to TET2 (TET2C)
or to TET2 and TET3 (TET2?+?3). Fold change in the knockdown was calculated relative
to the shCtrl. (c) qRT-PCR for TET2 and TET3. (d) Global levels of 5hmC and 5mC by LCMS. (e) DNA methylation changes by Infinium arrays after depletion of TET2.

Survival outcomes estimated from publicly available colorectal cancer datasets 52],53] further indicate that TET2 expression levels do not significantly associate with patient survival, which is
consistent with the small effect that we see in these in vitro TET2 studies. TET2 therefore seems to play a moderate role in controlling cytosine
modifications during gut tumourigenesis.

Promoters with high levels of 5hmC in normal colon overlap with bivalently marked
promoters in human embryonic stem cells that do not become methylated in colon cancer

If tumours arise from intestinal cells in the crypt and if 5hmC is a mark of terminally
differentiated cells, then how do we explain the resistance of 5hmC promoters to methylation
gain in tumours prior to their accumulating 5hmC in normal tissue? TET2 depletion
only has a moderate effect on DNA methylation in cancer cells, suggesting that the
protective mechanism is unlikely to be due to continuous TET2 binding at target promoters.
Although TET2 may not be involved in maintaining the unmethylated state of its target
promoters, we cannot exclude that other proteins within a TET-complex may be involved.
However there may be alternative explanations, one of which is that 5hmC promoters
are epigenetically marked during early development to make them intrinsically unlikely
to develop characteristics such as H3K27me3 in the soma that predispose to DNA methylation
gain.

Precedents for early epigenetic marking include genomic imprinting and X-inactivation,
but may also include the recently described instructive process for gain of methylation
in cancer which occurs at promoters containing histone H3K4 and H3K27 tri-methylation
(so-called bivalent promoters) in human embryonic stem cells (hESC) 54]-58]. ESCs unlike most other proliferating cells already have high levels of 5hmC. In
mouse ESCs Tet1 is found either at the TSS of bivalent promoters together with silencing
complexes independent of 5hmC or downstream of the TSS together with 5hmC and the
PRC2 complex 59],60]. In human ESC 5hmC has been found more at active gene promoters and enhancers than
at poised (bivalent) enhancers 61].

A comparison of our dataset of 5hmC marked promoters to a published dataset of hESC
bivalent promoters 57] confirmed that approximately 65% of promoters that gain methylation in our colon
cancer cohort are also bivalently marked in hESC (Figure 6a and b). Consequently we also examined the extent to which promoters marked by 5hmC
in normal colon overlap with bivalently marked promoters in hESCs. We found that 30%
of all 5hmC promoters overlapped with bivalent genes in hESCs (Figure 6a and b). Interestingly, these mostly coincided with bivalent promoters that do not
become hypermethylated in colorectal cancer. This observation indicates that bivalent
promoters can be broadly separated into discrete instructive categories: one for silencing
after tissue differentiation and susceptible to methylation gain in cancer; and another
for poised activation and acquisition of 5hmC with resistance to methylation gain
in cancer. If 5hmC is acquired as an end point of instructive activation, this would
fit with our data where we see 5hmC accumulating in terminally differentiated cells
at genes that are active in both cancer and normal tissue.

Figure 6. 5hmC marked promoters are not subject to histone-bivalency-mediated methylation gain. Venn diagrams to illustrate a high incidence of promoter methylation gain in our
cohort at promoters with H3K4me3/K27me3 bivalency in human embryonic stem cells (hESCbiv).
The incidence of methylation gain is low at hESCbiv promoters marked by 5hmC in normal
colon. (a) For narrow and (b) broad 5hmC promoters.