We first set out to identify genes marked by 5hmC in colon by hmeDIP-seq in order
\n to ultimately follow their methylation fate in cancer. Initial hmeDIP-seq on five
\n DNA samples from normal mucosa of affected patients showed 5hmC enrichment at promoters,
\n absent at the transcription start site (TSS), abundant within the body of genes and
\n underrepresented within intergenic regions (Figure\u00c2\u00a01a and b).<\/p>\n
Figure 1.<\/strong> 5hmC promoter profiles and their association with active genes in normal colon. (a)<\/strong> hmeDIP-seq profile for all genes around the TSS in normal colon tissue (n?=?5). (b)<\/strong> Quantification of 5hmC enrichments in genomic features. (c)<\/strong> Two distinct promoter profiles were identified. Left panel: high 5hmC within a promoter From the profiling of 5hmC content across genes we identified two types of enrichments The \u00e2\u20ac\u02dcnarrow\u00e2\u20ac\u2122 and \u00e2\u20ac\u02dcbroad\u00e2\u20ac\u2122 profiles were distinct in terms of promoter CpG content (Figure\u00c2\u00a01d) and in distribution of 5hmC around promoter CpG islands (Figure\u00c2\u00a01e). Promoters with the \u00e2\u20ac\u02dcnarrow\u00e2\u20ac\u2122 profile were enriched for intermediate CpG content Together these data show that the content and distribution of 5hmC within promoters Next we examined DNA methylation content with respect to the 5hmC profiles by comparing The heatmaps also identified the \u00e2\u20ac\u02dcnarrow\u00e2\u20ac\u2122 promoters as typified by clusters 3 and We then compared the meDIP-seq methylation clusters to the methylation levels assessed Thus in our normal colon tissues, the Infinium arrays concur with meDIP-seq enrichment Having established profiles for 5hmC and 5mC in normal colon we next analysed their Figure 2.<\/strong> Reduced 5hmC in tumours without global changes in<\/strong>TET<\/em><\/strong>s transcripts. (a)<\/strong> Global content of 5hmC and 5mC in normal (N), adenoma (Ad) and adenocarcinoma (T) Mutation at the Fe2 and a-KG binding pockets could account for a lack of TET activity We do not have TET protein data associated with our sample set and therefore we cannot We profiled 5hmC in four matching adenocarcinomas. The hmeDIP-seq read content in Illumina expression array data generated from four normals and 14 tumours showed a To ascertain whether promoters normally marked by 5hmC undergo DNA methylation changes Methylation changes in our patient cohort showed both gain and loss of promoter DNA Figure 3.<\/strong> Promoters marked by 5hmC in normal colon resist DNA methylation gain in tumours. (a)<\/strong> DNA methylation changes in adenocarcinoma (n?=?17) relative to matched normal tissues Importantly, the reciprocal pattern of high\/low 5hmC in normal with loss\/gain of methylation Together these results indicate that gene promoters marked with 5hmC in normal rarely There is strong evidence from cell labelling experiments that colon cancer can originate In order to examine whether TETs are bound to DNA in cancer cells we turned to the Chromatin immunoprecipitation sequencing (ChIP-seq) revealed that TET2 preferentially Figure 4.<\/strong> TET2 binds promoters of active genes in cancer cells. (a)<\/strong> Example of TET2 binding profile in HCT116 colorectal cancer cells. (b)<\/strong> TET2 binds close to TSSs and (c, d)<\/strong> primarily at CpG islands within HCP promoters. (e)<\/strong> TET2 bound islands are largely unmethylated and (f, g)<\/strong> associate with active genes.\n <\/p>\n If the TETs bind to DNA and protect against hypermethylation in tumours, then it would Figure 5.<\/strong> Pervasive maintenance of a methylation-free state at TET2 bound promoters. (a)<\/strong> DNA methylation gain in primary tumours was remarkably scarce at the TET2 bound promoters Survival outcomes estimated from publicly available colorectal cancer datasets 52],53] further indicate that TET2<\/em> expression levels do not significantly associate with patient survival, which is If tumours arise from intestinal cells in the crypt and if 5hmC is a mark of terminally Precedents for early epigenetic marking include genomic imprinting and X-inactivation, A comparison of our dataset of 5hmC marked promoters to a published dataset of hESC Figure 6.<\/strong> 5hmC marked promoters are not subject to histone-bivalency-mediated methylation gain.<\/strong> Venn diagrams to illustrate a high incidence of promoter methylation gain in our 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 […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[],"tags":[],"class_list":["post-8297","post","type-post","status-publish","format-standard","hentry"],"_links":{"self":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/8297","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/comments?post=8297"}],"version-history":[{"count":0,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/8297\/revisions"}],"wp:attachment":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/media?parent=8297"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/categories?post=8297"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/tags?post=8297"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
\n window (-1\u00c2\u00a0kb to +0.5\u00c2\u00a0kb) with a \u00e2\u20ac\u02dcnarrow\u00e2\u20ac\u2122 promoter profile. Right panel: high 5hmC
\n within gene bodies (from the TSS to the TTS) with a \u00e2\u20ac\u02dcbroad\u00e2\u20ac\u2122 promoter profile. Below
\n are examples of each type of profile. (d)<\/strong> 5hmC and CpG content in the promoter. High, intermediate and low CpG content (HCP,
\n ICP and LCP, respectively). Inset numbers represent the number of promoters for each
\n category (LCP numbers not shown). (e)<\/strong> 5hmC content at promoter CpG islands. The levels represent an average of the population
\n for each promoter type, thus individual loci may not necessarily display the full
\n profile. Additional file 2 shows further examples. (f)<\/strong> Expression levels (log2 microarray intensity) of genes associated with 5hmC promoter
\n profiles (P<\/em> values were obtained by a Wilcox test).\n <\/p>\n
\n at gene promoters (Figure\u00c2\u00a01c). A \u00e2\u20ac\u02dcnarrow\u00e2\u20ac\u2122 type was observed after ranking 5hmC read content inside a window of
\n -1\u00c2\u00a0kb to +0.5\u00c2\u00a0kb of the TSS and a \u00e2\u20ac\u02dcbroad\u00e2\u20ac\u2122 type after ranking by 5hmC read content
\n in the gene body (from TSS to the TTS). We identified 2,156 unique \u00e2\u20ac\u02dcnarrow\u00e2\u20ac\u2122 and 2,199
\n unique \u00e2\u20ac\u02dcbroad\u00e2\u20ac\u2122 promoters (listed in Additional file 1).\n <\/p>\n
\n promoters (ICP) whereas the \u00e2\u20ac\u02dcbroad\u00e2\u20ac\u2122 promoters where mostly high in CpG content (Figure\u00c2\u00a01d). Both promoter types showed that 5hmC is enriched within the shores of promoter
\n CpG islands, more so within the upstream shore, and a higher overall content of 5hmC
\n for the \u00e2\u20ac\u02dcnarrow\u00e2\u20ac\u2122 type (Figure\u00c2\u00a01e). Note, however, that the enrichment of 5hmC in the downstream shore of ACTN2<\/em> is lower than that for AGAP1<\/em>. The levels measured over the islands represent an average of the population for
\n each type of promoter, and thus individual loci may not necessarily display the full
\n enrichment profile across the associated promoter CpG island. Additional file 2 shows further examples to illustrate this. Interestingly, comparison of the 5hmC
\n profiles with Illumina expression array data from four normal cases showed that \u00e2\u20ac\u02dcnarrow\u00e2\u20ac\u2122
\n promoter genes are less active than the \u00e2\u20ac\u02dcbroad\u00e2\u20ac\u2122 type (Figure\u00c2\u00a01f), in accordance with previous correlations made for higher 5hmC content at promoters
\n and reduced gene activity in mouse and human ES cells 41],42]. Biological processes also typified the 5hmC promoters; gene ontology categories
\n indicative of gut function were enriched for the \u00e2\u20ac\u02dcnarrow\u00e2\u20ac\u2122 type whereas cell differentiation
\n and development where enriched for the \u00e2\u20ac\u02dcbroad\u00e2\u20ac\u2122 type (Additional file 2).\n <\/p>\n
\n and gene bodies correlates with gene activities involved in normal gut epithelial
\n function and differentiation.\n <\/p>\n5hmC enrichment is similar to 5mC enrichment at genic regions<\/h4>\n
\n 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\u00c2\u00a0kb to +20\u00c2\u00a0kb
\n around the TSS (Additional file 3a). Ten clusters were generated based on the distribution of 5hmC and 5mC within this
\n window. Overall we found that where 5hmC-specific enrichment is observed, the enrichment
\n profiles are similar for 5mC (Additional file 3a). The exception was cluster 2 where there was more DNA methylation near the TSS
\n than 5 hmC. Further comparison of 5hmC and 5mC profiles closer to the TSS (-3\u00c2\u00a0kb to
\n +3\u00c2\u00a0kb) of all loci suggest that the differences in enrichment patterns for 5hmC and
\n 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.\n <\/p>\n
\n 8 whereas the \u00e2\u20ac\u02dcbroad\u00e2\u20ac\u2122 promoters fell within clusters 5, 6, 7 and 9 (Additional file
\n 3c). With the exception of clusters 2 and 3 that showed an enrichment for LCP promoters,
\n most of the 5hmC\/5mC clusters fell with promoters of an intermediate or high CpG content
\n (Additional file 3e).\n <\/p>\n
\n by the Infinium27k arrays in 17 normal samples from our patient cohort (Additional
\n file 3e). For the loci plotted in the heatmap the maximal distance of the Infinium probes
\n to the TSS is 1499\u00c2\u00a0bp. The highest methylation levels for these probes were around
\n the promoters grouped within clusters 1 and 2, which correspond to the meDIP-seq data
\n 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
\n around the TSS by meDIP-seq also had lower levels of DNA methylation at the corresponding
\n Infinium probes (Additional file 3a and e).\n <\/p>\n
\n patterns proximal to the TSS of genes.\n <\/p>\nReduced levels of 5hmC in colon tumours do not correlate with changes in TET transcript
\n levels
<\/h4>\n
\n behaviour in neoplasia. Our colon cancer cohort is composed of 47 normal tissues,
\n 36 adenomas and 31 adenocarcinomas (Additional file 1). We confirmed that 5hmC and 5mC are globally reduced during colon cancer progression
\n using liquid chromatography mass spectrometry (LCMS) and immunofluorescence (IF) (Figure\u00c2\u00a02a and b). The IF also shows that 5hmC is concentrated in the differentiated colon
\n epithelium and is low in the base of the crypts and tumours consistent with previous
\n reports 21]. Importantly, we observed TET1<\/em>, TET2<\/em> and TET3<\/em> were consistently transcribed in normal and tumour tissue and that the absolute levels
\n of TET1<\/em> were low relative to TET2<\/em> and TET3<\/em> by Sybr-Green qRT-PCR (Figure\u00c2\u00a02c). Further analysis of TET<\/em> expression in normal-tumour matched cases by Taqman qRT-PCR showed no correlation
\n with the changes in global levels of 5hmC (Additional file 4). Moreover, mining of recently published data sets 31],44] indicates that TET<\/em>s are present in normal crypt and differentiated epithelium and tumours.<\/p>\n
\n DNA by mass spectrometry (P<\/em> values were obtained by a Wilcox test). (b)<\/strong> Representative images from a colon cancer tissue microarray immunofluorescence. Arrows
\n indicate the epithelium, arrowheads the stroma. (c)<\/strong> Absolute levels of TET<\/em>s (standard curve method) in selected cases from our colon cancer cohort. Orange vertical
\n bands represent the median. Negative values indicate TET<\/em>s transcripts are less abundant than B2M<\/em> transcripts. There was no significant change in levels across tissues but considerable
\n variation within tissues.\n <\/p>\n
\n 30] but these were specifically excluded in our sample set through targeted exonic sequencing
\n (Additional file 5a and Additional methods). We identified non-synonymous mutations elsewhere in the
\n catalytic domains of TET<\/em>s but their presence did not correlate with the changes in global 5hmC levels (Additional
\n file 5b). Reduction of 5hmC in tumours may also be due to inhibition of TETs by metabolites
\n that accumulate through mutation of IDH1\/2, Fumarate hydratase (FH) or Succinate dehydrogenase
\n (SDH) 39],45]. In our study IDH1\/2 mutations were excluded in a subset of samples (not shown) and
\n recent larger studies have shown IDH1\/2, FH or SDH mutation is rare or absent in colon
\n cancer 31],40].\n <\/p>\n
\n exclude that the global reduction in 5hmC could be due to post-transcriptional events
\n with an impact on variations in the stability or activity of TETs. However, the detection
\n of mRNA at levels similar to the normal tissues suggests that the reduced levels of
\n 5hmC that we uncover in all our colon tumours is unlikely to be due to an absence
\n or mutation of TETs or an inhibition by currently recognised onco-metabolites.\n <\/p>\n5hmC is reduced across the genome of tumours with a small effect on gene transcription<\/h4>\n
\n tumours showed an overall similar distribution to the normal tissue but with markedly
\n reduced 5hmC levels across the genome as assessed by 5hmC content within repetitive
\n 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
\n selected loci by a glycosylase-restriction enzyme sensitive assay (gluc-MS-qPCR \u00e2\u20ac\u201c
\n Additional file 7c) indicating that genes continue to be marked by a reduced amount of 5hmC in tumours.\n <\/p>\n
\n small but statistically significant reduction in gene activity for genes with \u00e2\u20ac\u02dcbroad\u00e2\u20ac\u2122
\n 5hmC promoters (Additional file 7d). Thus, although 5hmC associates with active gene transcription, the reduction of
\n 5hmC in tumours were accompanied by very small expression level changes. These results
\n indicate that genes that acquire 5hmC in normal colon are transcriptionally active
\n in tumours and suggest that low levels of 5hmC do not hinder transcription.\n <\/p>\nLoci marked by 5hmC in normal have an innate resistance to DNA hypermethylation in
\n cancer
<\/h4>\n
\n in colon cancer, we assessed DNA methylation in 17 tumours matched to the normal tissues
\n using Infinium methylation arrays. The Infinium27k arrays are a robust platform for
\n quantitative measurement of the DNA methylation status of 27,578 CpG sites located
\n at the promoter regions of 14,495 protein-coding genes 43],46]. Infinium technology is based on bisulfite conversion that does not distinguish between
\n 5mC and 5hmC. However, 5hmC only makes up a small percentage of modified cytosines
\n in normal colon and an even smaller percentage in colon cancer tissue. Based on the
\n median levels of 5hmC detected by LCMS (Figure\u00c2\u00a02), only about 2.4% of 5mC reported in the Infinium data is likely to be undistinguishable
\n from 5hmC in normal cells, and about 0.7% in tumours.\n <\/p>\n
\n methylation (Figure\u00c2\u00a03a). To refine our analysis of 5hmC content to changes in DNA methylation at the promoters
\n assessed by the Infinium platform, we counted the hmeDIP-seq reads from normals in
\n 200\u00c2\u00a0bp windows around the Infinium probes (Figure\u00c2\u00a03b). After ranking by read content we identified the top 3,000 5hmC enriched loci (5hmC-high)
\n as well as 3,000 loci where 5hmC was low or undetected (5hmC-low). Interestingly,
\n by this measurement of read counts around the Infinium probes, we observed that promoters
\n with high 5hmC in normal are either resistant to methylation change or are prone to
\n methylation loss (79% loss vs. 21% gain from 676 probes with significant change out
\n of 3,000) and that 5hmC marked promoters more frequently associate with a range of
\n intermediate levels of methylation in normal (Figure\u00c2\u00a03c left panel and d). 5hmC low promoters more frequently associated with low levels
\n of methylation in normal and showed an increased propensity to methylation gain, albeit
\n methylation loss was also observed (56% gain vs. 44% loss from 379 probes with significant
\n change out of 3,000) (Figure\u00c2\u00a03c right panel and d). We also find that the methylation-prone genes that lack 5hmC
\n in normal have a low level of expression in the normal tissue (Figure\u00c2\u00a03d, right panel), in agreement with a recent report where propensity to methylation
\n gain in tumours is frequent at promoters of genes with low expression in the normal
\n tissue 47].<\/p>\n
\n (n?=?17) (Infinium arrays). Each dot represents a CpG (grey dots are changes with
P<\/em> 0.01). (b)<\/strong> 5hmC read content measured in windows around the Infinium probes (black bars). CpG
\n island (CpGi) as orange bar. (c)<\/strong> Overlay of 5hmC high or 5hmC low promoters on the methylation states. (d)<\/strong> Left panel: 5hmC content around the Infinium probes of promoters with a significant
\n change in methylation. High 5hmC promoters are prone to loss of DNA methylation in
\n tumours whereas low 5hmC promoters are prone to methylation gain in tumours (limma
\n geneSetTest). Middle panel: 5hmC content in normal and levels of DNA methylation in
\n normal to show that methylation gain or loss occurs across a range of methylation
\n levels in normal (P<\/em> values from a Wilcox test). Right panel: 5hmC content in normal and expression levels
\n in normal. DNA methylation prone genes (5hmC low) have low expression in the normal
\n tissue (P<\/em> values from a Wilcox test). (e)<\/strong> Heatmap comparing 5hmC and 5mC levels in normal to the 5mC changes in tumours at
\n selected loci.\n <\/p>\n
\n 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
\n 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
\n (Figure\u00c2\u00a03e and Additional file 9).\n <\/p>\n
\n become hypermethylated when 5hmC is reduced in tumours. Indeed these promoters have
\n a tendency to lose DNA methylation in cancer. We also identified 117 promoters where
\n 5hmC was still detected in adenocarcinomas, albeit at very low levels, and found that
\n these where three times more likely to have lost methylation rather than gain (27%
\n vs. 8.5%, respectively) (Additional file 10). These results may suggest that DNA demethylation at a subset of proximal promoters
\n could be mediated via hydroxymethylation and\/or that the presence of 5hmC helps to
\n repel DNA methylating complexes as previously suggested 48],49].\n <\/p>\n
\n 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
\n tissues may suggest that 5hmC is not lost in colon cancer. Rather, 5hmC may not accumulate
\n due to an aberrant progenitor-like proliferative state. One explanation for why the
\n loci that would accumulate 5hmC upon terminal differentiation are seemingly more resistant
\n to gain of DNA methylation in cancer, in contrast with loci that do not accumulate
\n 5hmC, could be that the TETs in cancer cells are bound to their target promoters to
\n prevent de novo<\/em> DNA methylation.\n <\/p>\nTET2 marks promoters in cancer cells that resist DNA methylation gain in primary tumours
\n but is not required to maintain a demethylated state
<\/h4>\n
\n colorectal cancer cell line HCT116. This cell line shows low global levels of 5hmC
\n and TET2<\/em> and TET3<\/em> transcript levels comparable to that observed in normal and adenocarcinoma tissue
\n (Additional file 11a to c). Despite the extremely low global content of 5hmC in these cells, lower than
\n that seen in the primary tissues, TET2 and TET3 proteins can be detected in the nuclear
\n 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
\n and tumours by immunohistochemistry (Additional file 12).\n <\/p>\n
\n binds to gene promoters within 1\u00c2\u00a0kb of the TSS (Figure\u00c2\u00a04a and b). Overall 3,144 promoters were identified as TET2 targets (Additional file
\n 1) of which the large majority were CpG island-containing promoters of the HCP type
\n (Figure\u00c2\u00a04c and d). CpG islands bound by TET2 were largely unmethylated as measured by Infinium450k
\n arrays (from GSE29290) and CpG island shores showed lower methylation levels at the
\n TET2 bound sites relative to those not bound by TET2 (Figure\u00c2\u00a04e). We validated a number of loci identified in the TET2 ChIP-seq by ChIP-qPCR (Additional
\n file 13). Interestingly, presence of TET2 associated with active genes measured by expression
\n arrays (GSE36133) or evidenced by a considerable overlap with RNA Pol2 binding sites
\n (ENCODE Pol2 ChIP-seq) (Figure\u00c2\u00a04f and g).<\/p>\n
\n be expected that promoters susceptible to DNA methylation gain in colon tumours would
\n form a distinct group with a minimal overlap with TET target promoters. We therefore
\n examined whether loci that gained DNA methylation in our primary tumours (1,597 probes
\n for 1,077 promoters) were likely TET2 target promoters (4,201 probes for 3,144 promoters).
\n This analysis showed less than 1% overlap between loci that gain DNA methylation in
\n tumours and the TET2 bound promoters (Figure\u00c2\u00a05a). These results could suggest that TET2 might be part of a mechanism that protects
\n promoters from de novo<\/em> DNA methylation. To examine this we depleted TET2 in HCT116 cells by stable transfection
\n of shRNAs (Figure\u00c2\u00a05b and c). In one instance we used shRNA against TET2 alone (TET2C) and in the other
\n shRNA against TET2 and TET3 (TET2?+?3 where TET3 mRNA was not affected and therefore
\n treat this sample as a TET2 only knockdown) (Figure\u00c2\u00a05c). LCMS after TET2 depletion showed a marked reduction in the global level of 5hmC
\n (Figure\u00c2\u00a05d), confirming TET2 oxygenase activity in HCT116, without changes in global levels
\n of 5mC (Figure\u00c2\u00a05d) but this could be due to the small contribution of promoter methylation to the
\n methylome. Infinium arrays identified several loci with changes in DNA methylation
\n (Figure\u00c2\u00a05e) that were for the most part low in magnitude (median of change was 10.4%; not shown).
\n Similar changes in levels of DNA methylation were recently observed after TET1 depletion
\n in differentiated cells 51]. However in our study, methylation levels at TET2 bound CpG islands were largely
\n unaffected after TET2 depletion (less than 1%, Figure\u00c2\u00a05e), suggesting that these promoters do not require high levels of TET2 to maintain
\n the methylation free state and are intrinsically resistant to methylation changes.<\/p>\n
\n identified in HCT116 cells (P<\/em> 0.0001, binomial test). (b)<\/strong> Western blot for TET2 and beta TUBULIN from whole cell extracts of HCT116 cells stably
\n transfected with a non-targeting shRNA control (shCtrl.) or with shRNA to TET2 (TET2C)
\n or to TET2 and TET3 (TET2?+?3). Fold change in the knockdown was calculated relative
\n to the shCtrl. (c)<\/strong> qRT-PCR for TET2<\/em> and TET3<\/em>. (d)<\/strong> Global levels of 5hmC and 5mC by LCMS. (e)<\/strong> DNA methylation changes by Infinium arrays after depletion of TET2.\n <\/p>\n
\n consistent with the small effect that we see in these in vitro<\/em> TET2 studies. TET2 therefore seems to play a moderate role in controlling cytosine
\n modifications during gut tumourigenesis.\n <\/p>\nPromoters with high levels of 5hmC in normal colon overlap with bivalently marked
\n promoters in human embryonic stem cells that do not become methylated in colon cancer
<\/h4>\n
\n differentiated cells, then how do we explain the resistance of 5hmC promoters to methylation
\n gain in tumours prior to their accumulating 5hmC in normal tissue? TET2 depletion
\n only has a moderate effect on DNA methylation in cancer cells, suggesting that the
\n protective mechanism is unlikely to be due to continuous TET2 binding at target promoters.
\n Although TET2 may not be involved in maintaining the unmethylated state of its target
\n promoters, we cannot exclude that other proteins within a TET-complex may be involved.
\n However there may be alternative explanations, one of which is that 5hmC promoters
\n are epigenetically marked during early development to make them intrinsically unlikely
\n to develop characteristics such as H3K27me3 in the soma that predispose to DNA methylation
\n gain.\n <\/p>\n
\n but may also include the recently described instructive process for gain of methylation
\n in cancer which occurs at promoters containing histone H3K4 and H3K27 tri-methylation
\n (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
\n mouse ESCs Tet1 is found either at the TSS of bivalent promoters together with silencing
\n complexes independent of 5hmC or downstream of the TSS together with 5hmC and the
\n PRC2 complex 59],60]. In human ESC 5hmC has been found more at active gene promoters and enhancers than
\n at poised (bivalent) enhancers 61].\n <\/p>\n
\n bivalent promoters 57] confirmed that approximately 65% of promoters that gain methylation in our colon
\n cancer cohort are also bivalently marked in hESC (Figure\u00c2\u00a06a and b). Consequently we also examined the extent to which promoters marked by 5hmC
\n in normal colon overlap with bivalently marked promoters in hESCs. We found that 30%
\n of all 5hmC promoters overlapped with bivalent genes in hESCs (Figure\u00c2\u00a06a and b). Interestingly, these mostly coincided with bivalent promoters that do not
\n become hypermethylated in colorectal cancer. This observation indicates that bivalent
\n promoters can be broadly separated into discrete instructive categories: one for silencing
\n after tissue differentiation and susceptible to methylation gain in cancer; and another
\n for poised activation and acquisition of 5hmC with resistance to methylation gain
\n in cancer. If 5hmC is acquired as an end point of instructive activation, this would
\n fit with our data where we see 5hmC accumulating in terminally differentiated cells
\n at genes that are active in both cancer and normal tissue.<\/p>\n
\n cohort at promoters with H3K4me3\/K27me3 bivalency in human embryonic stem cells (hESCbiv).
\n The incidence of methylation gain is low at hESCbiv promoters marked by 5hmC in normal
\n colon. (a)<\/strong> For narrow and (b)<\/strong> broad 5hmC promoters.\n <\/p>\n","protected":false},"excerpt":{"rendered":"