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A pre-neoplastic epigenetic field defect in HCV-infected liver at transcription factor binding sites and polycomb targets

View Article: PubMed Central - PubMed

ABSTRACT

The predisposition of patients with Hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) involves components of viral infection, inflammation and time. The development of multifocal, genetically distinct tumors is suggestive of a field defect affecting the entire liver. The molecular susceptibility mediating such a field defect is not understood. One potential mediator of long-term cellular reprogramming is heritable (epigenetic) regulation of transcription, exemplified by DNA methylation. We studied epigenetic and transcriptional changes in HCV-infected livers in comparison with control, uninfected livers and HCC, allowing us to identify pre-neoplastic epigenetic and transcriptional events.

We find the HCV-infected liver to have a pattern of acquisition of DNA methylation targeted to candidate enhancers active in liver cells, enriched for the binding sites of the FOXA1, FOXA2 and HNF4A transcription factors. These enhancers can be subdivided into those proximal to genes implicated in liver cancer or to genes involved in stem cell development, the latter distinguished by increased CG dinucleotide density and polycomb-mediated repression, manifested by the additional acquisition of histone H3 lysine 27 trimethylation (H3K27me3). Transcriptional studies on our samples showed that the increased DNA methylation at enhancers was associated with decreased local gene expression, results validated in independent samples from The Cancer Genome Atlas (TCGA). Pharmacological depletion of H3K27me3 using the EZH2 inhibitor GSK343 in HepG2 cells suppressed cell growth and also revealed that local acquired DNA methylation was not dependent upon the presence of polycomb-mediated repression.

The results support a model of HCV infection influencing the binding of transcription factors to cognate sites in the genome, with consequent local acquisition of DNA methylation, and the added repressive influence of polycomb at a subset of CG-dense cis-regulatory sequences. These epigenetic events occur before neoplastic transformation, resulting in what may be a pharmacologically-reversible epigenetic field defect in HCV-infected liver.

No MeSH data available.


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The genomic contexts of loci gaining or losing DNA methylation. In panel (a) we show the observed/expected ratio for overlap of the loci with distinctive DNA methylation with different genomic features. The loci with increasing DNA methylation are enriched at candidate enhancers (with histone H3 lysine 4 monomethylation H3K4me1 and H3K27ac but not co-incident H3K4me3 that would indicate promoter function). These loci are also enriched at regions likely to be transcribed (with H3K36me3 and at RefSeq gene bodies). These enrichments were tested by permutation analyses (b), revealing them to be non-random (black). Loci where DNA methylation is lost during disease progression is enriched at several genomic features (a) but only that for intergenic sequences survives the permutation analysis of significance in panel (b).
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Figure 3: The genomic contexts of loci gaining or losing DNA methylation. In panel (a) we show the observed/expected ratio for overlap of the loci with distinctive DNA methylation with different genomic features. The loci with increasing DNA methylation are enriched at candidate enhancers (with histone H3 lysine 4 monomethylation H3K4me1 and H3K27ac but not co-incident H3K4me3 that would indicate promoter function). These loci are also enriched at regions likely to be transcribed (with H3K36me3 and at RefSeq gene bodies). These enrichments were tested by permutation analyses (b), revealing them to be non-random (black). Loci where DNA methylation is lost during disease progression is enriched at several genomic features (a) but only that for intergenic sequences survives the permutation analysis of significance in panel (b).

Mentions: We studied the loci defined in Figure 2a as having significantly increased or decreased DNA methylation. The loci with increases in DNA methylation are significantly enriched at enhancers, characterized by local enrichment of H3K4me1 and H3K27ac (but not the promoter-associated mark H3K4me3), as well as loci likely to be undergoing active transcription (gene bodies and H3K36me3-enriched loci, Figure 3). In contrast, decreased DNA methylation is only significantly enriched at intergenic regions and not at any candidate regulatory elements (Figure 3). As increasing DNA methylation at enhancer sequences is potentially reflective of local TF binding30,32, the permutation analysis was repeated using ChIP-seq data for transcription factor binding sites (TFBSs) mapped in the HepG2 HCC cell line as part of the ENCODE project74, the best available hepatocyte surrogate with extensive TF mapping. No significant associations were observed for loci with loss of DNA methylation, but permutation testing revealed significant enrichment for several TFs at loci that gain DNA methylation: FOXA1, FOXA2, HNF4A, MAFK, MAFF, CEBPB and RXRA (Figure 4). No significant changes in levels of expression of these genes were found at alpha=0.05 (Figure S4), so this acquisition of DNA methylation is not attributable to a simple model of down-regulation of production of these TFs. All seven TFs are involved in normal liver development and physiology75–80, with CEBPB, RXRA and MAF TFs also involved with inflammation and cellular stress81–87. The most strongly perturbed FOXA1/2 and HNF4A TFs have also been implicated in WNT signaling pathways that promote carcinogenesis and epithelial-to-mesenchymal (EMT) transition88–91, which links the DNA methylation changes to the risk of neoplastic progression.


A pre-neoplastic epigenetic field defect in HCV-infected liver at transcription factor binding sites and polycomb targets
The genomic contexts of loci gaining or losing DNA methylation. In panel (a) we show the observed/expected ratio for overlap of the loci with distinctive DNA methylation with different genomic features. The loci with increasing DNA methylation are enriched at candidate enhancers (with histone H3 lysine 4 monomethylation H3K4me1 and H3K27ac but not co-incident H3K4me3 that would indicate promoter function). These loci are also enriched at regions likely to be transcribed (with H3K36me3 and at RefSeq gene bodies). These enrichments were tested by permutation analyses (b), revealing them to be non-random (black). Loci where DNA methylation is lost during disease progression is enriched at several genomic features (a) but only that for intergenic sequences survives the permutation analysis of significance in panel (b).
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Related In: Results  -  Collection

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Figure 3: The genomic contexts of loci gaining or losing DNA methylation. In panel (a) we show the observed/expected ratio for overlap of the loci with distinctive DNA methylation with different genomic features. The loci with increasing DNA methylation are enriched at candidate enhancers (with histone H3 lysine 4 monomethylation H3K4me1 and H3K27ac but not co-incident H3K4me3 that would indicate promoter function). These loci are also enriched at regions likely to be transcribed (with H3K36me3 and at RefSeq gene bodies). These enrichments were tested by permutation analyses (b), revealing them to be non-random (black). Loci where DNA methylation is lost during disease progression is enriched at several genomic features (a) but only that for intergenic sequences survives the permutation analysis of significance in panel (b).
Mentions: We studied the loci defined in Figure 2a as having significantly increased or decreased DNA methylation. The loci with increases in DNA methylation are significantly enriched at enhancers, characterized by local enrichment of H3K4me1 and H3K27ac (but not the promoter-associated mark H3K4me3), as well as loci likely to be undergoing active transcription (gene bodies and H3K36me3-enriched loci, Figure 3). In contrast, decreased DNA methylation is only significantly enriched at intergenic regions and not at any candidate regulatory elements (Figure 3). As increasing DNA methylation at enhancer sequences is potentially reflective of local TF binding30,32, the permutation analysis was repeated using ChIP-seq data for transcription factor binding sites (TFBSs) mapped in the HepG2 HCC cell line as part of the ENCODE project74, the best available hepatocyte surrogate with extensive TF mapping. No significant associations were observed for loci with loss of DNA methylation, but permutation testing revealed significant enrichment for several TFs at loci that gain DNA methylation: FOXA1, FOXA2, HNF4A, MAFK, MAFF, CEBPB and RXRA (Figure 4). No significant changes in levels of expression of these genes were found at alpha=0.05 (Figure S4), so this acquisition of DNA methylation is not attributable to a simple model of down-regulation of production of these TFs. All seven TFs are involved in normal liver development and physiology75–80, with CEBPB, RXRA and MAF TFs also involved with inflammation and cellular stress81–87. The most strongly perturbed FOXA1/2 and HNF4A TFs have also been implicated in WNT signaling pathways that promote carcinogenesis and epithelial-to-mesenchymal (EMT) transition88–91, which links the DNA methylation changes to the risk of neoplastic progression.

View Article: PubMed Central - PubMed

ABSTRACT

The predisposition of patients with Hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) involves components of viral infection, inflammation and time. The development of multifocal, genetically distinct tumors is suggestive of a field defect affecting the entire liver. The molecular susceptibility mediating such a field defect is not understood. One potential mediator of long-term cellular reprogramming is heritable (epigenetic) regulation of transcription, exemplified by DNA methylation. We studied epigenetic and transcriptional changes in HCV-infected livers in comparison with control, uninfected livers and HCC, allowing us to identify pre-neoplastic epigenetic and transcriptional events.

We find the HCV-infected liver to have a pattern of acquisition of DNA methylation targeted to candidate enhancers active in liver cells, enriched for the binding sites of the FOXA1, FOXA2 and HNF4A transcription factors. These enhancers can be subdivided into those proximal to genes implicated in liver cancer or to genes involved in stem cell development, the latter distinguished by increased CG dinucleotide density and polycomb-mediated repression, manifested by the additional acquisition of histone H3 lysine 27 trimethylation (H3K27me3). Transcriptional studies on our samples showed that the increased DNA methylation at enhancers was associated with decreased local gene expression, results validated in independent samples from The Cancer Genome Atlas (TCGA). Pharmacological depletion of H3K27me3 using the EZH2 inhibitor GSK343 in HepG2 cells suppressed cell growth and also revealed that local acquired DNA methylation was not dependent upon the presence of polycomb-mediated repression.

The results support a model of HCV infection influencing the binding of transcription factors to cognate sites in the genome, with consequent local acquisition of DNA methylation, and the added repressive influence of polycomb at a subset of CG-dense cis-regulatory sequences. These epigenetic events occur before neoplastic transformation, resulting in what may be a pharmacologically-reversible epigenetic field defect in HCV-infected liver.

No MeSH data available.


Related in: MedlinePlus