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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.


Related in: MedlinePlus

Functional properties of genes targeted for DNA methylation and transcriptional changes. We focus in (a) on the 644 genes with gain of DNA methylation during disease progression at promoter-proximal enhancers, but breaking down the associated pathways depending on whether the hypermethylated enhancer loci with H3K4me1 in normal liver continued to have this mark in the HepG2 HCC cells, and whether the repressive H3K27me3 polycomb mark is added at those sites in HepG2 cells. We show that the associated functional pathways are segregated into two groups (red boxes) when these patterns of change of histone modifications are studied. Genes acquiring DNA methylation at nearby H3K4me1-defined enhancers that do not lose this mark or gain polycomb repression are those associated with cancers in general and liver cancers in particular. The genes that acquire H3K27me3 are those encoding transcription factors and with stem cell properties. In (b) we show a result from our SMITE analysis based on integration of transcriptional information with genomic context-dependent DNA methylation changes. The module shown is highly enriched for proteins involved in WNT signaling, consistent with the targeting of genomic dysregulation to genes with stem cell functions.
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Figure 6: Functional properties of genes targeted for DNA methylation and transcriptional changes. We focus in (a) on the 644 genes with gain of DNA methylation during disease progression at promoter-proximal enhancers, but breaking down the associated pathways depending on whether the hypermethylated enhancer loci with H3K4me1 in normal liver continued to have this mark in the HepG2 HCC cells, and whether the repressive H3K27me3 polycomb mark is added at those sites in HepG2 cells. We show that the associated functional pathways are segregated into two groups (red boxes) when these patterns of change of histone modifications are studied. Genes acquiring DNA methylation at nearby H3K4me1-defined enhancers that do not lose this mark or gain polycomb repression are those associated with cancers in general and liver cancers in particular. The genes that acquire H3K27me3 are those encoding transcription factors and with stem cell properties. In (b) we show a result from our SMITE analysis based on integration of transcriptional information with genomic context-dependent DNA methylation changes. The module shown is highly enriched for proteins involved in WNT signaling, consistent with the targeting of genomic dysregulation to genes with stem cell functions.

Mentions: To understand the potential cellular consequences of altering transcription levels and dysregulating genes by DNA methylation changes, Gene Set Enrichment Analysis (GSEA)93 was performed focusing on a comparison between control and malignant samples. Three lists of genes were generated – the 309 downregulated and the 193 upregulated genes, and the 644 genes where increased DNA methylation was found at H3K4me1-defined enhancers within 5 kb of the transcription start site (Table S4). All three gene lists showed concordance for enrichment in gene sets related to altered expression in liver cancer, fetal liver expression and polycomb repressive complex 2 (PRC2) targeting in ES cells (Figure S7). Interestingly, two of the gene sets were published as targets of the HNF4A and FOXA2 TFs in liver94,95, supporting the possibility illustrated in Figure 4 that the regulation exerted by these TFs is specifically targeted in HCV infection and HCC. Because we found pathway-enrichment for PRC2 gene targets associated H3K27me3 in embryonic stem cells, the pathway analysis for 644 genes with H3K4me1-defined enhancers in normal adult liver was stratified by the presence of H3K4me1 and H3K27me3 in HepG2, using publicly available ChIP-seq data. Of the 644 genes defined to have increasing DNA methylation at promoter-proximal enhancers, 326 retained those enhancers in HepG2 cells, but another 137 added the repressive H3K27me3 mark at those enhancers, while 63 genes gained H3K27me3 and lost the H3K4me1 mark altogether. This sub-categorization allowed further insights – the genes that do not gain the H3K27me3 mark are more likely to represent those previously associated with liver and other cancers, while those that do gain the H3K27me3 modification represent known targets of polycomb and other transcriptional regulators, especially in stem cells and early development (Figure 6a). When the CG dinucleotide content of the enhancers in each sub-category was tested, it became strikingly apparent that the acquisition of H3K27me3 and the loss of H3K4me1 were positively associated with increased CG content (Figure S8). The cancer gene signature is therefore associated with acquisition of DNA methylation at CG-depleted loci, and the stem cell signature with acquisition of DNA methylation and H3K27me3 at CG-dense loci.


A pre-neoplastic epigenetic field defect in HCV-infected liver at transcription factor binding sites and polycomb targets
Functional properties of genes targeted for DNA methylation and transcriptional changes. We focus in (a) on the 644 genes with gain of DNA methylation during disease progression at promoter-proximal enhancers, but breaking down the associated pathways depending on whether the hypermethylated enhancer loci with H3K4me1 in normal liver continued to have this mark in the HepG2 HCC cells, and whether the repressive H3K27me3 polycomb mark is added at those sites in HepG2 cells. We show that the associated functional pathways are segregated into two groups (red boxes) when these patterns of change of histone modifications are studied. Genes acquiring DNA methylation at nearby H3K4me1-defined enhancers that do not lose this mark or gain polycomb repression are those associated with cancers in general and liver cancers in particular. The genes that acquire H3K27me3 are those encoding transcription factors and with stem cell properties. In (b) we show a result from our SMITE analysis based on integration of transcriptional information with genomic context-dependent DNA methylation changes. The module shown is highly enriched for proteins involved in WNT signaling, consistent with the targeting of genomic dysregulation to genes with stem cell functions.
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Related In: Results  -  Collection

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Figure 6: Functional properties of genes targeted for DNA methylation and transcriptional changes. We focus in (a) on the 644 genes with gain of DNA methylation during disease progression at promoter-proximal enhancers, but breaking down the associated pathways depending on whether the hypermethylated enhancer loci with H3K4me1 in normal liver continued to have this mark in the HepG2 HCC cells, and whether the repressive H3K27me3 polycomb mark is added at those sites in HepG2 cells. We show that the associated functional pathways are segregated into two groups (red boxes) when these patterns of change of histone modifications are studied. Genes acquiring DNA methylation at nearby H3K4me1-defined enhancers that do not lose this mark or gain polycomb repression are those associated with cancers in general and liver cancers in particular. The genes that acquire H3K27me3 are those encoding transcription factors and with stem cell properties. In (b) we show a result from our SMITE analysis based on integration of transcriptional information with genomic context-dependent DNA methylation changes. The module shown is highly enriched for proteins involved in WNT signaling, consistent with the targeting of genomic dysregulation to genes with stem cell functions.
Mentions: To understand the potential cellular consequences of altering transcription levels and dysregulating genes by DNA methylation changes, Gene Set Enrichment Analysis (GSEA)93 was performed focusing on a comparison between control and malignant samples. Three lists of genes were generated – the 309 downregulated and the 193 upregulated genes, and the 644 genes where increased DNA methylation was found at H3K4me1-defined enhancers within 5 kb of the transcription start site (Table S4). All three gene lists showed concordance for enrichment in gene sets related to altered expression in liver cancer, fetal liver expression and polycomb repressive complex 2 (PRC2) targeting in ES cells (Figure S7). Interestingly, two of the gene sets were published as targets of the HNF4A and FOXA2 TFs in liver94,95, supporting the possibility illustrated in Figure 4 that the regulation exerted by these TFs is specifically targeted in HCV infection and HCC. Because we found pathway-enrichment for PRC2 gene targets associated H3K27me3 in embryonic stem cells, the pathway analysis for 644 genes with H3K4me1-defined enhancers in normal adult liver was stratified by the presence of H3K4me1 and H3K27me3 in HepG2, using publicly available ChIP-seq data. Of the 644 genes defined to have increasing DNA methylation at promoter-proximal enhancers, 326 retained those enhancers in HepG2 cells, but another 137 added the repressive H3K27me3 mark at those enhancers, while 63 genes gained H3K27me3 and lost the H3K4me1 mark altogether. This sub-categorization allowed further insights – the genes that do not gain the H3K27me3 mark are more likely to represent those previously associated with liver and other cancers, while those that do gain the H3K27me3 modification represent known targets of polycomb and other transcriptional regulators, especially in stem cells and early development (Figure 6a). When the CG dinucleotide content of the enhancers in each sub-category was tested, it became strikingly apparent that the acquisition of H3K27me3 and the loss of H3K4me1 were positively associated with increased CG content (Figure S8). The cancer gene signature is therefore associated with acquisition of DNA methylation at CG-depleted loci, and the stem cell signature with acquisition of DNA methylation and H3K27me3 at CG-dense loci.

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