Dynamic changes in 5-hydroxymethylation signatures underpin early and late events in drug exposed liver

Aberrant DNA methylation is a common feature of neoplastic lesions, and early detection of such changes may provide powerful mechanistic insights and biomarkers for carcinogenesis. Here, we investigate dynamic changes in the mouse liver DNA methylome associated with short (1 day) and prolonged (7, 28 and 91 days) exposure to the rodent liver non-genotoxic carcinogen, phenobarbital (PB). We find that the distribution of 5mC/5hmC is highly consistent between untreated individuals of a similar age; yet, changes during liver maturation in a transcriptionally dependent manner. Following drug treatment, we identify and validate a series of differentially methylated or hydroxymethylated regions: exposure results in staged transcriptional responses with distinct kinetic profiles that strongly correlate with promoter proximal region 5hmC levels. Furthermore, reciprocal changes for both 5mC and 5hmC in response to PB suggest that active demethylation may be taking place at each set of these loci via a 5hmC intermediate. Finally, we identify potential early biomarkers for non-genotoxic carcinogenesis, including several genes aberrantly expressed in liver cancer. Our work suggests that 5hmC profiling can be used as an indicator of cell states during organ maturation and drug-induced responses and provides novel epigenetic signatures for non-genotoxic carcinogen exposur

Computational modeling identifies key gene regulatory interactions underlying phenobarbital-mediated tumor promotion

Gene regulatory interactions underlying the early stages of non-genotoxic carcinogenesis are poorly understood. Here, we have identified key candidate regulators of phenobarbital (PB)-mediated mouse liver tumorigenesis, a well-characterized model of non-genotoxic carcinogenesis, by applying a new computational modeling approach to a comprehensive collection of in vivo gene expression studies. We have combined our previously developed motif activity response analysis (MARA), which models gene expression patterns in terms of computationally predicted transcription factor binding sites with singular value decomposition (SVD) of the inferred motif activities, to disentangle the roles that different transcriptional regulators play in specific biological pathways of tumor promotion. Furthermore, transgenic mouse models enabled us to identify which of these regulatory activities was downstream of constitutive androstane receptor and β-catenin signaling, both crucial components of PB-mediated liver tumorigenesis. We propose novel roles for E2F and ZFP161 in PB-mediated hepatocyte proliferation and suggest that PB-mediated suppression of ESR1 activity contributes to the development of a tumor-prone environment. Our study shows that combining MARA with SVD allows for automated identification of independent transcription regulatory programs within a complex in vivo tissue environment and provides novel mechanistic insights into PB-mediated hepatocarcinogenesi

Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques

The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hm

Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome.

BACKGROUND: Induction and promotion of liver cancer by exposure to non-genotoxic carcinogens coincides with epigenetic perturbations, including specific changes in DNA methylation. Here we investigate the genome-wide dynamics of 5-hydroxymethylcytosine (5hmC) as a likely intermediate of 5-methylcytosine (5mC) demethylation in a DNA methylation reprogramming pathway. We use a rodent model of non-genotoxic carcinogen exposure using the drug phenobarbital. RESULTS: Exposure to phenobarbital results in dynamic and reciprocal changes to the 5mC/5hmC patterns over the promoter regions of a cohort of genes that are transcriptionally upregulated. This reprogramming of 5mC/5hmC coincides with characteristic changes in the histone marks H3K4me2, H3K27me3 and H3K36me3. Quantitative analysis of phenobarbital-induced genes that are involved in xenobiotic metabolism reveals that both DNA modifications are lost at the transcription start site, while there is a reciprocal relationship between increasing levels of 5hmC and loss of 5mC at regions immediately adjacent to core promoters. CONCLUSIONS: Collectively, these experiments support the hypothesis that 5hmC is a potential intermediate in a demethylation pathway and reveal precise perturbations of the mouse liver DNA methylome and hydroxymethylome upon exposure to a rodent hepatocarcinogen.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Xenobiotic CAR activators induce Dlk1-Dio3 locus non-coding RNA expression in mouse liver

Predicting the impact of human exposure to chemicals such as pharmaceuticals and agrochemicals requires the development of reliable and predictive biomarkers suitable for the detection of early events potentially leading to adverse outcomes. In particular, drug-induced non-genotoxic carcinogenesis (NGC) during preclinical development of novel therapeutics intended for chronic administration in humans is a major challenge for drug safety. We previously demonstrated Constitutive Androstane Receptor (CAR) and WNT signaling-dependent up-regulation of the pluripotency associated Dlk1-Dio3 imprinted gene cluster non-coding RNAs (ncRNAs) in the liver of mice treated with tumorpromoting doses of phenobarbital (PB). Here, to explore the sensitivity and the specificity of this candidate liver tumor promotion ncRNAs signature we compared phenotypic, transcriptional and proteomic data from wild-type, CAR/PXR double knock-out and CAR/PXR double humanized animals treated with tumor-promoting doses of PB or chlordane, both well-established CAR activators. We further investigated selected transcriptional profiles from mouse liver samples exposed to seven NGC compounds working through different mode of actions, overall suggesting CAR-activation specificity of the Dlk1-Dio3 long ncRNAs activation. We propose that Dlk1-Dio3 long ncRNAs up-regulation is an early CAR-activation dependent transcriptional signature during xenobiotic-induced mouse liver tumor promotion. This signature may further contribute mode of action-based ‘weight of evidence’ cancer risk assessment for xenobiotic-induced rodent liver tumors

Drug-induced chromatin accessibility changes associate with sensitivity to liver tumor promotion

Liver cancer susceptibility varies amongst humans and between experimental animal models due to multiple genetic and epigenetic factors. The molecular characterization of such susceptibilities has the potential to enhance cancer risk assessment of xenobiotic exposures and disease prevention strategies. Here, using DNase I hypersensitivity mapping coupled with transcriptomic profiling, we investigate perturbations in cis-acting gene regulatory elements associated with the early stages of phenobarbital (PB)- mediated liver tumor promotion in susceptible versus resistant mouse strains (B6C3F1 versus C57BL/6J). Integrated computational analyses of strain-selective changes in liver chromatin accessibility underlying PB-response reveal differential epigenetic regulation of molecular pathways associated with PB-mediated tumor promotion, including Wnt/-catenin signalling. Complementary transcription factor motif analyses reveal mouse strain-selective gene regulatory networks and a novel role for Stat, Smad and Fox transcription factors in the early stages of PB-mediated tumor promotion. Mapping perturbations in cis-acting gene regulatory elements provides novel insights into the molecular basis for susceptibility to xenobiotic-induced rodent liver tumor promotion and has the potential to enhance mechanism-based cancer risk assessments of xenobiotic exposures

Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques

The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hmC

L’hétérochromatine constitutive dans tous ses états

Les centromères, lieu d’assemblage des kinétochores, sont cruciaux pour la ségrégation des chromosomes lors de la mitose et de la méiose. Leur altération peut conduire à l’aneuploïdie, une anomalie génétique très fréquemment observée dans les tumeurs humaines. Ces régions sont organisées sous forme d’hétérochromatine dite « constitutive », et restent condensées tout au long du cycle cellulaire. L’hétérochromatine constitutive a longtemps été perçue comme une structure stable et inerte. Cette conception a depuis largement évolué, de nombreuses études montrant sa plasticité et lui associant de multiples fonctions dans les cellules en interphase. Cet article présente certains aspects de cette plasticité de l’hétérochromatine constitutive et souligne l’importance de l’intégrité épigénétique des centromères pour la stabilité du génome

Characterisation of set-1 , a conserved PR/SET domain gene in Caenorhabditis elegans

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