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

Liver cancer susceptibility varies amongst humans and between experimental animal models because of 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/ß-catenin signaling. 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.

Pharmacological BACE1 and BACE2 inhibition induces hair depigmentation by inhibiting PMEL17 processing in mice.

Melanocytes of the hair follicle produce melanin and are essential in determining the differences in hair color. Pigment cell-specific MELanocyte Protein (PMEL17) plays a crucial role in melanogenesis. One of the critical steps is the amyloid-like functional oligomerization of PMEL17. Beta Site APP Cleaving Enzyme-2 (BACE2) and γ-secretase have been shown to be key players in generating the proteolytic fragments of PMEL17. The ß-secretase (BACE1) is responsible for the generation of amyloid-ß (Aß) fragments in the brain and is therefore proposed as a therapeutic target for Alzheimer's disease (AD). Currently BACE1 inhibitors, most of which lack selectivity over BACE2, have demonstrated efficacious reduction of amyloid-ß peptides in animals and the CSF of humans. BACE2 knock-out mice have a deficiency in PMEL17 proteolytic processing leading to impaired melanin storage and hair depigmentation. Here, we confirm BACE2-mediated inhibition of PMEL17 proteolytic processing in vitro in mouse and human melanocytes. Furthermore, we show that wildtype as well as bace2(+/-) and bace2(-/-) mice treated with a potent dual BACE1/BACE2 inhibitor NB-360 display dose-dependent appearance of irreversibly depigmented hair. Retinal pigmented epithelium showed no morphological changes. Our data demonstrates that BACE2 as well as additional BACE1 inhibition affects melanosome maturation and induces hair depigmentation in mice.

Identification of Dlk1-Dio3 imprinted gene cluster noncoding RNAs as novel candidate biomarkers for liver tumor promotion.

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for ß-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and ß-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice.

Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.

Morpholino oligonucleotide-triggered knockdown reveals a role for maternal E-cadherin during early mouse development.

We report that gene silencing via intracytoplasmic microinjections of morpholino-modified antisense oligonucleotides is an effective and reproducible method to study both maternal and zygotic gene functions during early and late stages of mouse preimplantation development. The zygotic expression of the beta-geo transgene in the ROSA26 mouse strain could be inhibited until at least the early blastula stages. Thus morpholino-triggered gene inactivation appears to be a useful method to study the functional role of genes in preimplantation development. Using this approach, we have investigated a potential role of maternal expression of Cdh1, the gene encoding the cell-adhesion molecule E-cadherin. Inhibition of translation of maternal E-cadherin mRNA causes a developmental arrest at the two-cell stage. BrUTP incorporation assays indicated that this developmental defect cannot be explained by a general failure in transcriptional activity. This defect is reversible since E-cadherin mRNA can rescue the affected embryos, suggesting that a functional adhesion complex, present at the junction between blastomeres, is a prerequisite for the normal development of the mouse preimplantation embryo. Our study thus reveals a previously unanticipated role of maternal E-cadherin during early stages of mouse development.