Paradoxical whole genome DNA methylation dynamics of 5’aza-deoxycytidine in chronic low-dose exposure in mice

ABSTRACT

Decitabine (5-aza-2ʹdeoxycytidine; DAC) is a DNA methyltransferase inhibitor used to hypomethylate the epigenome. Current dosing regimens of DAC for use in mice vary widely and their hypomethylating ability has not been robustly characterized, despite reliable results of hypomethylation of the epigenome with cell lines in vitro and tissue specificity in vivo. We investigated the effects on the DNA methylome and gene expression within mice exposed to chronic low doses of DAC ranging from 0 to 0.35 mg/kg over a period of 7 weeks without causing toxicity. Our dose paradigm resulted in no cytotoxic effects within target tissues, although testes weight and sperm concentration significantly reduced as dose increased (p-value <0.05). By whole genome bisulphite sequencing (WGBS), we identify tissue and dose-specific differentially methylated CpGs (DMCs) and regions (DMRs) in testes and liver. Testes methylation is more sensitive to DAC exposure when compared to liver, cortex, and hippocampus. Gene expression was dysregulated in testes and liver, targeting non-specific pathways as dose increases. Together our data suggest DNA methylation and gene expression are disrupted by in vivo DAC treatment in a non-uniform manner contrary to expectations, and that no dose level or regimen is sufficient to cause systemic hypomethylation in whole mice.

KEYWORDS:

• Decitabine
• DNA methylation
• epigenetics
• chronic low-dose
• Whole Genome Bisulfite Sequencing
• transcriptome

Acknowledgments

The authors would like to thank the University of Michigan Epigenomics and Advanced Genomics Cores for the assistance with whole genome bisulfite sequencing data generation and analysis. The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.

Accession numbers

Liver and Testes RNA-seq raw fastq reads for doses 0.0 mg/kg, 0.15 mg/kg, and 0.35 mg/kg have been deposited with the Sequence Read Archived submission: SUB7033954 under accession number PRJNA608917. Liver and Testes WGBS raw fastq reads for doses 0.0 mg/kg, 0.15 mg/kg, and 0.35 mg/kg have been deposited with the Sequence Read Archived submission: SUB6853480 under accession number PRJNA603069.

Disclosure statement

The authors have no conflicts of interest.

Supplementary material

Supplemental data for this article can be accessed here.

Availability

Fastq RNA-seq and WGBS reads are available in the SRA repository. (https://www.ncbi.nlm.nih.gov/sra)

Additional information

Funding

This work was supported by National Institute of Environmental Health Sciences R01ES026877 (DD, CF); National Institute of Environmental Health Sciences U01 Supplement U01 ES026697 (DD); National Institutes of Health Office of the Director T32OD010993 (NW); and National Institute of Environmental Health Sciences Pathways to Independence Award R00ES022221 (CF).NIH Office of the Director [T32OD010993]; National Institute of Environmental Health Sciences [R00ES022221]; National Institute of Environmental Health Sciences [R01ES026877]; National Institute of Environmental Health Sciences [U01 ES026697].