DNA methylation and hydroxymethylation have distinct genome-wide profiles related to axonal regeneration

ABSTRACT

Alterations in environmentally sensitive epigenetic mechanisms (e.g., DNA methylation) influence axonal regeneration in the spinal cord following sharp injury. Conventional DNA methylation detection methods using sodium bisulphite treatment do not distinguish between methylated and hydroxymethylated forms of cytosine, meaning that past studies report a composite of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). To identify the distinct contributions of DNA methylation modifications to axonal regeneration, we collected spinal cord tissue after sharp injury from untreated adult F3 male rats with enhanced regeneration of injured spinal axons or controls, derived from folate- or water-treated F0 lineages, respectively. Genomic DNA was profiled for genome-wide 5hmC levels, revealing 658 differentially hydroxymethylated regions (DhMRs). Genomic profiling with whole genome bisulphite sequencing disclosed regeneration-related alterations in composite 5mC + 5hmC DNA methylation levels at 2,260 differentially methylated regions (DMRs). While pathway analyses revealed that differentially hydroxymethylated and methylated genes are linked to biologically relevant axon developmental pathways, only 22 genes harbour both DhMR and DMRs. Since these differential modifications were more than 60 kilobases on average away from each other, the large majority of differential hydroxymethylated and methylated regions are unique with distinct functions in the axonal regeneration phenotype. These data highlight the importance of distinguishing independent contributions of 5mC and 5hmC levels in the central nervous system, and denote discrete roles for DNA methylation modifications in spinal cord injury and regeneration in the context of transgenerational inheritance.

KEYWORDS:

• Folate
• transgenerational
• DNA methylation
• transcriptome
• epigenetics
• axonal regeneration

Acknowledgments

The authors would like to thank the UW biotechnology centre. This work was supported in part by the University of Wisconsin-Madison department of Neurological Surgery (RSA, BJI), the University of Wisconsin Fall Competition Award (BJI), March of Dimes Gene Discovery and Translational Research Grant #6-FY14-435 (BJI), NICHD 1R01HD047516 (BJI), NIH 3R01HD047516-04S1 ARRA Supplement (BJI), NARSAD Young Investigator Grant from the Brain & Behavioral Research Foundation #22669 (LP), and a Ruth L. Kirschstein National Research Service Award (MH113351-02) (AM). The authors declare no competing financial interests.

Disclosure statement

No potential conflict of interest was reported by the authors.

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Funding

This work was supported by the Brain and Behavior Research Foundation [22669]; March of Dimes Foundation [6-FY14-435]; National Institute of Child Health and Human Development [1R01HD047516]; National Institutes of Health [MH113351-02]; National Institutes of Health [3R01HD047516-04S1].