# Concurrent Alterations in DNA Methylation and RNA m6A Methylation During Epigenetic and Transcriptomic Reprogramming Induced by Tail Docking Stress in Fat-Tailed Sheep

## TL;DR

Tail docking in fat-tailed sheep causes coordinated changes in DNA and RNA methylation, helping the animals adapt to stress and improve productivity.

## Contribution

This study reveals how tail docking induces coordinated DNA hypomethylation and RNA m6A hypermethylation to reprogram gene expression in fat-tailed sheep.

## Key findings

- Tail docking leads to DNA hypomethylation and RNA m6A hypermethylation in fat-tailed sheep.
- 143 genes show concurrent methylation and expression changes, with 41 regulated by both DNA and RNA methylation.
- Genes like CITED4 and ZNF644 are significantly altered across DNA methylation, RNA methylation, and mRNA expression.

## Abstract

This study examined the molecular mechanisms underlying the improved health and productivity observed in fat-tailed sheep following tail docking—an economically important breed in which tail fat functions as an adaptive energy reserve. By analyzing post-procedure molecular changes, we found that tail docking decreases DNA methylation while elevating RNA m6A methylation. These coordinated epigenetic adjustments reprogram genes associated with tissue development and stress response, thereby facilitating physiological adaptation. Our findings elucidate the molecular basis of this adaptive process and offer valuable insights for optimizing breeding and management approaches to enhance both animal welfare and production efficiency.

Tail docking, serving as an important management intervention in animal husbandry, plays a significant role in regulating tail fat deposition and improving production performance and health status in fat-tailed sheep. This study systematically revealed the reprogramming effects of tail docking on the epigenetic landscape and transcriptome of fat-tailed sheep by integrating whole-genome bisulfite sequencing (WGBS) and RNA m6A methylated immunoprecipitation sequencing (MeRIP-seq). At the DNA level, the tail-docked group exhibited a pronounced trend of hypomethylation across multiple functional genomic regions, including promoters, exons, and introns. Differentially methylated regions (DMRs) were significantly enriched in pathways related to tissue development and stress response, such as the Hippo signaling pathway and adherens junctions. Pyrosequencing validation of the promoter region of the key gene DGAT1 further confirmed the reliability of the WGBS data. At the RNA level, RNA m6A modifications showed an overall up-regulated pattern: the tail-docked group displayed higher numbers of m6A peaks, greater total peak length, and increased genomic coverage compared to the control group, along with better overall prediction of modification sites. Genes associated with differential m6A peaks were closely related to processes such as stem cell pluripotency and cytoskeleton regulation. qPCR validation of several methylation-related enzyme genes (e.g., METTL3, FTO, YTHDF1) yielded results consistent with the sequencing trends. Through integrated analysis of DNA methylation and RNA methylation, we identified 143 genes with concurrent changes in methylation and mRNA expression, among which 41 genes were regulated by both DNA and RNA methylation. These genes were primarily enriched in the adherens junction pathway. Notably, two core genes CITED4 and ZNF644 showed significant changes across all three levels: DNA methylation, RNA methylation, and mRNA expression. This study systematically elucidates the epigenetic mechanism by which tail docking stress induces coordinated DNA hypo-methylation and RNA m6A hyper-methylation to regulate transcriptomic reprogramming in response to environmental intervention. The findings provide novel insights into the molecular basis of trait formation in livestock.