Whole-genome methylation reveals tissue-specific differences in non-CG methylation in bovine

DNA methylation at non-CG dinucleotides(mCH, H=A, C,T) widely occurs and plays an important role in specific cell types, including pluripotent, neural, and germ cells.However, the functions and regulatory mechanisms of mCH, particularly in species other than humans and mice,remain inadequately explored. In this study, we analyzed the distribution of mCH across different bovine tissues,identifying significantly elevated mCH levels in bovine embryonic stem cells(bESCs), as well as brain, spleen,and ileum tissues compared to other tissues. Marked differences in mCH patterns between somatic cells and bESCs were observed, reflecting distinct base preferences and the differential expression of DNA methyltransferases.We also identified exon methylation in both CG and nonCG contexts, resembling gene-associated methylation patterns observed in plants. To characterize tissue-specific variations in mCH, we developed a novel method for differential mCH analysis. Results indicated that mCH is not randomly distributed but tends to be enriched in tissuespecific functional regions. Furthermore, regression models demonstrated a positional correlation between CG methylation and mCH. This study enhances our understanding of mCH distribution and function in bovine somatic and stem cells, providing new insights into its potential roles across species and tissues. These findings advance knowledge of epigenetic mechanisms, shedding light on the potential involvement of mCH in development and disease processes.