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 non-CG 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 tissue-specific 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.
非CG二核苷酸（mCH，H=A、C、T）中的DNA甲基化已被证实广泛存在，在特定细胞类型（如多能细胞、大脑和生殖细胞）中发挥重要作用。目前，对于mCH的功能和机制，尤其是在人类和小鼠以外的物种中，仍知之甚少。在本研究中，我们分析了牛不同组织中mCH的分布，发现在牛胚胎干细胞（bESCs）、大脑、脾脏和回肠组织中，mCH水平显著高于其他组织，而且体细胞和bESCs之间的mCH模式存在显著差异，包括碱基偏好性不同以及DNA甲基转移酶的差异性表达。我们还发现在某些外显子中既有CG也有非CG甲基化的情况，类似于植物中某些基因的甲基化情况。为了识别不同组织中的差异性CH甲基化，我们开发了一种用于差异性mCH分析的方法。差异分析结果表明，mCH不是随机分布的，而是倾向于在组织特异性功能区域中富集。此外，我们通过回归模型证明了CG甲基化（mCG）与mCH之间的定位相关性。牛体细胞和干细胞中mCH分布及功能的研究，可以为阐明mCH在不同物种和组织中的作用提供新见解。本研究不仅推进了我们对表观遗传机制的理解，还揭示了mCH在发育和疾病发生中的潜在作用。.
Keywords: CH-DMRs; Comparative Genomics; Developmental Biology; Epigenetics; Non-CG Methylation.