Table_1_Developmental Accumulation of Gene Body and Transposon Non-CpG Methylation in the Zebrafish Brain.xlsx

DNA methylation predominantly occurs at CG dinucleotides in vertebrate genomes; however, non-CG methylation (mCH) is also detectable in vertebrate tissues, most notably in the nervous system. In mammals it is well established that mCH is targeted to CAC trinucleotides by DNMT3A during nervous system development where it is enriched in gene bodies and associated with transcriptional repression. However, the conservation of developmental mCH accumulation and its deposition by DNMT3A is largely unexplored and has yet to be functionally demonstrated in other vertebrates. In this study, by analyzing DNA methylomes and transcriptomes of zebrafish brains, we identified enrichment of mCH at CAC trinucleotides (mCAC) at defined transposon motifs as well as in developmentally downregulated genes associated with developmental and neural functions. We further generated and analyzed DNA methylomes and transcriptomes of developing zebrafish larvae and demonstrated that, like in mammals, mCH accumulates during post-embryonic brain development. Finally, by employing CRISPR/Cas9 technology, we unraveled a conserved role for Dnmt3a enzymes in developmental mCAC deposition. Overall, this work demonstrates the evolutionary conservation of developmental mCH dynamics and highlights the potential of zebrafish as a model to study mCH regulation and function during normal and perturbed development.

DNA甲基化主要发生在脊椎动物基因组中的CG二核苷酸处；然而，在脊椎动物组织中，尤其是神经系统组织中，也可检测到非CG甲基化（mCH）。在哺乳动物中，mCH在神经系统发育过程中被DNMT3A靶向至CAC三核苷酸，这一过程在基因体中富集，并与转录抑制相关。然而，发育过程中mCH积累的保守性及其由DNMT3A沉积的特性尚未得到充分探索，且在其他脊椎动物中尚未见功能性的证实。在本研究中，通过对斑马鱼大脑的DNA甲基组和转录组进行分析，我们发现在特定的转座子基序以及与发育和神经功能相关的发育下调基因中，mCH在CAC三核苷酸（mCAC）处富集。进一步地，我们生成并分析了发育中斑马鱼幼虫的DNA甲基组和转录组，证明了与哺乳动物类似，mCH在胚胎后脑发育过程中积累。最后，通过运用CRISPR/Cas9技术，我们揭示了Dnmt3a酶在发育过程中mCAC沉积中的保守作用。总体而言，本研究展示了发育过程中mCH动态的进化保守性，并突出了斑马鱼作为研究mCH调控及其在正常和异常发育过程中功能的一个潜在模型的潜力。