Complementary Roles of DNA Methylation and miRNA in Regulating Gene Expression Under Environmental Stress in...

Biological invasions significantly threaten global biodiversity and disrupt the stability of ecosystems worldwide. Effective responses to environmental stressors are crucial for invasion success; however, the underlying epigenetic regulatory mechanisms remain poorly understood, especially regarding the interplay among multiple regulatory layers such as DNA methylation and microRNAs (miRNAs). Here we employed an integrative multi-omics approach to investigate the model invasive Ciona robusta subjected to repeated salinity stress. Focusing on canonical osmotic regulation pathways, we revealed a dynamic and coordinated regulation of stress-responsive gene expression, with miRNAs and DNA methylation playing distinct yet complementary roles across functional pathways/genes and distinct regions within the same genes. Regulating osmolyte shifts during repeated stress, miRNAs emerged as dominant regulators through widespread and flexible targeting of genes, whereas DNA methylation contributed more selectively. Notably, both mechanisms co-regulated certain genes via spatially distinct genomic regions, supporting a multilayered model of gene regulation. Furthermore, we observed significantly reduced methylation levels in miRNA-targeted genes, suggesting an evolutionary structural complementarity between the two epigenetic systems. Moreover, the permutation test revealed that dual regulation was a non-random event. Interestingly, miRNAs and DNA methylation did not converge on a limited set of stress-related pathways; instead, they provided complementary regulation across multiple functions, while dual regulation did not directly amplify gene expression changes. Together, these findings underscore the critical role of complex interplay among epigenetic processes in enabling rapid phenotypic plasticity and provide novel insights into the molecular mechanisms underlying invasion success under environmental stress.