Context-dependent crosstalk between DNA methylation and histone modifications shapes durable CRISPR/dCas9-based epigenetic editing

Understanding how histone modifications and DNA methylation cooperate within defined chromatin contexts to shape long-term transcriptional outcomes remains a major challenge in epigenetic engineering. Here, we dissect locus-specific crosstalk between DNMT3A-dCas9 and distinct dCas9-fused histone modifiers co-targeted to the CpG islands of ZEB1 and SNAI1, two promoters with similar basal activity but distinct chromatin architectures in HepG2 cells. Deposition of H3K9me2/3 or removal of H3K4 methylation enhanced DNMT3A-mediated cytosine methylation in an effector- and locus-dependent manner. DNMT3A combined with G9a or G9a-me3 induced robust and durable DNA methylation at SNAI1, resulting in long-term repression, but failed to synergize at ZEB1. Interestingly, cooperative DNMT3A–G9a activity at SNAI1 occurred independently of histone acetylation and was correlated with locus-specific enrichment of H2A.Z and PRC2/EZH2-associated factors BCLAF1 and THRAP3, as shown by protein profiling. In contrast, the distribution of active marks throughout the large ZEB1 CpG island likely restricted stable G9a-mediated H3K9 methylation, possibly through antagonism with preexisting H3K4me3, which prevented additional DNA methylation. Synergy of DNMT3A with LSD1 or RIOX1 further demonstrated that a fully demethylated H3K4 state, rather than deacetylation, is critical for the effectors cooperation. Collectively, our findings show that chromatin context dictates the compatibility and efficacy of combinatorial epigenetic editors, providing a framework for designing stable CRISPR-based transcriptional reprogramming strategies.