p-LoopF is associated with chromatin looping in large introns of Ginkgo biloba.

In gymnosperms such as Ginkgo biloba, the regulatory role of large introns remains unclear. To address this, we conducted integrative multi-omics analyses of chromatin accessibility, three-dimensional chromosomal architecture, histone modifications, DNA methylation, RNA polymerase II occupancy, and gene expression in Ginkgo, complemented by laboratory experiments. We first identified a plant-specific factor, plant-Loop Factor (p-LoopF), which shares ~40% sequence similarity with human CCCTC-binding factor (CTCF). p-LoopF binds specifically to the consensus motif recognized by human CTCF and is significantly associated with chromatin looping, but it lacks canonical CTCF functional features, including motif orientation dependence, chromatin insulation activity, and statistically significant correlations with cohesin subunits. Through integrative multi-omics analyses, we propose a regulatory hypothesis in which p-LoopF-associated chromatin loops are correlated with the recruitment of distal enhancer-like regulatory regions, with large introns serving as a key regulatory context for these interactions. p-LoopF also localizes to promoters and distal intergenic regions, correlating with transcriptional regulation and local chromatin organization. We characterized large introns as regions enriched for chromatin loops, p-LoopF binding sites, and enhancer-like elements, which are strongly associated with the transcriptional regulation of their host genes. Additionally, active histone marks and DNA demethylation were enriched near the boundaries of large introns, particularly around splice sites, suggesting that splicing regulation differs between large and small introns.