Heat Shock Transcription Factors (HSFs) Mediate Multilayer Regulations of Heat-Induced Chromatin Dynamics in Plants [DAP-seq]

Eukaryotic organisms remodel chromatin landscapes to regulate gene expression in response to environmental stress. In plants, heat stress (HS) induces widespread chromatin changes, yet the role of early responsive Heat Shock Transcription Factors (HSFs) in chromatin remodeling and their evolutionary conservation remain unclear. Here, we use chromatin accessibility profiling and transcriptomics in Marchantia polymorpha hsf mutants to reveal HSFA1 as a key determinant in positioning cis-regulatory elements (CREs) for stress-induced gene activation. Comparative analysis suggests that this HSFA1-dependent chromatin remodeling mechanism is evolutionarily conserved across land plants. Multi-layered gene regulatory network (GRN) analysis further identifies MpWRKY10 and MpABI5B as HSF-independent regulators of HS responses, providing insights into parallel transcriptional subnetworks. We develop a cross-species and cross-condition machine learning framework that accurately predicts chromatin accessibility and gene expression, demonstrating a conserved regulatory logic of stress-responsive transcription. Our findings establish a scalable computational approach for decoding gene regulatory networks and provide a conceptual framework for how TFs coordinate chromatin architecture to drive stress adaptation in plants and other eukaryotes. Overall design: DAP-seq of Marchantia polymorpha HSFA1 in Tak-1 genomic DNA at Day14 of germmalings. We investigated the binding sites and locations of MpHSFA1 in regulating chromatin dynamics and gene expression under heat stress (HS) using DNA Affinity Purification Sequencing (DAP-seq). The HSFA1_DAP sample consists of in vitro translated MpHSFA1 proteins incubated with Tak-1 genomic DNA. The sequencing results represent the MpHSFA1-bound DNA fragments. For peak calling, we used the Golden Gate expression vector pICH47732 (which was used to express the MpHSFA1 CDS) as a negative control to remove non-specific binding sites. The Empty_DAP sample represents this control.