High-resolution mapping of chromatin conformation in HBeAg-treated macrophage provides insights into pathogenesis of HBV-related liver diseases.

Hepatitis B virus (HBV) e antigen (HBeAg) plays a critical role in inducing macrophage activation and subsequent immune tolerance, which facilitates viral immune escape. However, the underlying spatial 3D genomic and epigenetic mechanisms driving this macrophage dysfunction remain largely unknown. To map the topological and transcriptional regulatory landscape, we integrated RNA-sequencing (RNA-seq), high-throughput chromosome conformation capture (Hi-C), and chromatin immunoprecipitation-sequencing (ChIP-seq) to comprehensively analyze control and HBeAg-stimulated macrophages. Key findings were cross-validated using human HBV-infected datasets. HBeAg robustly activated pro-inflammatory transcriptional programs, prominently featuring the TNF signaling pathway as a central node. Hi-C analysis revealed profound global 3D chromatin reorganization accompanying this phenotypic shift. Specifically, inactive-to-active (B-to-A) compartment switching, coupled with de novo H3K27ac enhancer accumulation, directly drove the upregulation of functional genes such as MET and FHOD3. Furthermore, topologically associating domain (TAD) restructuring, particularly TAD merging, exposed new regulatory elements to upregulate FLNB and SESN2. Conversely, the disruption of specific intrachromosomal loops resulted in the targeted downregulation of genes like KBTBD11 and BLVRB. Mechanistically, targeted epigenetic reprogramming drove this 3D structural rewiring: H3K27ac enhancer deposition was enriched for AP-1/STAT motifs, CTCF coordinated with FOX-family factors to alter structural boundaries, and HLTF mediated targeted H3K27me3-associated gene silencing. HBeAg orchestrates a highly coordinated hierarchical restructuring of the 3D genome and targeted epigenetic reprogramming to induce macrophage dysfunction. The identified structural variants and core spatial-target genes (such as MET, FLNB, and BLVRB) provide novel mechanistic insights and represent potential therapeutic targets for HBV-related liver diseases.