Many enhancers exist as clusters in the genome and control cell identity and disease genes. However, it remains largely unexplored how spatial gene expression patterns are orchestrated by enhancers in the genome. Here we introduce eSpatial, a computational framework that deciphers the spatially resolved enhancer regulation on gene expression through integrating spatial profiling of gene expression and chromatin accessibility. Briefly, eSpatial detects spatial domains, identifies spatial-specific cis-regulatory elements and enhancer clusters for each gene, and depicts spatial patterns of enhancer regulation. We apply eSpatial to diverse spatial datasets derived from various technologies and biological contexts, including mouse embryo and brain development, as well as studies on human melanoma and breast cancer diseases. We uncover the prevalence of a spatial enhancer code, where spatially divergent combinations of enhancers determine spatial gene expression. We validate spatial-specific enhancers and their target genes using public datasets such as VISTA, Allen in situ hybridization (ISH), and H3K27ac MERFISH data. Moreover, we conduct transgenic reporter assays and in vivo CRISPR/Cas9-mediated perturbation experiments to confirm the Atoh1 spatial enhancer code determining Atoh1 spatial expression in mouse embryonic spinal cord and brain. Collectively, our study establishes a framework to decipher the spatial enhancer code elucidating how spatially divergent combinations of enhancers intricately regulate gene expression in development and disease contexts.
 

enhancer cluster, Hi-C, spatial enhancer code, gene expression