Cis-regulatory role of alternative promoters in cancer

Alternative promoters are increasingly recognized as critical regulators in cancer, with differential promoter usage driving transcriptional shifts that can support oncogenic activity. While promoters are traditionally viewed as transcription initiation sites, emerging evidence suggests they can also act as cis-regulatory elements, enhancing or compensating for the expression of neighboring genes. Our 3D genomics data reveal that alternative promoters of the same gene interact with each other at the chromatin level, yet it remains unknown whether these interactions play a functional cis-regulatory role in regulating the gene's expression. Through analysis of TCGA data, we discovered that minor alternative promoters exhibit enhancer-like histone modification signatures and cancer-type-specific chromatin accessibility. To mechanistically interrogate their cis-regulatory potential, we employed CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) to modulate minor promoter activity and assess the impact on major isoform expression in cancer-related genes such as EGFR. We also found that this cis-regulatory enhancer function of minor promoters is driven by lineage-specific transcriptional master regulators. Additionally, CRISPRi-mediated repression of the major promoter led to increased minor isoform expression, suggesting a compensatory function of minor promoters. We will further investigate this compensatory activity using high-resolution chromatin interaction mapping (HiChIP) to determine whether shifts in enhancer interactions contribute mechanistically to compensation. Currently, the field lacks a clear understanding of how alternative promoters function as cis-regulatory elements, limiting our ability to define how cancer cells exploit these promoters to promote oncogenic activity. These studies will delineate mechanisms by which alternative promoters regulate oncogenic gene expression through enhancer-like and compensatory functions and will generate a resource of cancer-type-specific alternative promoters with cis-regulatory activity, offering potential therapeutic targets and a new framework for modulating oncogene expression.