Amyotrophic Lateral Sclerosis-associated 3′ UTR enhancer embedded within CAV1 risk gene

Enhancer elements that reside within 3′ untranslated regions (UTRs) are an understudied phenomenon. Given the independent regulatory functions of enhancers and 3′ UTRs - enhancers governing pre-transcriptional control of gene expression and 3′ UTRs mediating post-transcriptional regulation of messenger RNA (mRNA) fate - 3′ UTR-associated enhancers may integrate these complementary layers to coordinate gene expression across multiple regulatory stages. Non-coding variation, impacting regulatory DNA, underpins the genetic architecture of disease. Indeed, the vast majority of single nucleotide polymorphisms (SNPs) associated with human complex diseases map to the non-coding genome, with causal variants particularly enriched within enhancers. Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder associated with non-coding risk variants, many of which are increasingly linked to enhancer disruption. The CAV1 gene, encoding the neuroprotective protein Caveolin-1, is a known ALS risk gene, yet the functional consequences of ALS-associated variation in its regulatory elements remain largely unexplored. Here, we combine genome-wide enhancer profiling with targeted experimental validation to define a previously uncharacterised ALS-associated enhancer embedded within the CAV1 3′ UTR, systematically assess its regulatory potential, and evaluate the impact of ALS-associated SNPs on enhancer function. We show that an individual ALS-associated SNP within this 3′ UTR-associated enhancer may disrupt function on multiple levels: at the DNA and chromatin level, by altering transcription factor binding with potential effects on recruitment of epigenetic co-regulators; and at the RNA level, by reshaping the structure and stability of a novel enhancer RNA transcribed from this locus. Collectively, our findings highlight this proximal CAV1/CAV2 enhancer as a functionally important regulatory element embedded with the 3′ UTR of an ALS risk gene, illustrate how non-coding variants can impact multiple layers of gene regulation, and provide mechanistic insight into how intragenic enhancers contribute to ALS risk. More broadly, this work underscores the importance of 3′ UTR-associated enhancers as modulators of risk gene expression and underexplored contributors to human complex disease.