Supplementary Data - Exonic enhancers are a widespread class of dual-function regulatory elements

Description This repository accompanies our manuscript, “Exonic enhancers are a widespread class of dual-function regulatory elements” in which we redefine the role of exonic regions in gene regulation. We demonstrate that many protein-coding exons also function as candidate Exonic Enhancers (cEEs) : previously underappreciated regulatory elements embedded within exons. By integrating TF ChIP-seq, chromatin accessibility data (DNase-seq/ATAC-seq), studying the impact of variants (common and cancer variants), high-throughput enhancer-reporter assays (STARR-seq, luciferase), and CRISPR-based validations, we show that cEEs play critical roles in cis-regulatory networks while retaining protein-coding functionality. This repository provides the processed datasets, nd supporting materials used throughout the study, organized into thematic folders to facilitate easy navigation and reproducibility. Analysis scripts are both in Github and in another Zenodo. Key findings Identification of cEEs Across Multiple Species Systematic discovery of cEEs using TF ChIP-seq, chromatin accessibility, and STARR-seq data. Many protein-coding exons exhibit enhancer activity. Dual Coding and Regulatory Roles EEs retain protein-coding functions while simultaneously acting as cis-regulatory elements. Both synonymous and nonsynonymous variants can disrupt EE activity and downstream gene expression. Long-Range Interactions and Target Gene Regulation Promoter capture Hi-C and eQTL analyses confirm interactions between EEs and gene promoters. CRISPR-based inactivation of EEs demonstrates regulatory effects on both host and distal target genes. Clinical and Evolutionary Implications Pan-cancer (TCGA) analyses show that mutations in EEs correlate with altered gene expression and clinical outcomes. Evolutionary conservation indicates that EEs are functionally constrained yet contribute to species-specific regulatory innovations. Repository Contents Below are the main folders included in this Zenodo repository. Each folder provides data or scripts relevant to the analyses described in the manuscript. 1_easy_accessContains the principal output files that may be of primary interest, including: ExonsEnhancers_all_species.tar.gz interactions_robust_EE_target_genes.tsv.gz STARR-seq_catalogs_all_species.tar.gz tcga_deseq_significative_results.tsv.gz tcga_variants_in_ee_per_patients_per_cancers_hg19.tsv.gz EE_selection/Scripts used to define candidate exonic enhancers (cEEs) based on TF ChIP-seq peaks and additional filtering criteria. Control_selection/Procedures for generating negative/positive control sets, ensuring unbiased comparisons with cEEs. Chromatin_accessibility/Scritps to curate DNase-seq, ATAC-seq and histones marks datasets assessing open chromatin in exons and cEEs across multiple species. Conservation_and_structure/Scripts for multi-species and pairwise alignment, phyloP scores, AlphaFold predictions, MobiDB disorder rates and gene-age analyses for cEEs. Randomisation/Scripts used to validate enrichment within cEEs through randomization and permutation tests. TFBS_in_EE/Motif analysis pipeline (e.g., JASPAR-based TFBS predictions) overlapping with candidate exonic enhancers. STARR-seq_experiment/Analysis scripts for STARR-seq data, including the reads pipeline and SNPs analysis. STARR-seq_catalog/Data curation of STARR-seq peaks from public data sources, as well as cEEs biotype signature definition. G-quadruplex/Scripts examining G4-forming sequences (G-quadruplex) in cEEs vs. control exons. Interaction_data/Integration of promoter capture Hi-C, eQTL (GTEx), and ENCODE-rE2G resources to identify robust cEE–target gene interactions. gnomADv3_analysis/Variant filtering/annotation pipelines and constraint analyses for common variants in gnomAD v3 that intersect with candidate exonic enhancers. GWAS_analysis/Overlaps of known GWAS loci with cEEs to reveal potential trait- and disease-associated variants within coding enhancer regions. PanCancer_analysis/Curation and analysis scripts (differential expression and survival analysis) for the TCGA PanCanAtlas. Genes_specificity/Scripts used to infer the cEEs host/target genes expression tendencies. CRISPRi_plots/Scripts used to generate the CRISPRi figures. UCSC_trackhub/Configurations for easily visualizing cEEs, TF binding, and variant positions in the UCSC Genome Browser. How to Cite If you use this dataset or associated methods in your research, please cite both this Zenodo repository and the corresponding manuscript: Mouren J-C, Torres M, Van-Ouwerkerk A, Manosalva I, Gallardo F, Spicuglia S, Ballester B. Exonic enhancers are a widespread class of dual-function regulatory elements. (In prep - journal). [https://www.biorxiv.org/content/10.1101/2025.03.27.645641v1]Mouren J-C, Ballester B. Supplementary Data - Exonic enhancers are a widespread class of dual-function regulatory elements. Zenodo. [https://doi.org/10.5281/zenodo.15079251]Mouren J-C, Ballester B. Code - Exonic enhancers are a widespread class of dual-function regulatory elements. Zenodo. [https://zenodo.org/records/17208475] Contact For questions regarding the data, analysis scripts, or methods, please contact:Jean-Christophe Mouren & Benoit BallesterAix Marseille University, INSERM, TAGC, UMR [email protected] & [email protected] We hope this repository serves as a valuable resource for further research into exonic enhancers and their roles in gene regulation, evolution, and disease.