Erfei Chen, Qiqi Yang, Haoyang Dai, Yixin Chen, Yihui Zhang et al.
Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, yet the functional impact of noncoding variants on enhancer activity remains largely unexplored. In this study, we adapted and applied two high-throughput techniques, SNP-STARR-seq and Methyl-STARR-seq, to systematically evaluate the influence of 30,790 noncoding SNPs and more than 134,000 CpG sites on enhancer activity in primary and metastatic CRC cells. We identified 922 SNPs and 487 CpG-containing elements modulating enhancer activity in primary cells and found 3136 SNPs and 3008 methylation-sensitive elements with metastasis-specific regulatory effects. Multi-omics integration linked these variants to target genes, and CRISPR editing validated their roles in driving tumorigenic and metastatic phenotypes. Furthermore, we identified two CRC-specific hypomethylated loci, cg08640619 and cg25982657, as exceptional tissue-based early detection biomarkers (AUROC > 0.96). Mechanistically, hypermethylation at cg08640619 disrupts RUNX2 binding, leading to inhibition of KIRREL1 and ETV3. Our study provides a comprehensive platform for understanding how genetic and epigenetic variants disrupt transcriptional programs in CRC, offering insights into disease susceptibility and identifying potential diagnostic and therapeutic targets.
Erfei Chen, Qiqi Yang, Haoyang Dai, Yixin Chen, Yihui Zhang, Qianglong Wang, Rongrong Hou, Ming Chen, Jie Wang, Qianwen Xie, Wenju Sun, Yong-Qiang Ning, Ligang Fan, Jian Yan. Systematic analysis of functional genetic and epigenetic variants in colorectal cancer. 2026-02-20. https://doi.org/10.1126/sciadv.aeb2473
Targeted Methyl-STARR-seq in human HEK293T cells used 120-bp genomic fragments centered on H3K27ac summits or CRC DMR CpGs cloned into pmSTARRseq1. M.SssI-treated methylated and mock-treated unmethylated plasmid libraries were compared after transfection, with RNA collected 36 hours later.
Targeted Methyl-STARR-seq in human SW620 cells used 120-bp genomic fragments centered on H3K27ac summits or CRC DMR CpGs cloned into pmSTARRseq1. M.SssI-treated methylated and mock-treated unmethylated plasmid libraries were compared after transfection, with RNA collected 36 hours later.
Targeted SNP-STARR-seq in human HEK293T cells used 120-bp genomic fragments centered on candidate noncoding SNPs, with synthesized reference and alternative alleles cloned into the hORI-STARR-seq reporter. Allele-specific enhancer activity was quantified from normalized RNA/DNA EAS 36 hours after transfection.
Targeted Methyl-STARR-seq in human HCT116 cells used 120-bp genomic fragments centered on H3K27ac summits or CRC DMR CpGs cloned into pmSTARRseq1. M.SssI-treated methylated and mock-treated unmethylated plasmid libraries were compared after transfection, with RNA collected 36 hours later.
Targeted SNP-STARR-seq in human SW480 cells used 120-bp genomic fragments centered on candidate noncoding SNPs, with synthesized reference and alternative alleles cloned into the hORI-STARR-seq reporter. Allele-specific enhancer activity was quantified from normalized RNA/DNA EAS 36 hours after transfection.
Targeted SNP-STARR-seq in human HCT116 cells used 120-bp genomic fragments centered on candidate noncoding SNPs, with synthesized reference and alternative alleles cloned into the hORI-STARR-seq reporter. Allele-specific enhancer activity was quantified from normalized RNA/DNA EAS 36 hours after transfection.
Targeted Methyl-STARR-seq in human SW480 cells used 120-bp genomic fragments centered on H3K27ac summits or CRC DMR CpGs cloned into pmSTARRseq1. M.SssI-treated methylated and mock-treated unmethylated plasmid libraries were compared after transfection, with RNA collected 36 hours later.
Targeted SNP-STARR-seq in human SW620 cells used 120-bp genomic fragments centered on candidate noncoding SNPs, with synthesized reference and alternative alleles cloned into the hORI-STARR-seq reporter. Allele-specific enhancer activity was quantified from normalized RNA/DNA EAS 36 hours after transfection.
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