Cross-Dataset Genomic Proximity and Functional Association of FOXA1 and TET2 at Hypomethylated Regulatory Regions in Colorectal Cancer

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Background/Objectives: Aberrant DNA methylation plays a critical role in the development and progression of colorectal cancer (CRC). However, how pioneer transcription factors are associated with DNA demethylases at cis-regulatory elements and influence tumor-associated transcriptional programs remains poorly understood. This study investigated the functional relationship between FOXA1 and TET2, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and its association with epigenetic features and malignant phenotypes in CRC. Methods: Public DNA methylation, ChIP-seq, and transcriptomic datasets were integrated with genome-wide genomic-proximity analyses. The association between FOXA1 and TET2 was evaluated by co-immunoprecipitation. FOXA1 and TET2 were individually or simultaneously depleted in HCT116 and SW480 cells, followed by proliferation and invasion assays. In addition, RNA-seq profiles were generated from control, FOXA1-KD, TET2-KD, and Double-KD HCT116 cells to characterize the resulting transcriptional changes. Results: CRC-associated hypomethylated regions showed preferential enrichment of FOXA1- and TET2-associated ChIP-seq signals. At a 500 bp summit-to-summit threshold, FOXA1–TET2 genomic proximity was 30.7-fold higher than expected under chromosome-wise circular permutation (empirical p = 0.000999). FOXA1/TET2-associated loci also showed lower methylation than FOXA1-only loci in TCGA-COAD tumors (median within-patient Δβ = −0.051; paired Wilcoxon p = 5.86 × 10−50), and FOXA1 and TET2 expression was positively correlated in TCGA-COAD tumors (Spearman’s ρ = 0.40 and p = 8.5 × 10−10). Co-immunoprecipitation supported the presence of FOXA1 and TET2 within the same protein complex but did not establish direct physical binding. FOXA1 depletion reduced cell-growth-associated and invasion-associated readouts in both CRC cell lines, whereas TET2 depletion increased growth-associated readouts and showed cell-line-dependent effects on invasion. Combined FOXA1/TET2 depletion produced an intermediate growth phenotype under the conditions examined. RNA-seq revealed broadly concordant transcriptome-wide changes among the knockdown conditions, with pairwise log2 fold-change correlations ranging from r = 0.835 to 0.920, together with shared and comparison-specific transcriptional responses.