NFYA regulates the expression of CD44 via binding to upstream cis-element in breast cancer cells

CRISPR/Cas9 has emerged as a powerful tool for editing of non-coding sequence, such as promoters and enhancers. Creating indels in enhancer sequences alter their function and provide valuable insights into their role in gene expression under normal and pathological conditions. This study tested the involvement of CD44-upstream cis-element in regulating CD44 expression and evaluated the potential of CRISPR/Cas9 system to manipulate the transcription factor (TF) binding site within this element. Bioinformatic tools predicted two binding sites (designated P1 and P2) for NFYA within the cis-element sequence. CRISPR/Cas9-mediated genome editing was employed to knockout NFYA gene and disrupt the NFYA-binding site at P2 in MDA-MB-231 breast cancer cells, then downstream analysis was performed using the mixed-edited cell population. Both genome editing strategies resulted in a significant reduction in CD44 gene expression at mRNA level. Notably, disruption of P2 sequence produced a greater reduction in the CD44⁺ CD24^- cell subpopulation than NFYA knockout. Both editing approaches impaired the migration capacity of MDA-MB-231 cells. Unlike NFYA knockout, altering P2 sequence induced apoptosis. ChIP assay revealed that NFYA binds to both P1 and P2 sequences, with greater enrichment observed at P2 than at P1. Site-directed mutagenesis and luciferase assay confirmed the regulatory contribution of both P1 and P2 to gene expression regulation, with stronger regulatory effect in case of P2 than in P1. Collectively, the findings demonstrated the regulation of CD44 by NFYA and highlighted the potential CRISPR/Cas9-mediated targeting of TF binding site as effective strategy for modulating CD44 expression and associated malignant phenotype.