Interaction with SP1, but not binding to the E-box motifs, is responsible for BHLHE40/DEC1-induced transcriptional suppression of CLDN1 and cell invasion in MCF-7 cells

Basic helix-loop-helix family member e40 (BHLHE40) is located in 3p26.1 and acts as a transcriptional repressor of the circadian rhythm by suppressing the expression of the clock genes and clock-controlled genes. Recent research indicated that BHLHE40 may be involved in regulating tumor cell progression. However the mechanism by which BHLHE40 regulates the invasion and metastasis of tumor cells is unclear. Our in vitro assays showed that BHLHE40 promoted tumor cell invasion while BHLHE40 silencing by siRNA suppressed tumor cell invasion of MCF-7 cells. BHLHE40 suppressed the mRNA and protein expression of CLDN1 CLDN4 and CDH1 and promoted the expression of SNAI1 and SNAI2. Reporter assays demonstrated that BHLHE40 suppressed CLDN1 transcription but not through direct binding to the E-box motifs in the CLDN1 promoter. Further studies demonstrated BHLHE40 suppressed CLDN1 transcription by preventing the interaction between SP1 and a specific motif within the promoter region of CLDN1. BHLHE40 could not further suppress CLDN1 transactivation after SP1 siRNA transfection that is, BHLHE40-induced suppression of CLDN1 relied on SP1. Furthermore our data indicated that SP1 was a major regulator of CLDN1 transcription by binding to a specific motif that was located at -233 to -61 bp upstream of the transcription start site. Immunoprecipitation and co-localization data revealed an interaction between BHLHE40 and SP1. By constructing deletion mutants we found that the BHLH and Orange regions are both essential for the BHLHE40-SP1 interaction. BHLHE40 probably acts as an inhibitory nuclear cofactor or perhaps recruits other inhibitory cofactors to inhibit the SP1-mediated CLDN1 transactivation. These results suggest that BHLHE40 facilitates cell invasion and may be used as a novel target for breast cancer prevention and treatment.