Fig 2: The sequence map of the human CNTNAP2 gene promoter. (A) 5′-RACE assay was conducted to map the CNTNAP2 transcription start site (TSS). The PCR product was analyzed on a 2% agarose gel. USP = universal primer, GSP = CNTNAP2 gene specific primer. (B) The 5′-RACE PCR product was cloned into the pRACE vector and sequenced to identify the TSS. The TSS is the first nucleotide after the SMARTer oligonucleotide, as indicated by the arrow. (C) The sequence of the human CNTNAP2 gene promoter. A 2,868 bp 5′-flanking region of the human CNTNAP2 gene was amplified from HEK genomic DNA. The TSS Adenine is designated +1. The putative binding sites for computationally predicted transcription factors are underlined.

Fig 3: Hypoxia upregulated CNTNAP2 promoter activity via functional HREs. (A) Hypoxia upregulated CNTNAP2 promoter activity. pCNTNAP2-A containing HRE1-5, pCNTNAP2-G containing HRE4-5, and pCNTNAP2-I without HREs were co-transfected with pCMV-Luc into HEK cells followed by hypoxia treatment (2% O2) for 24 h. Hypoxia increased the promoter activities of pCNTNAP2-A and pCNTNAP2-G while showing little effect on pCNTNAP2-I (n = 4, two-way ANOVA, treatment is considered as an independent variable, F (1, 18) = 38.05, p < 0.0001). (B) HIF-1α upregulated CNTNAP2 promoter activity. pCNTNAP2-A, G, and I were co-transfected with HIF-1α or vector and pCMV-Luc into HEK cells. Twenty-four hours post-transfection, cells were harvested for the dual-luciferase reporter assay. HIF-1α enhanced the promoter activities of pCNTNAP2-A and pCNTNAP2-G but did not affect pCNTNAP2-I activity [n = 3, two-way ANOVA, treatment effect, F (1, 12) = 13.98, p = 0.0028]. (C) Schematic diagram of CNTNAP2-HRE constructs. The name and construct of each plasmid were as illustrated. Triple HRE1-5 was cloned into the pGL-pL vector without any cis-acting enhancers. (D) Screening of functional HREs responsive to hypoxia treatment. HRE constructs and pGL-pL vector were co-transfected with pCMV-Luc into HEK cells and treated under hypoxia (2% O2) for 24 h. HRE3 and HRE4 were activated by hypoxia treatment, while HRE1, 2, and 5 were not reactive to the hypoxia condition [n = 3, two-way ANOVA, treatment effect, F (1, 24) = 11.7, p = 0.0022]. The values represent means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 by Sidak’s multiple comparisons test, compared with the control column of each plasmid.

Fig 4: Deletion analysis of the human CNTNAP2 gene promoter. (A) Schematic diagram of the human CNTNAP2 promoter deletion constructs. Various lengths of the human CNTNAP2 gene 5′ flanking region were cloned into the pGL3-Basic promoter upstream of the Firefly luciferase gene (Luc). The numbers represent each fragment’s start and end positions relative to the transcription start site (TSS). (B) The plasmids used in the luciferase reporter assay were confirmed by restriction enzyme digestion, and the digested products were analyzed on a 1.5% agarose gel. (C) Functional analysis of human CNTNAP2 gene promoter. pCNTNAP2 plasmids were co-transfected with pCMV-Luc into PC12 cells. 24 h post-transfection, cells were harvested for the dual-luciferase reporter assay. The relative luciferase activities were calculated by normalizing Firefly luciferase activities to the Renilla luciferase activities and represented by relative luciferase units (RLU). The activity of the pGL3-Basic vector (marked as V) was designated as 1 RLU, and the pGL3-Promoter (marked as P) served as the positive control. The values represent means ± SEM (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001 by Dunnett’s multiple comparisons test, compared with the pGL3-Basic control.