Figure 8. BORIS isoforms bind and activate the transcription of conserved human and mouse CST testis-specific promoter regions.

(A) Schematic representation of multiple mouse CST promoters, where exon 1f_s has been shown to be testis specific [48] and the BORIS binding site has been mapped to the testis-specific CST promoter [9]. The alignment of human and mouse BORIS binding site in CST promoter is shown at the bottom. The 5 cytosines boxed in the alignment are contact nucleotides for BORIS protein as it was determined by methylation interference assays [9]. Nucleotides that are 100% identical to the human nucleotide sequence are shaded in grey. Dashes indicate insertions or deletions. (B) and (C) Most BORIS isoforms bind to the human and mouse testis-specific CST promoter in EMSA. The number of ZFs and utilization of alternative N-and C-termini for each BORIS isoform are shown at the bottom of gel. The band shifts specific for particular N- and C-termini are indicated by arrows. Luciferase and CTCF were used as negative and positive controls, respectively. (D) The mouse CST promoter is transcriptionally activated by BORIS isoforms in HEK293T cells. HEK293T cells were either co-transfected with mouse Boris, mouse Ctcf, empty pCI vector (EV) or the human BORIS isoform constructs, as well as with pGL-3 containing 359 bp of wild type or mutant CST promoter. Luciferase assays were done 48 h after transfections. All luciferase activities were normalized for transfection efficiency by measuring the Renilla luciferase activity from the co-transfected pRL-TK vector. Error bars are standard deviations.