The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression

Truncated forms of CGGBP1 with (N-term) or without (C-term) the DNA-binding domain (DBD) have been used to to assay global gene expression. HEK293T cells with endogenous CGGBP1 knocked down were used to over-express truncated forms of CGGBP1 followed by RNA extraction and one-coloured global gene expression analysis. The data suggests that while the C-term of CGGBP1 is the major repressor of transcription, just the N-term containing the DBD fails to achieve so. Proximal promoters of CGGBP1-repressed genes, although significantly GC-poor, contain GC-rich transcription factor binding motifs and exhibit base compositions indicative of low C-T transition rates due to targeted prevention of cytosine methylation. Our findings suggest that CGGBP1 protects transcription factor binding sites (TFBS) from cytosine methylation-associated loss and thereby regulates gene expression. By analysing orthologous promoter sequences, we show that protection from cytosine methylation is a function of CGGBP1 progressively acquired during vertebrate evolution. Gene expression changes upon over-expression of truncated forms (N-term and C-term) and full-length CGGBP1 were measured after 48 hours of transfection in HEK293T cells. All the three experiments were performed in triplicates.