Characterisation of transcription factor binding site on the promoter of MIF and CSE1L that are involved in colorectal cancer

Abstract

Transcription factors are key cellular components that control gene expression. They play a crucial role in maintaining routine cellular functions but when deregulated can lead to tumorigenesis. Therefore, the aim of this study was to identify the transcription factor binding site (TFBS) that regulate the promoter of colorectal cancer (CRC) related-genes. The microarray data from 13 paired samples of CRC Dukes’ B, C and D were statistically analysed using GeneSpring GX12.5. Using K-means clustering, differentially expressed genes were classified according to their expression pattern. MATCH™ (TRANSFAC) was used to identify over-represented TFBS among the CRC genes. The initial in silico studies had narrowed the scope on the role of PAX4 binding sites in regulating MIF and CSE1L genes. Individual PAX4 binding sites within the promoter of MIF and CSE1L genes were initially evaluated by preparing a series of deletion constructs at fixed 3’-end (relative to the transcriptional start site (TSS)) but with different lengths of the 5’-end. Further involvement of PAX4 binding sites in MIF and CSE1L promoter activity was determined by inhibiting PAX4 expression using LY294002 (PAX4 inhibitor) and deleting individual PAX4 binding site using site directed mutagenesis. The promoter activity was measured using luciferase assay conducted in PAX4 wild type (COLO320DM) and mutant (HCT116) cell lines. Among the series of deletion constructs, the luciferase activity of MIF and CSE1L promoters was observed to increase as longer 5’-end was deleted. The DNA sequences deleted had covered the deletion of PAX4 binding sites at the position of -492 bp to -500 bp relative to the TSS in MIFp-2108bp, PAX4 binding sites at -753 bp to -764 bp in CSE1Lp 731bp and PAX4 binding sites at -569 bp to -580 bp and -550 bp to -561 bp relative to the TSS in CSE1Lp-495bp. However, during site directed mutagenesis, individual deletion of PAX4 binding sites in the promoter of MIF resulted in the up-regulation of the luciferase activity and down-regulated CSE1L promoter activity significantly when PAX4 binding site at tgggtggtggtg (-753 to -64 bp relative to TSS) and tgggtggggcca (- 67 to -78 bp relative to TSS) were deleted. Therefore, detailed molecular mechanism of PAX4 in the promoter region still need further elucidation to understand its function in regulating colorectal cancer related genes.