Characterization of the chicken CTCF genomic locus, and initial study of the cell cycle-regulated promoter of the gene

Abstract

CTCF is a multifunctional transcription factor encoded by a novel candidate tumor suppressor gene (Filippova, G. N., Lindbloma, A., Meinke, L. J., Klenova, E. M., Neiman, P. E., Collins, S. J., Doggett, N. D., and Lobanenkov, V. V. (1998) Genes Chromosomes Cancer 22, 26-36). We characterized genomic organization of the chicken CTCF (chCTCF) gene, and studied the chCTCF promoter. Genomic locus of chCTCF contains a GC-rich untranslated exon separated from seven coding exons by a long intron. The 2- kilobase pair region upstream of the major transcription start site contains a CpG island marked by a 'Not-knot' that includes sequence motifs characteristic of a TATA-less promoter of housekeeping genes. When fused upstream of a reporter chloramphenicol acetyltransferase gene, it acts as a strong transcriptional promoter in transient transfection experiments. The minimal 180-base pair chCTCF promoter region that is fully sufficient to confer high level transcriptional activity to the reporter contains high affinity binding element for the transcription factor YY1. This element is strictly conserved in chicken, mouse, and human CTCF genes. Mutations in the core nucleotides of the YY1 element reduce transcriptional activity of the minimal chCTCF promoter, indicating that the conserved YY1-binding sequence is critical for transcriptional regulation of vertebrate CTCF genes. We also noted in the chCTCF promoter several elements previously characterized in cell cycle-regulated genes, including the 'cell cycle-dependent element' and 'cell cycle gene homology region' motifs shown to be important for S/G2- specific up-regulation of cdc25C, cdc2, cyclin A, and Plk (polo-like kinase) gene promoters. Presence of the cell cycle-dependent element/cell cycle gene homology region element suggested that chCTCF expression may be cell cycle- regulated. We show that both levels of the endogenous chCTCF mRNA, and the activity of the stably transfected chCTCF promoter constructs, increase in S/G2 cells.