AN EXCEPTIONALLY CONSERVED TRANSCRIPTIONAL REPRESSOR, CTCF, EMPLOYS DIFFERENT COMBINATIONS OF ZINC FINGERS TO BIND DIVERGED PROMOTER SEQUENCES OF AVIAN AND MAMMALIAN C-MYC ONCOGENES

We have isolated and analyzed human CTCF cDNA clones and show here tha t the ubiquitously expressed 11-zinc-finger factor CTCF is an exceptio nally highly conserved protein displaying 93% identity between avian a nd human amino acid sequences. It binds specifically to regulatory seq uences in the promoter-proximal regions of chicken, mouse, and human c -myc oncogenes. CTCF contains two transcription repressor domains tran sferable to a heterologous DNA binding domain, One CTCF binding site, conserved in mouse and human c-myc genes, is found immediately downstr eam of the major P2 promoter at a sequence which maps precisely within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse and human cells show that CTCF is the predominant factor binding to this sequence, Mutational analysis of th e PZ-proximal CTCF binding site and transient-cotransfection experimen ts demonstrate that CTCF is a transcriptional repressor of the human c -myc gene, Although there is 100% sequence identity in the DNA binding domains of the avian and human CTCF proteins, the regulatory sequence s recognized by CTCF in chicken and human c-myc promoters are clearly diverged, Mutating the contact nucleotides confirms that CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are absent in the chicken c-myc CTCF binding site, Moreove r, proteolytic-protection assays indicate that several more CTCF Zn fi ngers are involved in contacting the human CTCF binding site than the chicken site, Gel shift assays utilizing successively deleted Zn finge r domains indicate that CTCF Zn fingers 2 to 7 are involved in binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF bin ding to the human promoter. This flexibility in Zn finger usage reveal s CTCF to be a unique ''multivalent'' transcriptional factor and provi des the first feasible explanation of how certain homologous genes (i. e., c-myc) of different vertebrate species are regulated by the same f actor and maintain similar expression patterns despite significant pro moter sequence divergence.