CTCF, A CONSERVED NUCLEAR FACTOR REQUIRED FOR OPTIMAL TRANSCRIPTIONALACTIVITY OF THE CHICKEN C-MYC GENE, IS AN 11-ZN-FINGER PROTEIN DIFFERENTIALLY EXPRESSED IN MULTIPLE FORMS

A novel sequence-specific DNA-binding protein, CTCF, which interacts w ith the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R. H. Nicolas, V. V. Adler, H. Pater son, E. M. Klenova, A. V. Polotskaja, and G. H. Goodwin, Oncogene 5:17 43-1753, 1990). In order to test directly whether binding of CTCF to o ne specific DNA region of the c-myc promoter is important for chicken c-myc transcription, we have determined which nucleotides within this GC-rich region are responsible for recognition of overlapping sites by CTCF and Sp1-like proteins. Using missing-contact analysis of all fou r nucleotides in both DNA strands and homogeneous CTCF protein purifie d by sequence-specific chromatography, we have identified three sets o f nucleotides which contact either CTCF or two Sp1-like proteins bindi ng within the same DNA region. Specific mutations of 3 of 15 purines r equired for CTCF binding were designed to eliminate binding of CTCF wi thout altering the binding of other proteins. Electrophoretic mobility shift assay of nuclear extracts showed that the mutant DNA sequence d id not bind CTCF but did bind two Sp1-like proteins. When introduced i nto a 3.3-kbp-long 5'-flanking noncoding c-myc sequence fused to a rep orter CAT gene, the same mutation of the CTCF binding site resulted in 10- and 3-fold reductions, respectively, of transcription in two diff erent (erythroid and myeloid) stably transfected chicken cell lines. I solation and analysis of the CTCF cDNA encoding an 82-kDa form of CTCF protein shows that DNA-binding domain of CTCF is composed of 11 Zn fi ngers: 10 are of C2H2 class, and 1 is of C2HC class. CTCF was found to be abundant and conserved in cells of vertebrate species. We detected six major nuclear forms of CTCF protein differentially expressed in d ifferent chicken cell lines and tissues. We conclude that isoforms of 11-Zn-finger factor CTCF which are present in chicken hematopoietic HD 3 and BM2 cells can act as a positive regulator of the chicken c-myc g ene transcription. Possible functions of other CTCF forms are discusse d.