Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF

CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclea r factor. Binding to various CTCF target sites (CTSs) is mediated by combin atorial contributions of different ZFs. Different CTSs mediate distinct CTC F functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessa ry and sufficient core sequences of diverse enhancer-blocking (insulator) e lements, including CpG methylation-sensitive ones, have recently been pinpo inted to CTSs. To determine whether a posttranslational modification may mo dulate CTCF functions, we studied CTCF phosphorylation. We demonstrated tha t most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sit es map to four serines within the (SKKEDSSDSE)-K-604-S-609-D-610-E-612 moti f that is highly conserved in vertebrates. Specific mutations of these seri nes abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylati on in vitro. In addition, we showed that completely preventing phosphorylat ion by substituting all serines within this site resulted in markedly enhan ced repression of the CTS-bearing vertebrate c-myc promoters, but did not a lter CTCF nuclear localization or in vitro DNA-binding characteristics assa yed with c-myc CTSs. Moreover, these substitutions manifested a profound ef fect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-i nteracting protein partners.