Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF

Abstract

CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S⁶⁰⁴KKEDS⁶⁰⁹S⁶¹⁰DS⁶¹²E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect 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-interacting protein partners.

ACKNOWLEDGMENTS

We are grateful to R. Marais for the kind gift of purified recombinant CKII and for sharing unpublished data; to M. Roth for the p6MycTags-D plasmid; to C. Heath for helpful advice and encouragement; to T. Zarkowska and M. Towatari for helping with the phosphoamino acid analyses; to H. Paterson for assistance with confocal microscopy; to J. Frampton for helping with a cell clonogenicity assay in HD3 cells; and to R. Nicolas, Y.-J. Hu, J. Moore, S. J. Thomas, G. Loring, L. Mueller, and S. Fagerlie for excellent technical assistance. We also thank D. Levens, J. Breen, and R. Ohlsson for critically reading the manuscript.

Initial work presented here was funded in part by the NCI/NIH RO1 grants CA20068 to P.E.N. and CA68360 and CA71732 to V.V.L., by The Cancer Research Campaign grants to G.H.G., by the Human Frontier Science Program long-term fellowship and The Royal Society research grants to E.M.K., by the UICC fellowship to I.C., and, more lately, by the NCI/NIH RO1 grant CA68360 to G.N.F., by the NIAID/NIH intramural research funding to V.V.L. and H.C.M. III, and by a research grant from The Research and Equipment Committee of the University of Oxford and a Royal Society research grant to E.M.K.