Mutational Analysis of the Poly(ADP-Ribosyl)ation Sites of the Transcription Factor CTCF Provides an Insight into the Mechanism of Its Regulation by Poly(ADP-Ribosyl)ation

Abstract

Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADP-ribosyl)ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank F. Docquier, A. Harrison, R. Meyer, M. Meyer-Ficca, and N. Curtin for helpful discussions. We are grateful to V. Lobanenkov for the gift of the anti-CTCF monoclonal antibody, L. Kraus for the gift of the anti-PARP-1 antibody, and M. Oshimura and A. Feinberg for A911M and A911P mouse-human hybrid cells.

Funding for this work was from the Medical Research Council (to D.F., S.R., and E.K.), the Breast Cancer Campaign (to S.R. and E.K.), the University of Essex (to E.K. and I.C.), the Swedish Science Research Council (to R.O.), the Swedish Cancer Research Foundation (to R.O.), the Swedish Pediatric Cancer Foundation (to R.O.), the Lundberg Foundation (to R.O.), HEROIC and ChILL (European Union integrated projects; to R.O.), a Cancer Research United Kingdom senior cancer research fellowship (to A.M.), the Wenner-Gren Foundation (to M.J.), and an Association of International Cancer Research grant (to A.M. and Y.I.). We acknowledge the support of the University of Cambridge, Cancer Research UK, and Hutchison Whampoa Limited (to A.M., M.J., and Y.I.).