The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8–ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.
Bibliographical noteFunding Information:
We would like to thank I. Zampeta for performing the Dnmt3a western blots, and R. van der Linden, R. Janssens and M. de Bruijn for performing flow cytometry. This work was supported by grants from the Netherlands Organisation for Scientific Research (ALW 822.02.018) and the Dutch Cancer Society (KWF EMCR 2008-4109). G.S. was supported by a Marie Skłodowska-Curie fellowship (H2020-MSCA-IF-2016, miRStem) and by the ‘Fundación Científica de la Asociación Española Contra el Cáncer’. R.S. is supported by the Netherlands Organization for Scientific Research (VENI 91617114) and an Erasmus MC Fellowship.
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