Exploration of CTCF post-translation modifications uncovers Serine-224 phosphorylation by PLK1 at pericentric regions during the G2/M transition

The zinc finger CCCTC-binding protein (CTCF) carries out many, at times contradictory, functions in the cell. Although previous studies have sought to explain CTCF multivalency based on sequence composition of binding sites, few have examined how CTCF post-translational modification (PTM) could contribute to the its many functions. Here, we performed CTCF mass spectrometry, identified a novel phosphorylation site at Serine 224 (Ser224-P), and demonstrate that phosphorylation is carried out by Polo kinase 1 (PLK1). CTCF Ser224-P is chromatin-associated, mapping to at least a subset of known CTCF sites. Interestingly, CTCF Ser224-P accumulates during the G2/M transition of the cell cycle and is enriched at pericentric regions. To test function, we mutated residue 224. The phospho-obviation mutant, S224A, appeared normal. However, the phospho-mimic mutant, S224E, is detrimental to mouse embryonic stem cell colonies. While ploidy and chromatin architecture appear unaffected, S224E mutants exhibit differential expression of hundreds of genes, including p53 and p21. We have thus identified a new CTCF PTM and provided evidence of function during cellular growth and gene regulation.