DNA methylation analysis of HCT116 after disruption of methyllysine reading and writing.

The chromatin-binding E3 ubiquitin ligase Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) maintains DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri- methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remains unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown in somatic cells, uptake of the CPP had no effect on the DNA methylation maintenance function of UHRF1. Further, we did not detect changes in bulk DNA methylation after chemical or genetic disruption of lysine methyltransferase activity. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro, implicate the UHRF1 TTD as a dispensable domain for DNA methylation maintenance, and call into question existing models of methyllysine-dependent DNA methylation maintenance in somatic cancer cells. HCT116 or HeLa cells were incubated for 7 days with high concentration (20 μM) of indicated methylated LIG1 peptides or water (- control). HCT116 cells were also treated with DMSO (control) or UNC0638 (1 uM) for 48 hrs. HCT116 cells were also passaged for 10 days with either H3.3 WT or H3.3 K9M transgene incorporation. Cells we were passaged to avoid confluence and peptide was refreshed at each passage. FAM, 6-carboxyfluorescein; CPP, cell penetrating peptide (-polyethylene glycol-kkkrkv). Bisulphite converted DNA from the 10 samples were hybridised to the Illumina Infinium MethylationEPIC BeadChip.