UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation [BiSulfite-seq]
The E3 ubiquitin ligase UHRF1 is an established cofactor for replication-coupled DNA methylation inheritance. The current model posits a stepwise pathway in which UHRF1 engages nucleosomes through histone and DNA binding. This multivalent nucleosome readout directs UHRF1 ubiquitin ligase activity toward several lysines on N-terminal tails of histone H3, binding sites for DNMT1 through tandem ubiquitin interacting motifs (UIM1 and UIM2). While this elegant chromatin regulatory mechanism is now taking shape, the extent to which ubiquitin signaling through UHRF1 contributes to DNMT1-dependent DNA methylation maintenance genome-wide is not known. Here, we present comparative, genome-scale analysis of DNA methylation inheritance in human colon cancer cells with compromised UHRF1 ubiquitin ligase and DNMT1 ubiquitin reading activities. We reveal that DNA methylation maintenance in contexts of low CpG density is particularly vulnerable to disruption of UHRF1 ubiquitin ligase activity. This low CpG density hypomethylation signature, a common feature of partially methylated domains (PMDs) in human cancers, is also seen when DNMT1 ubiquitin reading activity through UIM1 is disrupted. Notably, disrupting UIM2 function affects DNA methylation inheritance to a similar extent as a catalytically dead form of the enzyme. Collectively, these studies demonstrate that DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process and suggest that a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to the development of PMDs in human cancers. Overall design: Genomic DNA was isolated from HCT116 cells lines modified with stable integration of a doxycycline-inducible shRNA targeting the 3'UTR of either UHRF1 or DNMT1. Bulk (shDNMT1 = 2) and clonally expanded (shUHRF1 = 3 clones) cell populations were used. In all cases, Baseline (NoDox) samples were grown in culture for 2 weeks in the absence of doxycycline, and serve as the baseline methylome control for Knockdown and Recovery samples. Knockdown samples were treated with doxycycline (10 ng/mL) for two weeks to induce expression of the shRNA. Recovery samples were treated with doxycycline for two weeks (to induce knockdown of the target protein), and then doxycycline was removed for an additional two weeks to measure recovery methylation dynamics with re-expression of the either UHRF1 or DNMT1.