Chromatin modifiers Mdm2 and RNF2 prevent RNA:DNA hybrids that impair DNA replication

Proc Natl Acad Sci U S A. 2018 Nov 27;115(48):E11311-E11320. doi: 10.1073/pnas.1809592115. Epub 2018 Nov 9.

Abstract

The p53-Mdm2 system is key to tumor suppression. We have recently reported that p53 as well as Mdm2 are capable of supporting DNA replication fork progression. On the other hand, we found that Mdm2 is a modifier of chromatin, modulating polycomb repressor complex (PRC)-driven histone modifications. Here we show that, similar to Mdm2 knockdown, the depletion of PRC members impairs DNA synthesis, as determined in fiber assays. In particular, the ubiquitin ligase and PRC1 component RNF2/Ring1B is required to support DNA replication, similar to Mdm2. Moreover, the Ring finger domain of Mdm2 is not only essential for its ubiquitin ligase activity, but also for proper DNA replication. Strikingly, Mdm2 overexpression can rescue RNF2 depletion with regard to DNA replication fork progression, and vice versa, strongly suggesting that the two ubiquitin ligases perform overlapping functions in this context. H2A overexpression also rescues fork progression upon depletion of Mdm2 or RNF2, but only when the ubiquitination sites K118/K119 are present. Depleting the H2A deubiquitinating enzyme BAP1 reduces the fork rate, suggesting that both ubiquitination and deubiquitination of H2A are required to support fork progression. The depletion of Mdm2 elicits the accumulation of RNA/DNA hybrids, suggesting R-loop formation as a mechanism of impaired DNA replication. Accordingly, RNase H overexpression or the inhibition of the transcription elongation kinase CDK9 each rescues DNA replication upon depletion of Mdm2 or RNF2. Taken together, our results suggest that chromatin modification by Mdm2 and PRC1 ensures smooth DNA replication through the avoidance of R-loop formation.

Keywords: Mdm2; RNF2; histone; p53; ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Chromatin / genetics
  • Chromatin / metabolism*
  • DNA / genetics*
  • DNA / metabolism
  • DNA Replication*
  • Histones / genetics
  • Histones / metabolism
  • Humans
  • Polycomb Repressive Complex 1 / genetics
  • Polycomb Repressive Complex 1 / metabolism*
  • Protein Domains
  • Proto-Oncogene Proteins c-mdm2 / chemistry
  • Proto-Oncogene Proteins c-mdm2 / genetics
  • Proto-Oncogene Proteins c-mdm2 / metabolism*
  • RNA / genetics*
  • RNA / metabolism
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism
  • Ubiquitin Thiolesterase / genetics
  • Ubiquitin Thiolesterase / metabolism
  • Ubiquitination

Substances

  • BAP1 protein, human
  • Chromatin
  • Histones
  • Tumor Suppressor Proteins
  • RNA
  • DNA
  • MDM2 protein, human
  • Polycomb Repressive Complex 1
  • Proto-Oncogene Proteins c-mdm2
  • RNF2 protein, human
  • Ubiquitin Thiolesterase