Mdm4 supports DNA replication in a p53-independent fashion

Abstract

The Mdm4 (alias MdmX) oncoprotein, like its paralogue and interaction partner Mdm2, antagonizes the tumor suppressor p53. p53-independent roles of the Mdm proteins are emerging, and we have reported the ability of Mdm2 to modify chromatin and to support DNA replication by suppressing the formation of R-loops (DNA/RNA-hybrids). We show here that the depletion of Mdm4 in p53-deficient cells compromises DNA replication fork progression as well. Among various deletion mutants, only full-length Mdm4 was able to support DNA replication fork progression. Co-depletion of Mdm4 and Mdm2 further impaired DNA replication, and the overexpression of each partially compensated for the other's loss. Despite impairing replication, Mdm4 depletion only marginally hindered cell proliferation, likely due to compensation through increased firing of replication origins. However, depleting Mdm4 sensitized p53−/− cells to the nucleoside analog gemcitabine, raising the future perspective of using Mdm4 inhibitors as chemosensitizers. Mechanistically, Mdm4 interacts with members of the Polycomb Repressor Complexes and supports the ubiquitination of H2A, thereby preventing the accumulation of DNA/RNA-hybrids. Thus, in analogy to previously reported activities of Mdm2, Mdm4 enables unperturbed DNA replication through the avoidance of R-loops.

The integrated stress response induces R-loops and hinders replication fork progression

“…Dot blots were conducted as described previously 24 . Cells were seeded, treated with Thap, BEPP or CHX as indicated and harvested.…”

Non-targeting control for MISSION® shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

“…It has also been demonstrated that silencing of SNRPD3 promoted MDM4 exon 6 skipping leading to the expression of a shorter and more labile transcript variant (Siebring-van Olst et al, 2017), which in turn could affect the level of MDM4 and p53-dependent and p53-independent functions of the protein (Haupt, Mejia-Hernandez, Vijayakumaran, Keam, & Haupt, 2019). For example MDM4 has recently been shown to facilitate unperturbed DNA replication via suppressing the formation of RNA-DNA hybrids (Wohlberedt et al, 2020). We observed substantially diminished Mdm4 mRNA levels in SHC016-expressing cells but only in those of murine origin (Figure 1).…”

MDM2-Driven Ubiquitination Rapidly Removes p53 from Its Cognate Promoters

“…Chromatin immunoprecipitation was carried out as described by our group [ 16 , 17 ] but modified as follows. The cells were cultivated while adherent on 145 mm dishes (Cat.…”