The role of PR-Set7 in replication licensing depends on Suv4-20h

Abstract

PR-Set7 is the sole monomethyltransferase responsible for H4K20 monomethylation (H4K20me1) that is the substrate for further methylation by Suv4-20h1/h2. PR-Set7 is required for proper cell cycle progression and is subject to degradation by the CRL4(Cdt2) ubiquitin ligase complex as a function of the cell cycle and DNA damage. This report demonstrates that PR-Set7 is an important downstream effector of CRL4(Cdt2) function during origin of DNA replication licensing, dependent on Suv4-20h1/2 activity. Aberrant rereplication correlates with decreased levels of H4K20me1 and increased levels of H4K20 trimethylation (H4K20me3). Expression of a degradation-resistant PR-Set7 mutant in the mouse embryo that is normally devoid of Suv4-20 does not compromise development or cell cycle progression unless Suv4-20h is coexpressed. PR-Set7 targeting to an artificial locus results in recruitment of the origin recognition complex (ORC) in a manner dependent on Suv4-20h and H4K20me3. Consistent with this, H4K20 methylation status plays a direct role in recruiting ORC through the binding properties of ORC1 and ORCA/LRWD1. Thus, coordinating the status of H4K20 methylation is pivotal for the proper selection of DNA replication origins in higher eukaryotes.

Figure 1.

PR-Set7 is a critical substrate of CRL4^Cdt2 that regulates replication licensing. (A) Cell cycle progression of HeLa cells expressing PR-Set7^wt or PR-Set7^PIPM2 after FACS analysis measuring DNA and EdU incorporation. (B) Western blot of protein expression after various siRNA treatments in HeLa cells; H3 and PCNA served as loading controls. (C) FACS analysis showing the cell cycle profile of HeLa cells after treatment with various siRNAs, including control, Cdt2, Cdt1, and PR-Set7.

Figure 2.

PR-Set7^PIPM2 toxicity is dependent on Suv4-20 and H4K20me2/3 in MEFs. (A) Proliferation of wild-type (wt) and Suv4-20^−/− MEFs after inducible expression of Flag-HA PR-Set7^PIPM2. Induction of PR-Set7^PIPM2 after addition of doxycycline and dexamethasone at day 0. (B) Western blot of wild-type and Suv4-20^−/− MEF lysates after inducible expression of PR-Set7^PIPM2. (C) FACS analysis of cell cycle distribution of wild-type and Suv4-20^−/− MEFs before and after induction of PR-Set7^PIPM2. (D) FACS analysis of cell cycle distribution of wild-type and Suv4-20^−/− MEFs before and after treatment with siRNA against Cdt2. (E) DAPI staining and quantification of the nuclear size of wild-type and Suv4-20^−/− MEFs after induction of PR-Set7^PIPM2. n = 3 for proliferation and cell cycle experiments. (*) Statistical significance with P < .05.

Figure 3.

Both PR-Set7^PIPM2 and PR-Set7^−/− phenotypes are dependent on Suv4-20 in the early embryo. (A) Two-cell stage embryo injection experiment schematic depicting the possible outcomes for wild-type and heterozygous PR-Set7 embryos versus the null embryos. (B) PR-Set7^+/− and PR-Set7^−/− two-cell stage embryos were injected with PR-Set7^PIPM1M2 mRNA. Injected cells are marked with GFP; the polar body is labeled by arrowheads, and GFP⁺ cells are indicated by arrows. Embryos were monitored for growth and stained with H4K20me1 (red). Full Z-series projections and single sections are depicted. (C) PR-Set7^−/+ and PR-Set7^−/− were injected with mRNA for PR-Set7^PIPM1M2 in combination with Suv4-20h1 mRNA and GFP as a tracer. The arrows point to one of the injected cells. Embryos were stained with H4K20me3 (red) and γH2A.X (yellow). Insets show an injected cell stained for H4K20me3 (red) and DAPI (blue). (D) PR-Set7^−/+ and PR-Set7^−/− were injected with PR-Set7^PIPM1M2. Arrows point to injected cells, which were identified by the presence of GFP; the polar body is marked by arrowheads. Embryos were stained with γH2A.X, cortical actin, and DAPI (blue). (E) PR-Set7^−/− and PR-Set7^−/+ embryos were injected with Suv4-20h1 mRNA alone and stained for cortical actin (green) and γH2A.X (red) accumulation. Injected cells (marked by arrows) were identified by the presence of GFP (not shown). The genotypes of individual embryos were determined after confocal acquisition as described previously.

Figure 4.

PR-Set7 recruits the ORC to chromatin through H4K20me. (A) Diagram depicting the GAL4-UAS system in 293T-REx cells. (B) ChIP analysis at an integrated UAS-binding site in 293T-REx cells before and after inducible expression of GAL4-PR-Set7 using GAL4, PR-Set7, H3, H4K20me1, H4K20me3, ORC1, and ORCA antibodies. (C) Peptide pull-down for the GST-ORC1 BAH domain and His-ORCA binding to unmethylated, H4K20me1, H4K20me2, and H4K20me3 peptides. (*) Statistical significance with P < 0.05.

Figure 5.

Summary of function of PR-Set7 and Suv4-20 (A) along with their role in regulation of origin of replication licensing through CRL4^Cdt2 (B).