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. 2007 Dec 31;179(7):1337-45.
doi: 10.1083/jcb.200706150.

The histone methyltransferase SET8 is required for S-phase progression

Stine Jørgensen  1 , Ingegerd Elvers, Morten Beck Trelle, Tobias Menzel, Morten Eskildsen, Ole Nørregaard Jensen, Thomas Helleday, Kristian Helin, Claus Storgaard Sørensen
Affiliations

The histone methyltransferase SET8 is required for S-phase progression

Stine Jørgensen et al. J Cell Biol. .

Abstract

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.

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Figures

Figure 1.
Figure 1.
Removal of SET8 results in reduced cell proliferation. (A) U2OS cells were treated with either mock siRNA or siRNA against SET8. Cells were counted 48 and 96 h after siRNA treatment. Every time point represents three independent samples, and the experiment was repeated three times with similar results. (B) Pictures were taken of representative areas of the cell dishes before harvest. A fraction of the cells from A was collected and processed for Western blotting. MCM7 is a loading control. Molecular weights are indicated on the left side. Error bars represent SD. Bars: (top) 25 μm; (bottom) 4 μm.
Figure 2.
Figure 2.
SET8 depletion leads to a delay in S-phase progression. (A) SET8 was depleted in U2OS cells using siRNA for 52 h. Nocodazole was added 16 h before harvest to the indicated samples. The samples were stained with PI and analyzed by flow cytometry analysis (FACS). (B) Cells were collected from a similar experiment as in A and were processed for immunoblotting. (C) U2OS cells were treated with siRNA against SET8 or mock for 48 and 72 h. Cells were pulsed with BrdU for 5 min, fixed, stained for BrdU and PI, and analyzed by FACS. The distribution of cells corresponding to the boxed areas is presented in the table beneath the graph. A fraction of the cells was collected and processed for immunoblotting.
Figure 3.
Figure 3.
SET8 depletion leads to DSBs and checkpoint activation in S-phase cells. (A) SET8 was depleted in U2OS cells for 24 and 48 h. Cells were preextracted and fixed before staining with antibody toward γ-H2AX and Rad51. (B) U2OS cells were treated as in A and were fixed and stained for γ-H2AX and PI followed by FACS analysis. (C) Cells were treated as in A and stained with antibodies recognizing γ-H2AX and monomethylated H4 Lys20. Cells were also collected and processed for Western blotting with the indicated antibodies. Reduction of H4 Lys20 monomethylation upon SET8 depletion is quantified and indicated below the immunoblot. (D) Cells were treated as in A and were stained with 53BP1 and RPA antibodies. Bars, 10 μm.
Figure 4.
Figure 4.
SET8 depletion leads to replication-dependent DNA damage that activates Chk1. (A) SET8 was depleted in U2OS cells using siRNA for 52 h. Nocodazole was added for the last 16 h as indicated. Cells were collected and processed for immunoblotting. (B) U2OS cells were treated with siRNA against mock, SET8, or Chk1 for 48 h. Nocodazole was added 13 h before harvest. Cells were fixed, stained with PI, and analyzed by FACS. A graph was generated by overlaying the FACS profiles from SET8-depleted cells and cells codepleted for SET8 and Chk1. (C) SET8 was depleted in U2OS cells using siRNA for 52 h. Nocodazole (16 h) and the Chk1 inhibitor Gö6976 (3 h) were added before harvest. Samples were fixed, stained with PI, and analyzed by FACS. (B and C) A fraction of the cells was collected and processed for immunoblotting. (D) 48 h after SET8 siRNA transfection, cells were treated with 5 μM aphidicolin and/or the Chk1 inhibitor CEP-3891 for an additional 24 h. Cells were then processed for pulsed-field gel electrophoresis. (E) U2OS cells were treated with siRNA against mock, SET8, Cdc45, or Rad51 for 48 h. Cells were then fixed and stained for γ-H2AX and PI followed by FACS analysis.
Figure 5.
Figure 5.
SET8 interacts with PCNA and is required for replication fork progression. (A) U2OS cells were transfected with siRNA against mock or SET8 and treated with thymidine for 30 h. Cells in the bottom panel were released from G1 arrest by the addition of new media. Nocodazole was added as the cells were released 10 h before harvest. (B) 48 h after SET8 siRNA transfection, cells were pulse labeled with [3H]thymidine for 30 min and were allowed to progress with and without Chk1 inhibitor CEP-3891 for the indicated times. Replication fork elongation from the incorporated [3H]thymidine was then measured as described in Fig. S3 D. 100% labeled single-stranded DNA (ssDNA) is equivalent to no fork progression. (C) Colocalization between HA-FLAG–tagged SET8 and YFP-PCNA transfected in U2OS cells. Cells were preextracted before fixation and processed for immunostaining with HA antibody. (D) Immunoprecipitation of HA-FLAG–tagged SET8 wild type and PIP-box mutant (LTDFY mutated to ATDAA) from HEK293 cells. Cells were transfected with the indicated constructs followed by lysis, immunoprecipitation, and immunoblotting. (E) Endogenous SET8 from HEK293 cells was immunoprecipitated and processed for immunoblotting. Bars, 10 μm.
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