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. 2017 Sep 15;36(18):2726-2741.
doi: 10.15252/embj.201796541. Epub 2017 Aug 4.

Histone H4K20 Tri-Methylation at Late-Firing Origins Ensures Timely Heterochromatin Replication

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Free PMC article

Histone H4K20 Tri-Methylation at Late-Firing Origins Ensures Timely Heterochromatin Replication

Julien Brustel et al. EMBO J. .
Free PMC article

Abstract

Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2-7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes.

Keywords: DNA replication origins; heterochromatin; histone H4K20 methylation.

Figures

Figure 1
Figure 1. H4K20 mutation affects S‐phase progression and prevents DNA re‐replication induced by PR‐Set7 stabilization

Immunoblot analysis of histone H4 and FLAG‐tagged histone H4 protein levels in FLAG‐H4WT and FLAG‐H4K20A U2OS cells and subjected to biochemical fractionation: Cytosolic (S1) and nuclear (S2) are soluble supernatants and P3 is the chromatin‐enriched fraction. MEK1 was used as a control of soluble components and HCF‐1 protein was used for control of chromatin fraction.

Immunoblot analysis of PR‐Set7 and the levels of acetylation and methylation of endogenous H4 and FLAG‐tagged H4 in FLAG‐H4WT and FLAG‐H4K20A U2OS cells.

FACS analysis of DNA content and FLAG signal in FLAG‐H4WT or FLAG‐H4K20A cells. DNA content was analyzed according to the low (gate 1) and high (gate 2) levels of FLAG‐tagged histone H4 proteins.

FACS analysis of DNA content in cells expressing similar levels (gate 1) of FLAG‐tagged histone H4WT and H4K20A upon expression of the PR‐Set7PIPmut and PR‐Set7PIPmut+SETmut mutants. Quantitation of re‐replicating parental (No FLAG), FLAG‐H4WT, and H4K20A cells upon PR‐Set7PIPmut expression. Data are means ± SD, n = 3. (*) Statistical significance with P < 0.05 (t‐test).

Figure 2
Figure 2. H4K20 methylation enhances origin formation and activity

Schematic representation of EBN1A protein and studied EBV‐derived plasmids with the relative position of DNA fragments amplified during ChIP‐qPCR experiments.

Quantitation of replicating FR‐DS, FR‐UAS, and FR‐ORIRDH plasmids in cells expressing EBNA1 and either GAL4, GAL4‐PR‐Set7, or GAL4‐PR‐Set7SETmut. Data are means ± SEM (n = 4) with control FR‐DS plasmid arbitrarily set as one in every cell line (gray bars).

Quantitation of replicating FR‐UAS‐ORIRDH and FR‐ORIRDH in the same cell lines as above. Data are means ± SEM (n = 4) relative to FR‐ORIRDH (black bars). (*) Statistical significance (paired two‐tailed t‐test) with P < 0.05.

ChIP‐qPCR analysis at ORIRDH, UAS, and FR sequences in different GAL4 cell lines transfected with FR‐ORIRDH or FR‐UAS‐ORIRDH plasmids using antibodies as indicated. Data are means ± SEM (n = 4) as fold enrichment of the each antibody relative to isotype IgG control. (*) Statistical significance (paired two‐tailed t‐test) with P < 0.05.

Figure EV1
Figure EV1. H4K20 methylation promotes the replication of EBV‐derived episomes

Expression levels of GAL4, GAL4‐PR‐Set7, and GAL4‐PR‐Set7SETmut in EBNA1‐expressing HEK293 cell lines. Immunoblot analysis with GAL4 antibody.

ChIP‐qPCR analysis of the FR‐UAS plasmid with anti‐GAL4, anti‐H4K20me1, and anti‐H4K20me3 antibodies. Values are depicted relative to isotype control. Data are means ± SEM (n = 3).

Replication efficiency of FR‐DS and FR‐UAS‐DS plasmids in GAL4, GAL4‐PR‐Set7, and Gal4‐PRset7SETmut cells. Replication efficiencies are depicted relative to the FR‐DS plasmid arbitrarily set as 100% in every cell line. Data are means ± SEM (n = 4).

ChIP‐qPCR analysis at the FR, UAS, and DS sites with anti‐GAL4, anti‐MCM3, anti‐H4K20me1, and anti‐H4K20me3 using chromatin from GAL4, GAL4‐PR‐Set7, and GAL4‐PR‐Set7SETmut cells expressing EBNA1 and transfected with similar amounts of FR‐DS or FR‐UAS‐DS plasmids. The y‐axis represents the relative fold enrichment of the specific antibody versus isotype control. Data are means ± SEM (n = 5).

Figure 3
Figure 3. PR‐Set7 improves origin activity through Suv4‐20h activity on H4K20me1

ChIP‐qPCR analysis of H4K20me1 and H4K20me3 levels at ORIRDH, UAS, and FR in the HEK293 EBNA1+ Gal4‐PR‐Set7‐expressing cell line in the absence (left panel) and presence of the Suv4‐20h inhibitor A196 (right panels). Data are means ± SEM (n = 4).

ChIP‐qPCR analysis of MCM3 levels at ORIRDH, UAS, and FR in EBNA1/Gal4‐PR‐Set7‐expressing cell lines in the absence (left panel) and presence of A196 (right panel). Data are means ± SEM (n = 4).

Quantitation of replicating FR‐ORIRDH (black) and FR‐UAS‐ORIRDH (striped) plasmids in EBNA1/GAL4‐PR‐Set7‐expressing cells in the presence and absence of A196. The replication efficiency is relative to ORIRDH in untreated cells. Data are means ± SEM (n = 4).

Data information: Significance was determined employing an unpaired two‐tailed t‐test. *< 0.05.
Figure 4
Figure 4. Loss of Suv4‐20h impairs the timing of late‐replicating heterochromatin

Immunoblot analysis (left panel) of H4K20me1, H4K20me2, and H4K20me3 levels and replication‐timing profile (right panel) of a 23‐Mb fragment of chromosome 11 (cytogenetic coordinates qA31 to qB1.2) in MEFs364.2 untreated or 4 days after 4‐hydroxytamoxifen (4OHT) treatment. Arrows point to delayed domains in 4OHT‐treated cells.

Immunoblot analysis (left panel) of histone H4, H4K20me1, H4K20me2, and H4K20me3 levels and replication‐timing profile (right panel) of the same fragment of chromosome 11 as above in MEFs364.2 5 days after transduction with retrovirus encoding histone H4WT or H4K20A mutant. Arrows point to delayed domains in H4K20A‐expressing cells.

Box‐plot showing the percentage of delayed replication domains in each timing categories in 4OHT‐treated MEFs364.2.

Size distribution (megabase) of delayed domains in MEFs364.2 treated with 4OHT.

Box‐plot showing gene coverage (percentage) in unaffected early, mid‐, and late domains, in delayed domains, and in stochastic replication domains of 4OHT‐treated MEFs364.2.

Box‐plot showing H3K27ac coverage levels in unaffected early, mid‐, and late domains, and in delayed domains, and in stochastic replication domains of 4OHT‐treated MEFs364.2.

Box‐plot showing H3K9me2 coverage levels in unaffected early, mid‐, and late domains, in delayed domains, and in stochastic replication domains of 4OHT‐treated MEFs364.2.

Data information: (*) Statistical significance was detected when a Student's test (t‐test) was performed with < 10−3 (see Appendix Table S1 for detailed statistical analysis). Inside the box‐plot graphs, the thick line represents the median, the limit of the boxes corresponds to the 0.25 to 0.75 quartiles with whiskers extending to the maximum value of 1.5 times the interquartile range.
Figure EV2
Figure EV2. Loss of Suv4‐20h in MEFs affects the replication of late S‐phase

Proliferation rates of mouse embryonic fibroblasts MEFs364.2 (SUV4‐20H2 −/− , SUV4‐20H1 −/flox, Cre‐ER cells) untreated (blue curve) or treated with 4OHT (red curve) to induce the loss of H4K20me2/3. Data are means ± SD (n = 3).

FACS analysis of MEFs364.2 (SUV4‐20H1 flox/− ; SUV420H2 −/−) treated or not with 4OHT at the time of the replication‐timing analysis. The black arrow points to the accumulation of replicating (BrdU‐positive) 4OHT‐treated cells in late S‐phase.

Replication‐timing profiles of the whole chromosome 17 and at higher magnitude of the chromatin region 35979972‐44013079 (8 Mb, mm9) in wild‐type (black line) and constitutive SUV420H1/H2‐null MEFs (gray line). Arrows point to delayed mid/late domains. Lower panel is the replication‐timing profile of the same chromatin region of chromosome 17 in untreated and 4OHT‐treated (SUV4‐20H null) MEFs364.2 (SUV4‐20H1 flox/− ; SUV4‐20H2 −/−; CRE‐ER). The blue line indicates the same nucleotide position in all replication‐timing profiles.

Figure 5
Figure 5. Suv4‐20h‐mediated H4K20me3 is required for the appropriate licensing and activity of a subset of origins in late‐replicating heterochromatin

ChIP‐qPCR analysis of H4K20me3 levels at the control early‐firing MYC origin and at origins in delayed and non‐delayed late domains of chromosome 11 in untreated and 4OHT‐treated MEFs364.2 using anti‐H4K20me3 and an isotype non‐relevant antisera (IgG) as control. NEG corresponds to a negative control region 5 kb downstream of ORI‐1. Coordinates of studied ORIs are indicated in Appendix Table S2. Errors bars represent SD (n = 3).

ChIP‐qPCR analysis of MCM2 levels at the same origins as above in untreated and 4OHT‐treated MEFs364.2 stably expressing FLAG‐tagged MCM2 and using anti‐FLAG or IgG as ChIP control. Errors bars represent SD (n = 3).

Quantitation of the relative SNS enrichment of the same origins as above origin in untreated and 4OHT‐treated MEFs364.2 and arbitrarily normalized with respect to the control origin. Data are means ± SD (n = 3).

Data information: (*) Statistical significance with P < 0.001 (unpaired t‐test).
Figure EV3
Figure EV3. Replication‐timing alterations in Suv4‐20h‐null cells occur in late‐replication domains harboring high levels of H4K20me3 at origins

ChIP‐qPCR analysis in untreated and 4OHT‐treated MEFs364.2 of H4K20me3 levels at the control early‐firing MYC origin and at late‐firing origins located in delayed late‐replication domains (ORI‐5 to ORI‐14) and in non‐delayed late‐replication domains (ORI5‐ND to ORI14‐ND) using anti‐H4K20me3 or isotype non‐relevant antisera (IgG) as a control. Data are means ± SD (n = 3). Coordinates of origins are indicated in Appendix Table S2.

ChIP‐qPCR analysis in untreated and 4OHT‐treated MEFs364.2 of H4K20me1 levels at the control early‐firing MYC origin and at late‐firing origins located in delayed late‐replication domains (ORI‐1 to ORI‐4) of chromosome 11 with anti‐H4K20me1 antibody showing four late‐replicating heterochromatin origins (ORI‐1 to ORI‐4) using anti‐H4K20me1 or isotype non‐relevant antisera (IgG) as a control. Data are means ± SD (n = 3). Coordinates of origins are indicated in Appendix Table S2.

Figure EV4
Figure EV4. Expression of histone H4K20A mutant impairs the licensing and activation efficiency of H4K20me3‐associated origins

ChIP‐qPCR analysis of H4K20me3 levels at the control early‐firing MYC origin and at late‐firing origins in delayed and non‐delayed late domains of chromosome 11 in histone H4WT‐ and H4K20A‐expressing MEFs364.2 using anti‐H4K20me3 and an isotype non‐relevant antisera (IgG) as control. The y‐axis represents the relative enrichment between H4K20me3 and control IgG immunoprecipitate DNA. NEG corresponds to a negative control region at 5 kb downstream of ORI‐1. Coordinates of ORIs are indicated in Appendix Table S2. Errors bars represent SD (n = 3). (*) Statistical significance (unpaired t‐test) with P < 0.05.

ChIP‐qPCR analysis of FLAG‐MCM5 levels at the same origins as above in histone H4WT‐ and H4K20A‐expressing MEFs364.2 using anti‐FLAG antibody and an isotype non‐relevant antisera (IgG) as control. Errors bars represent SD (n = 3). (*) Statistical significance (unpaired t‐test) with P < 0.05. Noted that ectopically expressed histone H4 proteins contained a C‐terminal epitope FLAG, which was not recognized by FLAG antibody in the context of formaldehyde‐crosslinked chromatin (control data not shown).

Quantitation of the relative SNS enrichment of the same origins as above in histone H4WT‐ and H4K20A‐expressing MEFs364.2. SNS enrichment was arbitrarily normalized with respect to the control origin. Data are means ± SD (n = 3). (*) Statistical significance (unpaired t‐test) with P < 0.05.

ChIP‐qPCR analysis of FLAG‐MCM5 levels at the same origins as above in untreated and 4OHT‐treated MEFs364.2 using anti‐FLAG antibody and an isotype non‐relevant antisera (IgG) as control. Errors bars represent SD (n = 3). (*) Statistical significance (unpaired t‐test) with P < 0.01.

Figure 6
Figure 6. ORCA/LRWD1 binds to H4K20me3‐associated origins and promotes the licensing and activity of these origins

ChIP‐qPCR analysis of ORCA levels at the same origins as in Fig 5 in untreated and 4OHT‐treated MEFs364.2 stably expressing FLAG‐tagged ORCA and using anti‐FLAG or IgG as a control. NEG corresponds to a negative ORCA binding site. Errors bars represent SD (n = 3).

Immunoblot analysis of HCF‐1 and ORCA proteins in whole‐cell extracts (inputs) and in chromatin‐enriched fraction in untreated and 4OHT‐treated MEFs364.2. Chromatin‐bound HCF‐1 protein was used as control of chromatin purification. Quantitation of ORCA protein levels are indicated relative to HCF‐1 and normalized with respect to the ORCA levels in untreated cells. (*) indicates a ORCA cleavage product in chromatin fraction after biochemical fractionation.

Immunoblot analysis of ORCA and tubulin proteins in immortalized MEFs transduced with lentivirus encoding shRNA control or two independent shRNA ORCA. Quantitation of ORCA protein levels is indicated relative to tubulin and normalized with respect to the ORC levels in control cells.

Replication‐timing profile of the same 23‐Mb chromosome fragment as in Fig 5A in MEFs treated with shRNA control or shRNA (2) ORCA. Arrows point to delayed late domains in ORCA‐depleted cells.

ChIP‐qPCR analysis of MCM5 levels at the control early‐firing MYC origin and H4K20me3‐associated origins in control and ORCA‐depleted MEFs expressing FLAG‐tagged MCM5 and using anti‐FLAG or IgG as ChIP control. Errors bars represent SD (n = 3).

Quantitation of the relative SNS enrichment of the same origins as above. SNS enrichment was arbitrarily normalized with respect to the control origin. Errors bars represent SD (n = 2).

ChIP‐qPCR analysis of H4K20me3 levels at the same origins as above using anti‐H4K20me3 and IgG as ChIP control. Errors bars represent SD (n = 3).

Data information: (*) Statistical significance (unpaired t‐test) with P < 0.05 (for panels E–G) and P < 0.005 (for panel A).
Figure EV5
Figure EV5. ORCA binding to H4K20me3‐associated origins is reduced in histone H4K20A‐expressing cells
ChIP‐qPCR analysis of ORCA levels at the control early‐firing MYC origin and at late‐firing origins in delayed and non‐delayed late domains of chromosome 11 in histone H4WT‐ and H4K20A‐expressing MEFs364.2 using anti‐FLAG antibody and an isotype non‐relevant antisera (IgG) as control. NEG corresponds to a negative control region at 5 kb downstream of ORI‐1. Coordinates of ORIs are indicated in Appendix Table S2. Errors bars represent SD (n = 3). (*) Statistical significance (unpaired t‐test) with P < 0.05. Noted that ectopically expressed histone H4 proteins contained a C‐terminal epitope FLAG, which was not recognized by FLAG antibody in the context of formaldehyde‐crosslinked chromatin (control data not shown).

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