Methylation of histone H4 lysine 20 by PR-Set7 ensures the integrity of late replicating sequence domains inDrosophila

Abstract

The methylation state of lysine 20 on histone H4 (H4K20) has been linked to chromatin compaction, transcription, DNA repair and DNA replication. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7. PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which has been partially attributed to defects in origin selection and activation. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 and H4K20 methylation impact the replication program on a genomic scale. We employed genetic, cytological, and genomic approaches to better understand the role of PR-Set7 and H4K20 methylation in regulating DNA replication and genome stability in Drosophila cells. We find that deregulation of H4K20 methylation had no impact on origin activation throughout the genome. Instead, depletion of PR-Set7 and loss of H4K20me1 results in the accumulation of DNA damage and an ATR-dependent cell cycle arrest. Coincident with the ATR-dependent cell cycle arrest, we find increased DNA damage that is specifically limited to late replicating regions of the Drosophila genome, suggesting that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains.

Cell-cycle regulation of non-enzymatic functions of the Drosophila methyltransferase PR-Set7

“…The wild type and the GFP-PR-Set7 mutants were stably expressed in S2 cells that were then treated with either 7.2 dsRNA or an irrelevant LacZ dsRNA as a control. After 5 days, the efficacy of RNAi treatment was measured by immunoblotting and the progression through the cell cycle was examined by flow cytometry, as shown in Supplementary Figure S3 and in Figure 3B to E. In agreement with previous results ( 23 , 29 ), Drosophila PR-Set7 depletion in parental S2 cells triggered a decrease in H4K20me1 followed by an accumulation of cells with 4N or near 4N DNA content ( Supplementary Figure S3A ). These cells were likely arrested in late S and G2 phases as indicated by the low levels of the mitotic marker H3S10p in 7.2 treated cells compared to control cells ( Supplementary Figure S3A ).…”

“…Since the interaction between PR-Set7 and PCNA is conserved in Drosophila ( 28 ), we asked whether the PIP-motif is required for the proteolytic regulation of Drosophila PR-Set7. To address this question, we repeated cycloheximide chase experiments with S2 cell lines stably expressing either FLAG-tagged wild type PR-Set7 (FLAG-PR-Set7) or a FLAG-tagged PR-Set7 PIP-motif mutant (FLAG-PR-Set7 PIPmutant ) deficient for the interaction with PCNA ( 7 , 11 , 29 ). The results are shown in Figure 2A .…”

“…However, the use of histone replacement system to interrogate directly the function of specific lysine residues in Drosophila revealed that the mutation of histone H4 lysine 20 to alanine (H4K20A) has no impact on cell-cycle progression and on completion of development ( 31 ). Although exhibiting some DNA damage in wing discs, H4K20A mutant flies ( 29 ) are mainly viable in contrast to pr-set7 null flies that die at the pupal stage ( 20 , 24 ), thereby questioning whether the methylase activity of Drosophila PR-Set7 is required for cell-cycle progression. Our results show that a minimal functional version of Drosophila PR-Set7 (miniPR-Set7) harboring a mutation in the SET domain was unable to rescue the G2/M arrest induced by silencing of endogenous PR-Set7 (Figure 3 ).…”

DNA Replication Control During Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression

“…In Drosophila , decreased activity of PR-Set7, the methyltransferase responsible for H4K20 monomethylation, results in DNA damage checkpoint activation and a lengthened S phase in neuroblasts ( Sakaguchi and Steward 2007 ) and S2 cells ( Sakaguchi et al 2012 ). Consistent with these findings, Kc cells inhibited for H4K20 methylation exhibit a perturbed cell cycle with gross DNA damage, suggesting a defect in DNA replication ( Li et al 2016 ). Surprisingly, the inhibition of H4K20 methylation does not alter the genome-wide pattern of replication origin activation, but rather sensitizes late replicating domains to DNA damage.…”

Writing, erasing and reading histone lysine methylations

“… 259 , 272 Knockdown experiments in cell lines have revealed critical roles of SET8 in cell-cycle progression, DNA replication and genome stability. 273 , 274 , 275 Perinatal lethality and cell cycle defects resulting from Suv4-20h1/h2 double-knockout mice also support the function of H4K20 methylation in ensuring genomic integrity. 262 …”