DNA polymerase alpha interacts with PrSet7 and mediates H4K20 monomethylation in Drosophila

Abstract

BSTRACTIn human cells, appropriate monomethylation of histone H4 lysine 20 by PrSet7 (also known as SET8 and SETD7) is important for the correct transcription of specific genes and timely progression through the cell cycle. Over-methylation appears to be prevented through the interaction of PrSet7 with proliferating cell nuclear antigen (PCNA), which targets PrSet7 for destruction through the pathway mediated by CRL4Cdt2 (the cullin ring finger ligase-4 complex containing Cdt2).However, the factors involved in positive regulation of PrSet7 histone methylation remain undefined. Here, we present biochemical and genetic evidence for a previously undocumented interaction between Drosophila PrSet7 (dPrSet7) and DNA polymerase a in Drosophila. Depletion of the polymerase reduces H4K20 monomethylation suggesting that it is required for dPrSet7 histone methylation activity. We also show that the interaction between PCNA and PrSet7 is conserved in Drosophila, but is only detectable in chromatin fractions. Consistent with this, S2 cells show a significant loss of chromatin-bound dPrSet7 protein as S phase progresses. Based on these data we suggest that interaction with the DNA polymerase represents an important route for stimulation of PrSet7 histone methylase activity that is mediated by allowing loading of dPrSet7 onto chromatin or its subsequent activation.

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

“…Depletion of PR-Set7 for 72 h in Kc167 cells resulted in an accumulation of cells with 4N or near 4N DNA content, similar to previous reports in Drosophila S2 cells (13). Interestingly, we also observed an accumulation of cells with 4N DNA content following the simultaneous depletion of PR-Set7 and Suv4-20 despite the slight increase in H4K20me1.…”

“…PR-Set7 harbors a conserved PIP-box motif and undergoes PCNA- and CRL4 Cdt2 -mediated degradation during S phase (8–12). This interaction between the PR-Set7 PIP-box and PCNA is conserved in Drosophila cell lines, where the levels of PR-Set7 and H4K20me1 display a similar cell cycle oscillation pattern as seen in mammalian systems (13). …”

“…Interestingly, we also observed an accumulation of cells with 4N DNA content following the simultaneous depletion of PR-Set7 and Suv4-20 despite the slight increase in H4K20me1. Given that PR-Set7 activity is strictly coupled to the cell cycle and H4K20me1 is a transient epigenetic mark deposited in late S phase and converted to H4K20me2/3 by late mitosis (8,13), we hypothesize that the cell cycle arrest may not be due to the depletion of parental H4K20me1 levels, but rather the accumulation of nascent histone H4.…”

“…Cell cycle regulated levels of PR-Set7 start to peak in late S phase with the accumulation of H4K20 monomethylation (8,13). Recent reports indicate that Drosophila PR-Set7 is coupled to the replication fork via an interaction with DNA Pol alpha (13), which may suggest that the accumulation of H4K20 monomethylation in late S phase will be associated with recently replicated sequences.…”

“…Recent reports indicate that Drosophila PR-Set7 is coupled to the replication fork via an interaction with DNA Pol alpha (13), which may suggest that the accumulation of H4K20 monomethylation in late S phase will be associated with recently replicated sequences. We used cytological and genomic approaches to characterize and monitor the accumulation of H4K20 monomethylation throughout the cell cycle in Drosophila Kc167 cells.…”