Characterisation of the histone methyltransferase SET8 in cell cycle progression and the DNA damage response

TY - BOOK

T1 - Characterisation of the histone methyltransferase SET8 in cell cycle progression and the DNA damage response

AU - Jørgensen, Stine

PY - 2008

Y1 - 2008

N2 - Histone modifications and their catalysing enzymes have within the last few years proven to be essential players in many biological processes. Due to their ability to modulate chromatin structure and affect signalling pathways they are found to affect diverse processes such as transcription, DNA recombination and repair. I therefore initiated a mass spectrometry based study to identify changes in histone modifications after DNA damage. By using SILAC labelling of cells to quantatively measure the changes in histone modifications, we observed a marked reduction in the level of monomethylated Histone H4 lysine 20 (H4K20me1) after damage. H4K20me1 is catalysed by the histone methyltransferase SET8 (aka PR-SET7), and functional studies of this enzyme revealed that SET8 is important for S phase progression. We also showed that depletion of SET8 in several different cancer cell lines results in accumulation of DNA double strand breaks (DSBs) and activation of CHK1, an important mediator of the S phase checkpoint. Furthermore, we demonstrated that the generation of DSBs, seen upon depletion of SET8, was dependent on replication. Additionally, we identified an interaction between SET8 and PCNA, a key component of the replication fork, further supporting the involvement of SET8 in replication, suggesting that SET8 may be required to support replication fork progression. Finally, we showed that SET8 was rapidly degraded by DNA damage such as UV and ionizing radiation (IR), which was accompanied by a decrease in H4K20me1. The removal of SET8 could be rescued by addition of a proteasomal inhibitor. Combined with data showing in vivo ubiquitylation of SET8, we suggest that the degradation of SET8 is mediated via the ubiquitin-proteasomal pathway.Collectively, these data suggest that SET8, during normal cellular homeostasis, has a role in supporting the chromatin structure to facilitate DNA replication. However, when cells are challenged by DNA damage efficient repair may be dependent on rapid degradation of SET8 and a reduction of the monomethyl mark on histone H4 lysine 20.

AB - Histone modifications and their catalysing enzymes have within the last few years proven to be essential players in many biological processes. Due to their ability to modulate chromatin structure and affect signalling pathways they are found to affect diverse processes such as transcription, DNA recombination and repair. I therefore initiated a mass spectrometry based study to identify changes in histone modifications after DNA damage. By using SILAC labelling of cells to quantatively measure the changes in histone modifications, we observed a marked reduction in the level of monomethylated Histone H4 lysine 20 (H4K20me1) after damage. H4K20me1 is catalysed by the histone methyltransferase SET8 (aka PR-SET7), and functional studies of this enzyme revealed that SET8 is important for S phase progression. We also showed that depletion of SET8 in several different cancer cell lines results in accumulation of DNA double strand breaks (DSBs) and activation of CHK1, an important mediator of the S phase checkpoint. Furthermore, we demonstrated that the generation of DSBs, seen upon depletion of SET8, was dependent on replication. Additionally, we identified an interaction between SET8 and PCNA, a key component of the replication fork, further supporting the involvement of SET8 in replication, suggesting that SET8 may be required to support replication fork progression. Finally, we showed that SET8 was rapidly degraded by DNA damage such as UV and ionizing radiation (IR), which was accompanied by a decrease in H4K20me1. The removal of SET8 could be rescued by addition of a proteasomal inhibitor. Combined with data showing in vivo ubiquitylation of SET8, we suggest that the degradation of SET8 is mediated via the ubiquitin-proteasomal pathway.Collectively, these data suggest that SET8, during normal cellular homeostasis, has a role in supporting the chromatin structure to facilitate DNA replication. However, when cells are challenged by DNA damage efficient repair may be dependent on rapid degradation of SET8 and a reduction of the monomethyl mark on histone H4 lysine 20.

M3 - Ph.D. thesis

BT - Characterisation of the histone methyltransferase SET8 in cell cycle progression and the DNA damage response

ER -