The histone methyltransferase SET8 is required for S-phase progression

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.

Histone H4 lysine 20 monomethylation promotes transcriptional repression by L3MBTL1

“…Quantitative reverse transcriptase-PCR analysis confirmed an increase in cyclin E mRNA levels in the siPR-SET7, but not mock or siG9a, transfected cells ( Figure 6c). As we did not see any cell cycle defects in siPR-SET7-treated K562 cells, this change in cyclin E levels is unlikely due to difficulty in S-phase progression as reported for U2OS cells with knockdown of PR-SET7 (as reported by Jorgensen et al (2007) and Tardat et al (2007)). Using several siRNAs against L3MBTL1, we have been able to achieve less than a 50% knockdown in K562 cells.…”

“…The cell cycle-regulated chromatin association of L3MBTL1 mimics the emergence of the H4K20me1 mark (but not its peak), and the significant decrease in L3MBTL1 levels that precede the onset of mitosis is likely critical for proper cell cycle progression. Notably, the absence of PR-SET7 (and the H4K20me1 mark) also results in defects in cell cycle progression (Karachentsev et al, 2005;Jorgensen et al, 2007;Tardat et al, 2007). The absence of L3MBTL1 in the premitotic fraction 9 (Figure 3b) when H4K20me1 is abundant suggests that additional post-translational modifications of histones, or of L3MBTL1, may target the protein for premitotic destruction.…”

Catalytic Function of the PR-Set7 Histone H4 Lysine 20 Monomethyltransferase Is Essential for Mitotic Entry and Genomic Stability

“…Furthermore, we also defined that the cell cycle-dependent changes in monomethylated H4K20 occurred locally at specific genomic regions. Similar to recent reports, RNAi-mediated depletion of PR-Set7 and H4K20 monomethylation resulted in delayed S phase progression (29,30). However, we found that the PR-Set7 RNAi cells were capable of completing S phase and, in a p53-independent manner, arrested at prometaphase, when PR-Set7 and monomethylated H4K20 levels peak.…”

“…Besides functioning in transcription, it was recently found that PR-Set7 plays a role in mammalian cell cycle progression by binding proliferating cell nuclear antigen and localizing to rep-lication foci (28 -30). Although the loss of PR-Set7 by RNAi eventually resulted in defective replication fork activity and delayed S phase progression, several cell cycles were required to achieve this phenotype (29,30). Furthermore, we previously demonstrated that expression of PR-Set7 was tightly cell cycleregulated where it was undetectable at G 1 /S, slowly increased during late S phase, and peaked during mitosis, concomitant with its catalytic activity (31).…”

Nucleosome assembly and epigenetic inheritance

“…PCNA directly interacts with a series of chromatin modifying enzymes, and potentially recruits them to replication foci. These factors include CAF-1 (Shibahara and Stillman 1999;Moggs et al, 2000), HDACs (Milutinovic et al, 2002), ATP dependent chromatin remodeling complex WSTF-SNF2h (Poot et al, 2004), histone lysine methyltransferse PR-SET7 (Jørgensen et al, 2007;Huen et al, 2008) and DNA methyltransferase DNMT1 (Leonhardt et al, 1992;Chuang et al, 1997). CAF-1 is also reported to interact with chromatin factors, including MBD1 (methyl CpG-binding protein 1) and SETDB1 during heterochromatin DNA replication (Reese et al, 2003;Sarraf and Stancheva, 2004).…”