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Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae
Published on: December 29, 2017
The histone methyltransferase SET8 is required for S-phase progression.
Stine Jørgensen1, Ingegerd Elvers, Morten Beck Trelle
1Biotech Research and Innovation Centre and 2Centre for Epigenetics, University of Copenhagen, 2200 Copenhagen N, Denmark.
The Journal of Cell Biology
|January 2, 2008
Summary
SET8 (SETD8) protein is crucial for mammalian cell proliferation and genomic stability. Its depletion causes DNA damage during replication, leading to S-phase arrest via a Chk1-mediated checkpoint.
Area of Science:
- Epigenetics and Molecular Biology
- Cellular Biology
- Genomics
Background:
- Histone posttranslational modifications regulate chromatin structure and function.
- SET8 (SETD8) is a histone methyltransferase that monomethylates histone H4-K20.
- The role of SET8 in mammalian cell proliferation is not well understood.
Purpose of the Study:
- To investigate the function of SET8 in mammalian cell proliferation and genomic stability.
- To determine the molecular mechanisms underlying SET8's role in cell cycle regulation and DNA damage response.
Main Methods:
- Small interfering RNA (siRNA) for SET8 depletion.
- Cell proliferation assays and cell cycle analysis (S phase accumulation).
- DNA double-strand break (DSB) detection and recruitment of DNA repair proteins (RPA, Rad51, 53BP1).
- Analysis of SET8 interaction with proliferating cell nuclear antigen (PCNA).
- Assessment of DNA replication fork progression.
- Codepletion studies with Rad51.
Main Results:
- SET8 depletion decreased cell proliferation and caused S-phase accumulation.
- SET8 inhibition induced DNA double-strand breaks (DSBs) during replication.
- DNA damage triggered a Chk1-mediated S-phase checkpoint.
- SET8 interacts with PCNA and is essential for replication fork progression.
- Codepletion of Rad51 abrogated DNA damage induced by SET8 depletion.
Conclusions:
- SET8 is essential for maintaining genomic stability in mammalian cells.
- Reduced SET8 expression leads to replication-associated DNA damage and a Chk1-dependent S-phase arrest.
- SET8 plays a critical role in DNA replication and repair pathways.
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