The Molecular Mechanism Of SET8 Regulated UHRF1 In Cell Cycle

UHRF1 (Ubiquitin-like with PHD and Ring Finger domains 1) is an important epigenetic regulator. There are five function domains, UBL, TTD, PHD, SRA, and RING in UHRF1. It mainly has two functions, one is to promote cell proliferation. A lot of research proved that in cancer cells UHRF1 is highly expressed. The other is the maintenance of DNA methylation. The mechanism of UHRF1-mediated maintenance of DNA methylation is that its TTD domain can bind H3K9me2/3 and SRA domain can bind hemimethylated DNA, both recruit DNMT1 to maintain DNA methylation.UHRF1 is regulated in cell cycle. The protein level of UHRF1 is highest during S phase, lowest during G2/M phase. UHRF1 is an E3 ubiquitin ligase. It can catalyze ubiquitination of itself and other substrates. UHRF1 can also be phosphorylated. The phosphorylation inhibits UHRF1 ubiquitination, so the UHRF1 protein level is regulated during cell cycle. Whether there are other post-translational modifications that regulate UHRF1 should be investigated.SET8 (PR-SET7 or KMT5a) is a single subunit methylase. It monomethylates H4K20. If the level of H4K20me is high, DNA will be damaged, or chromatin will fold abnormally. In mouse model, high level of H4K20me could lead to death during embryonic period. SET8 can also methylate non-histone substrates such as p53. SET8 could regulate gene transcription by monoethylating p53 at K382. The protein level of SET8 is regulated by cell cycle. It is lowest during S phase and highest during G2/M phase. Since the protein levels of UHRF1 and SET8 are negatively correlated in cell cycle, whether there is any relationship between them should be investigated.Our project primary researched the mechanism of SET8 regulating UHRF1. We found SET8 could promote UHRF1 ubiquitin degradation. We also found SET8 could methylate UHRF1 on K385 site and the major ubiquitination site is K500. We also found when we knock out SET8, the level of DNA methylation increased in NITH3T3 cell line. Moreover UHRF1-K385A and UHRF1-K500A could not rescue DNA methylation in UHRF1-/-E14 cell line. Overall proved that K385 and K500 is very important.