The Molecular Mechanism Of SET8 Regulating UHRF1 And Genomic Methylation In Cell Cycle

SET8, so-called KMT5a or PR-SET7 is one of the lysine methyltransferase with SET domain as the enzymatic activity center. It specifically monomethylates H4K20 to H4K20me1, which is essential for DNA replication initiation. Non-histone proteins such as p53 and PCNA, can also be methylated by SET8. Methylation of p53 by SET8 blocks DNA damage response, which fails p53 to act as a tumor suppressor. Knockout of SET8 promotes the pro-apoptotic function of p53. PCNA is related to cell proliferation and methylation of PCNA by SET8 stablizes its protein level. High level of PCNA and SET8 is found in many tumor samples. Therefore, non-histone proteins methylation by SET8 is an important regulator in clinical therapy.The expression of SET8 is regulated by cell cycle. The protein level of SET8 is lower during S phase, while higher during G2/M phase. SET8 can be ubiquitinated by CRL4(CDT2) complex or APC complex. The high expression of SET8 blocks DNA replication, which causes cells to arrest at Gl/S phase. The degradation of SET8 in S phase is essential for normal operation of cells.UHRF1, so-called ICBP90 or NP95 plays a vital role in maintaining DNA methylation. It contains five domains, which are UBL, Tudor, PHD, SRA and RING domains. During S phase, UHRF1 binds to hemi-methylated DNA through its SRA domain to recruit DNMT1 for DNA methylation maintenance. Meanwhile, its Tudor domain specifically recognizes H3K9me2 and H3K9me3 and contributes to DNA maintenance methylation as well. UHRF1 is an E3 ligase with its RING domain. The expression of UHRF1 is also regulated by cell cycle. Its protein level is lower during G2/M phase, while higher in S phase. High level of UHRF1 is detected in some cancer cell lines and tumor samples, which may serve as a marker for early cancer diagnose.Our project focuses on whether SET8 can regulate UHRF1. Although the protein levels of SET8 and UHRF1 are reversely correlated in cell cycle, SET8 does promote UHRF1 ubiquitination dependent on its methyltransferase activity.We find that SET8 overexpression leads to decrease in UHRF1 protein level, while SET8 knockdown leads to increase in the level of UHRF1, and potentially increases overall genomic DNA methylation to some extent. Mass spectrometry analysis indicates that a methylation site in UHRFl is K385, and the main ubiquitination site is K500. The protein levels of both mutants of these two sites do not change during cell cycle. SET8 does not have effect on the degradation of these two mutants. Co-immunoprecipitation shows that SET8 interacts with UHRF1, and this interaction relies on PHD domain of UHRF1 instead of SET8 methyltransferase activity or the modification sites K385 and K500. Consistent with the potential regulation of UHRF1 by SET8, demethyltransferase LSD1 makes UHRF1 more stable in cell. These results illustrate SET8 as a regulator of UHRF1 and reveal their mutual interaction.