Study On The Stability Regulation Of Essential Protein UHRF1 In Epigenetics

Epigenetics refers to the study of heritable changes transmitted by mechanisms other than changes in the DNA sequence. Many phenomenoa, which couldn’t be explained by traditional geneticsincludinggenomic imprinting and maternal effects, are subjectsof investigation in the fields of epigenetics. The researches towards DNA methylation and histone modifications are two of most important subfields in epigenetics. DNA methylation is a biochemical process involving the addition of a methyl group to the cytosine or adenine DNA nucleotides (cytosine of CpG dinucleotides in mammal), which is mediated by DNA methyltransferases DNMT3A/B and an accessory factor DNMT3L, while DNMT1mainly plays a role in maintaining DNA methylation during DNA replication.Recent studies demonstrate that UHRF1(Ubiquitin-like, with PHD and RING finger domains1) is critical for the maintenance of DNA methylation patterns in the heritance of DNA methylation profiles. The SRA domain of UHRFl selectively binds hemimethylated CpG and recruits DNMT1to methylate the newly synthesized DNA strand, which ensures the faithful propagation of DNA methylation patterns. The epigenetic regulator UHRFl is composed of multiple functional domains, including the UBL, Tudor, PHD, SRA, and RING domains, which are responsible for the recognition of histone and DNA methylation to regulate gene expression in euchromatic region.Previous studies demonstrate that UHRF1is involved in the DNA damage response and the G1/S transition during cell cycle. However, it remains unclear about the function of UHRF1or the regulation of UHRF1in these processes. To address these questions, we purified UHRF1complex via TAP (Tandem Affinity Purification) and identified that the deubiquitylase USP7(HAUSP) interacts with UHRF1. Their interaction is dynamic during cell cycle, along with oscillation of UHRF1levels. Further experiments indicate that UHRF1Ser652is specifically phosphorylated in M phase of the cell cycle, which disrupts the interaction between UHRFl and USP7and subsequently destablizes UHRF1.As UHRFl is expected to be ubiquitylated before being deubiquitylated by USP7. We analyzed UHRF1protein sequence with bioinformatic and biochemical methods and found that a DSG motif in N-terminus of UHRF1is critical for UHRF1degradation by SCFβ-TxCP. It’s well documented that SCFβ-TrCP complex is targeted to its substrates by recognition of phosphorylated DSG, and we uncovered that CK18phosphorylates UHRF1in vitro and in vivo. Importantly, the phosphorylation is necessary for the ubiquitylation by SCF complex, which is triggered by DNA damage and induces subsequent UHRFl degradation.Taken together, we have identified asophisticated mechanism of positive and negative regulation of UHRF1protein level in vivo. More importantly, the demonstration of phosphorylation regulation of UHRF1stability sheds light on understanding how extracellular and intracellular stimuli influence the establishment and switch of epigenetic patterns through signaling pathways.