Using Mouse Models To Study The Function Of UHRF1 And UHRF2

Epigenetics refers to the study of heritable changes in gene activity not caused by DNA sequence variations. As one of the epigenetic mechanisms, DNA methylation pattern is established mainly by DNMT3A/B and maintained by DNMT1. UHRF1, a multi domain protein, can bind to hemimethylated DNA through SRA domain, then recruits DNMT1 to replication forks to convert the hemimethylated DNA to fully methylated DNA. Previous results in our lab showed that UHRF1 could also bind to H3K9me2/3 through TTD domain, and TTD domain mutant could only partially rescue the DNA methylation defect in Uhrfl-/-ES cells. To study the function of the binding of UHRF1 to H3K9me2/3 in DNA methylation maintenance, we constructed Uhrfl knock-in (KI) mice with a point mutation in the TTD domain. Pull-down assays confirmed that the mutant UHRF1 could not bind to H3K9me2/3, but the homozygous KI mice showed no overt abnormalities, indicating that the binding ability is not essential in mouse development. However, by HPLC and RRBS analysis, we found that global DNA methylation decreased at a significant level in both MEF cells and adult tissues (liver, lung and brain) to various degrees. These results indicate that the binding of UHRF1 to H3K9me2/3 is important for DNA methylation maintenance.UHRF2, a homolog of UHRF1, also possesses ubiquitin ligase activity but is not involved in DNA methylation maintenance. Through collaboration, we got gene modified UhrJ2-/- mice, which showed no overt abnormalities, demonstrating that UHRF2 is not essential for mouse development. However, by HPLC analysis we found global decrease in DNA hydroxyl-methylation level but not methylation level in the brain of Uhr/2-/- mice, while the transcription level of Tetl/2/3 remained nearly unchanged. These results indicate that UHRF2 may specifically regulate DNA hydroxymethylation by other unknown mechanisms.