Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.

Dual Functions of the RFTS Domain of Dnmt1 in Replication-Coupled DNA Methylation and in Protection of the Genome from Aberrant Methylation.

In mammals, DNA methylation plays important roles in embryogenesis and terminal differentiation via regulation of the transcription-competent chromatin state. The methylation patterns are propagated to the next generation during replication by maintenance DNA methyltransferase, Dnmt1, in co-operation with Uhrf1. In the N-terminal regulatory region, Dnmt1 contains proliferating cell nuclear antigen (PCNA)-binding and replication foci targeting sequence (RFTS) domains, which are thought to contribute to maintenance methylation during replication. To determine the contributions of the N-terminal regulatory domains to the DNA methylation during replication, Dnmt1 lacking the RFTS and/or PCNA-binding domains was ectopically expressed in embryonic stem cells, and then the effects were analyzed. Deletion of both the PCNA-binding and RFTS domains did not significantly affect the global DNA methylation level. However, replication-dependent DNA methylation of the differentially methylated regions of three imprinted genes, Kcnq1ot1/Lit1, Peg3, and Rasgrf1, was impaired in cells expressing the Dnmt1 with not the PCNA-binding domain alone but both the PCNA-binding and RFTS domains deleted. Even in the absence of Uhrf1, which is a prerequisite factor for maintenance DNA methylation, Dnmt1 with both the domains deleted apparently maintained the global DNA methylation level, whilst the wild type and the forms containing the RFTS domain could not perform global DNA methylation under the conditions used. This apparent maintenance of the global DNA methylation level by the Dnmt1 lacking the RFTS domain was dependent on its own DNA methylation activity as well as the presence of de novo-type DNA methyltransferases. We concluded that the RFTS domain, not the PCNA-binding domain, is solely responsible for the replication-coupled DNA methylation. Furthermore, the RFTS domain acts as a safety lock by protecting the genome from replication-independent DNA methylation.

Characterization of human papillomavirus infection in north Taiwan.

The detection of human papillomavirus (HPV) is very important for the evaluation of preventative strategies for cervical cancer. The major objective of this study was to characterize the prevalence of different genotypes of HPV in north Taiwan to contribute to the epidemiological knowledge of HPV infections. Papanicolaou (Pap) cervical smears were collected from 10,543 women aged between 14 and 87 years. The polymerase chain reaction (PCR) and DNA array hybridization techniques were used to genotype 51 different HPV strains. HPV was detected in 1,577 women, which gave an overall HPV prevalence rate of 15%. Forty-eight different genotypes were found in these patients, which included 9.7% that were high-risk HPV (HR-HPV) genotypes. The most common types of HR-HPV in patients, in descending order of frequency, were HPV 52, 16, 58, 56, 39, 51, 18, 68, 31, 33, 59, 45, and 35. HPV 52 was the most frequent type in every age group. The four most common HR-HPV types were found in 56.6% of the patients infected with HR-HPV. In cases that were infected with multiple HPV genotypes, 69.2% had at least one HR-HPV genotype. The rate of infection with HR-HPV was higher in the younger age groups than the older ones. In conclusion, 48 HPV genotypes were identified from a large study of cervical screening samples and the prevalence of HPV genotypes in different age groups was very diverse. The formulation of a public health strategy for HPV vaccination should take into account the prevalence of various HR-HPV/LR-HPV genotypes.