Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability.

Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.

Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism.

Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases. Molecular mechanisms of active DNA demethylation began to surface only recently with the discovery of the 5-methylcytosine (5mC)-directed hydroxylase and base excision activities of ten-eleven translocation (TET) proteins and thymine DNA glycosylase (TDG). This implicated a pathway operating through oxidation of 5mC by TET proteins, which generates substrates for TDG-dependent base excision repair (BER) that then replaces 5mC with C. Yet, direct evidence for a productive coupling of TET with BER has never been presented. Here we show that TET1 and TDG physically interact to oxidize and excise 5mC, and proof by biochemical reconstitution that the TET-TDG-BER system is capable of productive DNA demethylation. We show that the mechanism assures a sequential demethylation of symmetrically methylated CpGs, thereby avoiding DNA double-strand break formation but contributing to the mutability of methylated CpGs.

Oestrogen receptor ß regulates epigenetic patterns at specific genomic loci through interaction with thymine DNA glycosylase.

BACKGROUND: DNA methylation is one way to encode epigenetic information and plays a crucial role in regulating gene expression during embryonic development. DNA methylation marks are established by the DNA methyltransferases and, recently, a mechanism for active DNA demethylation has emerged involving the ten-eleven translocator proteins and thymine DNA glycosylase (TDG). However, so far it is not clear how these enzymes are recruited to, and regulate DNA methylation at, specific genomic loci. A number of studies imply that sequence-specific transcription factors are involved in targeting DNA methylation and demethylation processes. Oestrogen receptor beta (ERß) is a ligand-inducible transcription factor regulating gene expression in response to the female sex hormone oestrogen. Previously, we found that ERß deficiency results in changes in DNA methylation patterns at two gene promoters, implicating an involvement of ERß in DNA methylation. In this study, we set out to explore this involvement on a genome-wide level, and to investigate the underlying mechanisms of this function. RESULTS: Using reduced representation bisulfite sequencing, we compared genome-wide DNA methylation in mouse embryonic fibroblasts derived from wildtype and ERß knock-out mice, and identified around 8000 differentially methylated positions (DMPs). Validation and further characterisation of selected DMPs showed that differences in methylation correlated with changes in expression of the nearest gene. Additionally, re-introduction of ERß into the knock-out cells could reverse hypermethylation and reactivate expression of some of the genes. We also show that ERß is recruited to regions around hypermethylated DMPs. Finally, we demonstrate here that ERß interacts with TDG and that TDG binds ERß-dependently to hypermethylated DMPs. CONCLUSION: We provide evidence that ERß plays a role in regulating DNA methylation at specific genomic loci, likely as the result of its interaction with TDG at these regions. Our findings imply a novel function of ERß, beyond direct transcriptional control, in regulating DNA methylation at target genes. Further, they shed light on the question how DNA methylation is regulated at specific genomic loci by supporting a concept in which sequence-specific transcription factors can target factors that regulate DNA methylation patterns.