Summary DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.

Ranaviruses such as frog virus 3 ([FV3]family Iridoviridae) are increasingly prevalent pathogens that infect reptiles, amphibians, and fish worldwide. Whereas studies in the frog Xenopus laevis have revealed the critical involvement of CD8 T-cell and antibody responses in host resistance to FV3, little is known about the role played by innate immunity to infection with this virus. We have investigated the occurrence, composition, activation status, and permissiveness to infection of peritoneal leukocytes (PLs) in Xenopus adults during FV3 infection by microscopy, flow cytometry, and reverse transcription-PCR. The total number of PLs and the relative fraction of activated mononucleated macrophage-like cells significantly increase as early as 1 day postinfection (dpi), followed by NK cells at 3 dpi, before the peak of the T-cell response at 6 dpi. FV3 infection also induces a rapid upregulation of proinflammatory genes including arginase 1, interleukin-1␤, and tumor necrosis factor alpha. Although PLs are susceptible to FV3 infection, as evidenced by apoptotic cells, active FV3 transcription, and the detection of viral particles by electron microscopy, the infection is weaker (fewer infectious particles), more transitory, and involves a smaller fraction (less than 1%) of PLs than the kidney, the main site of infection. However, viral DNA remains detectable in PLs for at least 3 weeks postinfection, past the point of viral clearance observed in the kidneys. This suggests that although PLs are actively involved in anti-FV3 immune responses, some of these cells can be permissive and harbor quiescent, asymptomatic FV3.

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA–stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3′ overhang inhibits 5′→3′ resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo.

These findings provide a platform for fertility preservation in prepubertal boys undergoing gonadotoxic treatments.

ABSTRACT:Frog virus 3 (FV3) or FV3-like viruses (Iridoviridae) infect a wide range of amphibian species, and they are becoming increasingly and causally associated with amphibian disease outbreaks worldwide. We have established the frog Xenopus laevis as an experimental model to study host defense and pathogenesis of FV3 infection. Although X. laevis adults usually clear FV3 infection within a few weeks, viral DNA has been detected in the kidneys several months after they had been experimentally infected; virus also has been detected in seemingly healthy nonexperimentally infected adults. Based on this information, we hypothesized that covert FV3 infection may occur in Xenopus. We first conducted a survey that detected FV3 by polymerase chain reaction (PCR) in the kidneys (the main site of FV3 infection) in a significant fraction of X. laevis raised in five different locations in the United States. Asymptomatic FV3 carriers also were detected by initiation of an acute systemic FV3 infection in frogs that had been immunosupressed by sublethal c-irradiation. Finally, we focused on macrophages as a potential site for viral persistence, and we showed that FV3 can infect peritoneal macrophages in vitro as determined by reverse transcriptase-PCR detection of viral mRNAs. Unlike kidney cell lines that are readily killed by FV3, infected macrophages, like uninfected macrophages, survived up to 12 days. Viral transcription also was detected in macrophages from animals up to 12 days after infection. These results suggest that FV3 can become quiescent in resistant species such as Xenopus, thereby making these species potential viral reservoirs.