The cytoplasmic DNA sensor cGAS detects DNA in ruptured micronuclei and activates an innate immune response. Innate immune activation has been implicated in autoimmunity and cancer. Here, Andrew Jackson and colleagues provide evidence for an underlying mechanism whereby ruptured micronuclei, which result from endogenous or exogenous chromosomal damage, activate a cell-autonomous inflammatory response via the cytoplasmic DNA sensor cGAS. They conclude that cGAS recognition of micronuclei may be acting as a kind of immune surveillance system in cells. Elsewhere in this issue, Roger Greenberg and colleagues report a link between mitosis and DNA-damage-induced inflammatory signalling involving cGAS in cancer cells. DNA is strictly compartmentalized within the nucleus to prevent autoimmunity1; despite this, cyclic GMP–AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage2,3,4,5,6. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis7, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.

Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

Several organisms have retained methyltransferase 2 ( Dnmt2 ) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2 -only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2 -only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2 -deficient flies. Furthermore, genetically modified Dnmt2 -only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms.

Induced pluripotent stem cells (IPSCs), with their unlimited regenerative capacity, carry the promise for tissue replacement to counter age‐related decline. However, attempts to realize in vivo iPSC have invariably resulted in the formation of teratomas. Partial reprogramming in prematurely aged mice has shown promising results in alleviating age‐related symptoms without teratoma formation. Does partial reprogramming lead to rejuvenation (i.e., “younger” cells), rather than dedifferentiation, which bears the risk of cancer? Here, we analyse the dynamics of cellular age during human iPSC reprogramming and find that partial reprogramming leads to a reduction in the epigenetic age of cells. We also find that the loss of somatic gene expression and epigenetic age follows different kinetics, suggesting that they can be uncoupled and there could be a safe window where rejuvenation can be achieved with a minimized risk of cancer.