Andrea Leitch

Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus

Aicardi-Goutières syndrome (AGS) presents as a severe neurological brain disease and is a genetic mimic of the sequelae of transplacentally acquired viral infection. Evidence exists for a perturbation of innate immunity as a primary pathogenic event in the disease phenotype. Here, we show that TREX1, encoding the major mammalian 3′ → 5′ DNA exonuclease, is the AGS1 gene, and AGS-causing mutations result in abrogation of TREX1 enzyme activity. Similar loss of function in the Trex1−/− mouse leads to an inflammatory phenotype. Our findings suggest an unanticipated role for TREX1 in processing or clearing anomalous DNA structures, failure of which results in the triggering of an abnormal innate immune response.

Mutations in genes encoding ribonuclease H2 subunits cause Aicardi-Goutières syndrome and mimic congenital viral brain infection

Aicardi-Goutières syndrome (AGS) is an autosomal recessive neurological disorder, the clinical and immunological features of which parallel those of congenital viral infection. Here we define the composition of the human ribonuclease H2 enzyme complex and show that AGS can result from mutations in the genes encoding any one of its three subunits. Our findings demonstrate a role for ribonuclease H in human neurological disease and suggest an unanticipated relationship between ribonuclease H2 and the antiviral immune response that warrants further investigation.

Enzymatic Removal of Ribonucleotides from DNA Is Essential for Mammalian Genome Integrity and Development

Summary The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.

Mutations in the pre-replication complex cause Meier-Gorlin syndrome

Meier-Gorlin syndrome (ear, patella and short-stature syndrome) is an autosomal recessive primordial dwarfism syndrome characterized by absent or hypoplastic patellae and markedly small ears. Both pre- and post-natal growth are impaired in this disorder, and although microcephaly is often evident, intellect is usually normal in this syndrome. We report here that individuals with this disorder show marked locus heterogeneity, and we identify mutations in five separate genes: ORC1, ORC4, ORC6, CDT1 and CDC6. All of these genes encode components of the pre-replication complex, implicating defects in replication licensing as the cause of a genetic syndrome with distinct developmental abnormalities.

Mutations in ORC1, encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome.

Studies into disorders of extreme growth failure (for example, Seckel syndrome and Majewski osteodysplastic primordial dwarfism type II) have implicated fundamental cellular processes of DNA damage response signaling and centrosome function in the regulation of human growth. Here we report that mutations in ORC1, encoding a subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. We establish that these mutations disrupt known ORC1 functions including pre-replicative complex formation and origin activation. ORC1 deficiency perturbs S-phase entry and S-phase progression. Additionally, we show that Orc1 depletion in zebrafish is sufficient to markedly reduce body size during rapid embryonic growth. Our data suggest a model in which ORC1 mutations impair replication licensing, slowing cell cycle progression and consequently impeding growth during development, particularly at times of rapid proliferation. These findings establish a novel mechanism for the pathogenesis of microcephalic dwarfism and show a surprising but important developmental impact of impaired origin licensing.

cGAS surveillance of micronuclei links genome instability to innate immunity

DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP–AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. 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 chromothripsis, 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.

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

RNA:DNA hybrids are a novel molecular pattern sensed by TLR9.

The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen‐associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral‐derived sequences efficiently induce pro‐inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88‐dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease.

Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy

Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.

Stat3 is required for the development of skin cancer

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is constitutively activated in a variety of human malignancies, including prostate, lung, brain, breast, and squamous cell carcinomas. Inhibition of activated Stat3 leads to decreased proliferation and apoptosis of many cancer-derived cell lines, while the introduction of a constitutively activated form of Stat3 into immortalized human breast epithelial cells and rodent fibroblasts results in cellular transformation. Collectively, these data suggest a role for Stat3 in oncogenesis. A new study from Chan et al. is the first to demonstrate a requirement for Stat3 in de novo epithelial carcinogenesis in vivo. Using the two-step model of chemically induced skin carcinogenesis, the authors demonstrated that mice deficient in Stat3 were completely resistant to skin tumor development.