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Erratum in Publisher Correction: Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates. Szenker-Ravi E, Ott T, Khatoo M, Moreau de Bellaing A, Goh WX, Chong YL, Beckers A, Kannesan D, Louvel G, Anujan P, Ravi V, Bonnard C, Moutton S, Schoen P, Fradin M, Colin E, Megarbane A, Daou L, Chehab G, Di Filippo S, Rooryck C, Deleuze JF, Boland A, Arribard N, Eker R, Tohari S, Ng AY, Rio M, Lim CT, Eisenhaber B, Eisenhaber F, Venkatesh B, Amiel J, Crollius HR, Gordon CT, Gossler A, Roy S, Attie-Bitach T, Blum M, Bouvagnet P, Reversade B.Nat Genet. 2022 Jun;54(6):906. doi: 10.1038/s41588-022-01053-8.PMID: 35304595 No abstract available.; International audience; The vertebrate left-right axis is specified during embryogenesis by a transient organ: the left-right organizer (LRO). Species including fish, amphibians, rodents and humans deploy motile cilia in the LRO to break bilateral symmetry, while reptiles, birds, even-toed mammals and cetaceans are believed to have LROs without motile cilia. We searched for genes whose loss during vertebrate evolution follows this pattern and identified five genes encoding extracellular proteins, including a putative protease with hitherto unknown functions that we named ciliated left-right organizer metallopeptide (CIROP). Here, we show that CIROP is specifically expressed in ciliated LROs. In zebrafish and Xenopus, CIROP is required solely on the left side, downstream of the leftward flow, but upstream of DAND5, the first asymmetrically expressed gene. We further ascertained 21 human patients with loss-of-function CIROP mutations presenting with recessive situs anomalies. Our findings posit the existence of an ancestral genetic module that has twice disappeared during vertebrate evolution but remains essential for distinguishing left from right in humans.

Background Structural birth defects occur in approximately 3% of live births; most such defects lack defined genetic or environmental causes. Despite advances in surgical approaches, pharmacologic prevention remains largely out of reach. Methods We queried worldwide databases of 20,248 families that included children with neurodevelopmental disorders and that were enriched for parental consanguinity. Approximately one third of affected children in these families presented with structural birth defects or microcephaly. We performed exome or genome sequencing of samples obtained from the children, their parents, or both to identify genes with biallelic pathogenic or likely pathogenic mutations present in more than one family. After identifying disease-causing variants, we generated two mouse models, each with a pathogenic variant "knocked in," to study mechanisms and test candidate treatments. We administered a small-molecule Wnt agonist to pregnant animals and assessed their offspring. Results We identified homozygous mutations in WLS, which encodes the Wnt ligand secretion mediator (also known as Wntless or WLS) in 10 affected persons from 5 unrelated families. (The Wnt ligand secretion mediator is essential for the secretion of all Wnt proteins.) Patients had multiorgan defects, including microcephaly and facial dysmorphism as well as foot syndactyly, renal agenesis, alopecia, iris coloboma, and heart defects. The mutations affected WLS protein stability and Wnt signaling. Knock-in mice showed tissue and cell vulnerability consistent with Wnt-signaling intensity and individual and collective functions of Wnts in embryogenesis. Administration of a pharmacologic Wnt agonist partially restored embryonic development. Conclusions Genetic variations affecting a central Wnt regulator caused syndromic structural birth defects. Results from mouse models suggest that what we have named Zaki syndrome is a potentially preventable disorder. (Funded by the National Institutes of Health and others.).

Deficiencies in DNA repair and DNA degrading nucleases can lead to accumulation of cytosolic DNA and the activation of STING pathways in tumor cells. The DNA sensors that recognize cytosolic DNA in tumor cells are not known. Here we show that double-stranded DNA and RNA:DNA hybrids bind cGAS and activate the cGAS-STING pathway in TRAMP-C2 prostate and other cancer cells. STING function in TRAMP-C2 cells was critical for rejection and immune cell infiltration of TRAMP-C2 tumors. In contrast, STING agonists failed to active STING and induce type I interferon (IFN) expression in many tested tumor cells. Inhibition of JAK2 and STAT3 restored type I IFN expression in these tumor cells. Intratumoral injections of IL-6, a known activator of JAK2 and STAT3, significantly reduced the anti-tumor effects of cGAMP. In summary, we show that IL-6 suppresses recognition of cytosolic DNA by the cGAS-STING pathway in tumor cells, which contributes to anti-tumor responses. Funding Statement: This work was supported by the NRF grant HUJ-CREATE-Cellular and Molecular Mechanisms of Inflammation and a ministry of education grant. Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: Mice were housed according to the IACUC guidelines of the National University of Singapore (R14-0204).

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in flow cytometry and microscopy. Here we have investigated various PicoGreen variants for the ability to stain low amounts of cytosolic DNA present in many tumor cells. Analysis of stained cells by flow cytometry and fluorescent microscopy showed that certain variants improved the ability to stain low levels of cytosolic DNA when compared to the commercially available PicoGreen molecule.

Apoptosis is a controlled means of eliminating damaged cells without causing an inflammatory response or tissue damage. The mechanisms that contribute to the suppression of an inflammatory response upon apoptosis of cells are poorly understood. Here, we report that apoptotic cells release the interleukin-1 receptor antagonist (IL1RA). The release of IL1RA depended on the DNA damage response, caspase 9, and caspase 3. De novo translation, classical secretion pathways, or N-glycosylation was not required for the release of IL1RA. The amounts of IL1RA released by apoptotic cells impaired IL1-induced expression of IL6. In summary, we demonstrate that the release of IL1RA in response to genotoxic stress contributes to the immunosuppressive effects of apoptotic cells. Cancer Immunol Res; 4(4); 294–302. ©2016 AACR.

The DNA damage response (DDR) alerts the immune system to the danger posed by DNA damage through the induction of damage-associated molecular pattern molecules, chemokines, and ligands for activating immune receptors such as lymphocyte function-associated antigen 1 (LFA-1), NKG2D, and DNAX accessory molecule 1 (DNAM-1). Here we provide evidence that OVA(257-264) -pulsed fibroblasts gain the ability to activate naive OT-I CD8(+) T cells in response to DNA damage. The ability of fibroblasts to activate OT-I CD8(+) T cells depended on the upregulation of ICAM-1 on fibroblasts and DNAM-1 expression of CD8(+) T cells. OVA(257-264) -pulsed fibroblasts were able to induce a protective T-cell response against B16-OVA cells in a DDR-dependent manner. Hence, the DDR may alert the immune system to the presence of potentially dangerous cells by upregulating the expression of ligands that can induce the activation of innate and adaptive immune cells.

The four R-spondin secreted ligands (RSPO1–RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1–3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers. Independently of the LGR4/5/6 receptors, RSPO2 acts as a direct antagonistic ligand to RNF43 and ZNRF3 during embryogenesis, and specifies the position and number of limbs that an embryo should form.

The presence of damaged and microbial DNA can pose a threat to the survival of organisms. Cells express various sensors that recognize specific aspects of such potentially dangerous DNA. Recognition of damaged or microbial DNA by sensors induces cellular processes that are important for DNA repair and inflammation. Here, we review recent evidence that the cellular response to DNA damage and microbial DNA are tightly intertwined. We also discuss insights into the parameters that enable DNA sensors to distinguish damaged and microbial DNA from DNA present in healthy cells.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that catalyses the synthesis of cGAMP, which serves as a ligand for stimulator of interferon (IFN) genes (STING). Activation of STING results in production of type I IFNs through phosphorylation of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3). Type I IFNs are critical participants in the innate and adaptive immune recognition of cancer cells. Deficiencies in the cGAS-STING signalling pathway have been reported in many tumors. This mitigates expression of type I IFNs and may thus contribute to non-inflamed tumor microenvironment. In particular, a non-T-cell-inflamed tumor microenvironment correlates with poor patient survival. STING agonists may contribute to antitumor activity by upregulating proinflammatory cytokines and type I IFNs and various STING agonists are now being tested in clinical trials. The role of STING in immune cells is relatively well understood; however, its role in tumor cells has not yet been described in detail. Here we show that cGAS is able to bind to DNA present in the cytosol of tumor cells and subsequently induces STING signaling leading to expression of type I IFNs. Knockout of STING in mouse prostate TRAMP-C2 tumor cells resulted in higher tumor burden and reduced infiltration of immune cells such as dendritic and CD8+ T-cells into the tumor microenvironment. Consistently, treatment with the STING agonist cGAMP elevated type I IFNs levels in TRAMP-C2 cells and led to a reduced tumor volume compared to untreated control. This suggests a pivotal role of tumoral STING in antitumor immunity. However, despite intact STING expression, most tested human cancer cell lines were not responsive to various STING agonists and consequently failed to upregulate expression of type I IFNs. In contrast, all tested tumor cell lines responded to Poly(I:C)-induced TLR3 signaling, suggesting that failure to respond to cGAMP was due to a defect upstream of TBK1. Downregulation of cGAS did not render cells responsive to cGAMP, indicating that inability to respond to cGAMP is due to deficiencies of STING to activate TBK1. In the human and mouse prostate cancer cell lines DU145 and TRAMP-C2, respectively, autocrine IL-6 rendered cells unresponsive to STING agonists. While treatment with anti-IL-6 antibodies restored cGAMP responsiveness in DU145 cells, addition of recombinant IL-6 suppressed cGAMP-mediated upregulation of type I IFNs. In summary, our data suggest that cytosolic DNA activates the cGAS-STING signaling pathway. A functional STING is pivotal for eliciting an effective anticancer immune response. In most human cancer cell lines, however, STING signalling is inhibited. Since STING agonists are being evaluated in clinical trials, it is crucial to understand mechanisms that mediate STING unresponsiveness. We show that in tested prostate cancer cells, IL-6 signals contribute to unresponsiveness of STING and blocking of IL-6 can restore responsiveness towards STING agonists. Citation Format: Manuel Adrian Suter, Wendy Ya Ling Zhang, Muznah Bte Nazar Khan Khatoo, Nikki Ya Ling Tan, Chien Tei Too, Shubhita Tripathi, Paul A. MacAry, Veronique Angeli, Stephan Gasser. Tumoral STING is required for effective anticancer immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B189.

In this Letter, the surname of author Lena Vlaminck was misspelled 'Vlaeminck'. In addition, author Kris Vleminckx should have been associated with affiliation 16 (Center for Medical Genetics, Ghent University, Ghent, Belgium). These have been corrected online.

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

Self-DNA is present in the cytosol of many cancer cells and can promote effective immune rejection of tumor cells, but the mechanisms leading to the presence of cytosolic DNA are unknown. Here, we report that the cleavage of genomic DNA by DNA structure-specific endonuclease MUS81 and PARP-dependent DNA repair pathways leads to the accumulation of cytosolic DNA in prostate cancer cells. The number of nuclear MUS81 foci and the amount of cytosolic dsDNA increased in tandem from hyperplasia to clinical stage II prostate cancers and decreased at stage III. Cytosolic DNA generated by MUS81 stimulated DNA sensor STING-dependent type I interferon (IFN) expression and promoted phagocytic and T cell responses, resulting in type I and II IFN-mediated rejection of prostate tumor cells via mechanisms that partly depended on macrophages. Our results demonstrate that the tumor suppressor MUS81 alerts the immune system to the presence of transformed host cells.

We recently provided evidence that genome-derived DNA is present in the cytosol of many tumor cells. Genomic loci that give rise to cytosolic DNA can potentially form non-B DNA structures including triple-stranded RNA:DNA structures (R-loops). The RNA:DNA-specific endonuclease RNaseh1 reduced the levels of cytosolic DNA and type I interferon-dependent rejection of B-cell lymphoma suggesting that cytosolic DNA may contribute to immune surveillance of B-cell lymphoma.

Background: RNA:DNA hybrids exist in the nucleus and mitochondria but not in the cytosol, except in viral infection. Results: RNA:DNA hybrids exist in the cytosol of various human cells and are mediated by RNA polymerase III (Pol III), which regulates the microRNA machinery. Conclusion: Cytosolic RNA:DNA hybrids are regulated by Pol III. Significance: Previous unknown cytosolic RNA:DNA hybrids may have physiological relevance to miRNA machinery and RNA transport., RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells.