GTF3A mutations predispose to herpes simplex encephalitis by disrupting biogenesis of the host-derived RIG-I ligand RNA5SP141.

Herpes simplex virus 1 (HSV-1) infects several billion people worldwide and can cause life-threatening herpes simplex encephalitis (HSE) in some patients. Monogenic defects in components of the type I interferon system have been identified in patients with HSE, emphasizing the role of inborn errors of immunity underlying HSE pathogenesis. Here, we identify compound heterozygous loss-of-function mutations in the gene GTF3A encoding for transcription factor IIIA (TFIIIA), a component of the RNA polymerase III complex, in a patient with common variable immunodeficiency and HSE. Patient fibroblasts and GTF3A gene-edited cells displayed impaired HSV-1-induced innate immune responses and enhanced HSV-1 replication. Chromatin immunoprecipitation sequencing analysis identified the 5S ribosomal RNA pseudogene 141 (RNA5SP141), an endogenous ligand of the RNA sensor RIG-I, as a transcriptional target of TFIIIA. GTF3A mutant cells exhibited diminished RNA5SP141 expression and abrogated RIG-I activation upon HSV-1 infection. Our work unveils a crucial role for TFIIIA in transcriptional regulation of a cellular RIG-I agonist and shows that GTF3A genetic defects lead to impaired cell-intrinsic anti-HSV-1 responses and can predispose to HSE.

Modifying PCDH19 levels affects cortical interneuron migration.

PCDH19 is a transmembrane protein and member of the protocadherin family. It is encoded by the X-chromosome and more than 200 mutations have been linked to the neurodevelopmental PCDH-clustering epilepsy (PCDH19-CE) syndrome. A disturbed cell-cell contact that arises when random X-inactivation creates mosaic absence of PCDH19 has been proposed to cause the syndrome. Several studies have shown roles for PCDH19 in neuronal proliferation, migration, and synapse function, yet most of them have focused on cortical and hippocampal neurons. As epilepsy can also be caused by impaired interneuron migration, we studied the role of PCDH19 in cortical interneurons during embryogenesis. We show that cortical interneuron migration is affected by altering PCDH19 dosage by means of overexpression in brain slices and medial ganglionic eminence (MGE) explants. We also detect subtle defects when PCDH19 expression was reduced in MGE explants, suggesting that the dosage of PCDH19 is important for proper interneuron migration. We confirm this finding in vivo by showing a mild reduction in interneuron migration in heterozygote, but not in homozygote PCDH19 knockout animals. In addition, we provide evidence that subdomains of PCDH19 have a different impact on cell survival and interneuron migration. Intriguingly, we also observed domain-dependent differences in migration of the non-targeted cell population in explants, demonstrating a non-cell-autonomous effect of PCDH19 dosage changes. Overall, our findings suggest new roles for the extracellular and cytoplasmic domains of PCDH19 and support that cortical interneuron migration is dependent on balanced PCDH19 dosage.

Innovative mouse models for the tumor suppressor activity of Protocadherin-10 isoforms.

BACKGROUND: Nonclustered mouse protocadherin genes (Pcdh) encode proteins with a typical single ectodomain and a cytoplasmic domain with conserved motifs completely different from those of classic cadherins. Alternative splice isoforms differ in the size of these cytoplasmic domains. In view of the compelling evidence for gene silencing of protocadherins in human tumors, we started investigations on Pcdh functions in mouse cancer models. METHODS: For Pcdh10, we generated two mouse lines: one with floxed exon 1, leading to complete Pcdh10 ablation upon Cre action, and one with floxed exons 2 and 3, leading to ablation of only the long isoforms of Pcdh10. In a mouse medulloblastoma model, we used GFAP-Cre action to locally ablate Pcdh10 in combination with Trp53 and Rb1 ablation. From auricular tumors, that also arose, we obtained tumor-derived cell lines, which were analyzed for malignancy in vitro and in vivo. By lentiviral transduction, we re-expressed Pcdh10 cDNAs. RNA-Seq analyses were performed on these cell families. RESULTS: Surprisingly, not only medulloblastomas were generated in our model but also tumors of tagged auricles (pinnae). For both tumor types, ablation of either all or only long isoforms of Pcdh10 aggravated the disease. We argued that the perichondrial stem cell compartment is at the origin of the pinnal tumors. Immunohistochemical analysis of these tumors revealed different subtypes. We obtained several pinnal-tumor derived (PTD) cell lines and analyzed these for anchorage-independent growth, invasion into collagen matrices, tumorigenicity in athymic mice. Re-expression of either the short or a long isoform of Pcdh10 in two PTD lines counteracted malignancy in all assays. RNA-Seq analyses of these two PTD lines and their respective Pcdh10-rescued cell lines allowed to identify many interesting differentially expressed genes, which were largely different in the two cell families. CONCLUSIONS: A new mouse model was generated allowing for the first time to examine the remarkable tumor suppression activity of protocadherin-10 in vivo. Despite lacking several conserved motifs, the short isoform of Pcdh10 was fully active as tumor suppressor. Our model contributes to scrutinizing the complex molecular mechanisms of tumor initiation and progression upon PCDH10 silencing in many human cancers.

ADAR1 interaction with Z-RNA promotes editing of endogenous double-stranded RNA and prevents MDA5-dependent immune activation.

Loss of function of adenosine deaminase acting on double-stranded RNA (dsRNA)-1 (ADAR1) causes the severe autoinflammatory disease Aicardi-Goutières syndrome (AGS). ADAR1 converts adenosines into inosines within dsRNA. This process called A-to-I editing masks self-dsRNA from detection by the antiviral dsRNA sensor MDA5. ADAR1 binds to dsRNA in both the canonical A-form and the poorly defined Z conformation (Z-RNA). Mutations in the Z-RNA-binding Zα domain of ADAR1 are common in patients with AGS. How loss of ADAR1/Z-RNA interaction contributes to disease development is unknown. We demonstrate that abrogated binding of ADAR1 to Z-RNA leads to reduced A-to-I editing of dsRNA structures formed by base pairing of inversely oriented short interspersed nuclear elements. Preventing ADAR1 binding to Z-RNA triggers an MDA5/MAVS-mediated type I interferon response and leads to the development of lethal autoinflammation in mice. This shows that the interaction between ADAR1 and Z-RNA restricts sensing of self-dsRNA and prevents AGS development.

A new mouse model to study the role of ectopic Nanos3 expression in cancer.

BACKGROUND: NANOS3 is a gene conserved throughout evolution. Despite the quite low conservation of Nanos sequences between different organisms and even between Nanos paralogs, their role in germ cell development is remarkably universal. Human Nanos3 expression is normally restricted to the gonads and the brain. However, ectopic activation of this gene has been detected in various human cancers. Until now, Nanos3 and other Nanos proteins have been studied almost exclusively in germ cell development. METHODS: Transgenic mice were generated by targeted insertion of a human Nanos3 cDNA into the ROSA26 locus. The transgene could be spatiotemporally induced by Cre recombinase activity removing an upstream floxed STOP cassette. A lung tumor model with ectopic Nanos3 expression was based on the lung-specific activation of the reverse tetracycline transactivator gene, in combination with a tetO-CMV promoter controlling Cre expression. When doxycycline was provided to the mice, Cre was activated leading to deletion of TP53 alleles and activation of both oncogenic KRasG12D and Nanos3. Appropriate controls were foreseen. Tumors and tumor-derived cell cultures were analyzed in various ways. RESULTS: We describe the successful generation of Nanos3LSL/- and Nanos3LSL/LSL mice in which an exogenous human NANOS3 gene can be activated in vivo upon Cre expression. These mice, in combination with different conditional and doxycycline-inducible Cre lines, allow the study of the role of ectopic Nanos3 expression in several cancer types. The Nanos3LSL mice were crossed with a non-small cell lung cancer (NSCLC) mouse model based on conditional expression of oncogenic KRas and homozygous loss of p53. This experiment demonstrated that ectopic expression of Nanos3 in the lungs has a significant negative effect on survival. Enhanced bronchiolar dysplasia was observed when Nanos3-expressing NSCLC mice were compared with control NSCLC mice. An allograft experiment, performed with cell cultures derived from primary lung tumors of control and Nanos3-expressing NSCLC mice, revealed lymph node metastasis in mice injected with Nanos3-expressing NSCLC cells. CONCLUSIONS: A new mouse model was generated allowing examination of Nanos3-associated pathways and investigation of the influence of ectopic Nanos3 expression in various cancer types. This model might identify Nanos3 as an interesting target in cancer therapeutics.

GC Content of Early Metazoan Genes and Its Impact on Gene Expression Levels in Mammalian Cell Lines.

With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure-function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines.

NBPF1, a tumor suppressor candidate in neuroblastoma, exerts growth inhibitory effects by inducing a G1 cell cycle arrest.

BACKGROUND: NBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved. METHODS: Expression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21(CIP1/WAF1) were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-of-function approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool. RESULTS: We show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects. In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis. CONCLUSIONS: We demonstrated that NBPF1 exerts different tumor suppressive effects, depending on the cell line analyzed, and provide new clues into the molecular mechanism of the enigmatic NBPF proteins.

The human NANOS3 gene contributes to lung tumour invasion by inducing epithelial-mesenchymal transition.

We have explored the role of the human NANOS3 gene in lung tumour progression. We show that NANOS3 is over-expressed by invasive lung cancer cells and is a prognostic marker for non-small cell lung carcinomas (NSCLCs). NANOS3 gene expression is restricted in testis and brain and is regulated by epigenetic events. It is up-regulated in cultured cells undergoing epithelial - mesenchymal transition (EMT). NANOS3 over-expression in human NSCLC cell lines enhances their invasiveness by up-regulating EMT, whereas its silencing induces mesenchymal - epithelial transition. NANOS3 represses E-cadherin at the transcriptional level and up-regulates vimentin post-transcriptionally. Also, we show that NANOS3 binds mRNAs encoding vimentin and regulates the length of their poly(A) tail. Finally, NANOS3 can also protect vimentin mRNA from microRNA-mediated repression. We thus demonstrate a role for NANOS3 in the acquisition of invasiveness by human lung tumour cells and propose a new mechanism of post-transcriptional regulation of EMT.

The transcriptional repressor Kaiso localizes at the mitotic spindle and is a constituent of the pericentriolar material.

Kaiso is a BTB/POZ zinc finger protein known as a transcriptional repressor. It was originally identified through its in vitro association with the Armadillo protein p120ctn. Subcellular localization of Kaiso in cell lines and in normal and cancerous human tissues revealed that its expression is not restricted to the nucleus. In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody. We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex. We analyzed the functions of different domains of Kaiso by visualizing the subcellular distribution of GFP-tagged Kaiso fragments throughout the cell cycle. Our results indicate that two domains are responsible for targeting Kaiso to the centrosomes and microtubules. The first domain, designated SA1 for spindle-associated domain 1, is located in the center of the Kaiso protein and localizes at the spindle microtubules and centrosomes; the second domain, SA2, is an evolutionarily conserved domain situated just before the zinc finger domain and might be responsible for localizing Kaiso towards the centrosomal region. Constructs containing both SA domains and Kaiso's aminoterminal BTB/POZ domain triggered the formation of abnormal centrosomes. We also observed that overexpression of longer or full-length Kaiso constructs led to mitotic cell arrest and frequent cell death. Knockdown of Kaiso accelerated cell proliferation. Our data reveal a new target for Kaiso at the centrosomes and spindle microtubules during mitosis. They also strongly imply that Kaiso's function as a transcriptional regulator might be linked to the control of the cell cycle and to cell proliferation in cancer.

Chibby interacts with NBPF1 and clusterin, two candidate tumor suppressors linked to neuroblastoma.

The NBPF genes are members of a gene family that underwent a remarkable increase in their copy number during recent primate evolution. The NBPF proteins contain 5 to 40 copies of a domain known as the NBPF repeat or DUF1220. Very little is known about the function of these domains or about the NBPF proteins. We performed a yeast two-hybrid screening with the aminoterminal domain of NBPF11 and found that Chibby, a documented repressor of Wnt signaling, interacts with multiple NBPF proteins. More specifically, a coiled-coil region in the NBPF proteins interacts with the coiled-coil domain in the carboxyterminal region of Chibby. Nonetheless, this interaction did not influence the repressor function of Chibby in a TOPFLASH reporter assay. Using Chibby as bait in a new yeast two-hybrid screening, we identified clusterin as a binding protein. Chibby and clusterin were co-immunoprecipitated with NBPF1, suggesting the formation of a tri-molecular complex. Although we have not pinpointed the role of these mutual interactions, the possible formation of a macromolecular complex of three candidate tumor suppressor proteins, including the enigmatic NBPF1, points at important functional implications.

Expression of protocadherin-1 (Pcdh1) during mouse development.

Protocadherin-1 (Pcdh1) is a member of the delta-protocadherin subgroup of non-clustered protocadherins. We studied the expression of Pcdh1 from the early embryonic to the adult stage of mouse development by semi-quantitative RT-PCR and in situ hybridization. Pcdh1 can be detected as early as embryonic day 9.5. In early embryogenesis, expression is especially prominent in blood vessels. During later development and in the adult mouse, organs derived from the embryonic gut, such as the esophagus, intestines, liver, lung, and submandibular gland, contain epithelia and other types of tissues that are Pcdh1-positive. Other positive organs include the brain, spinal cord, retina, peripheral ganglia, the inner ear, hair follicles, kidney, vagina, uterus, placenta, testis, prostate, and the seminal gland. The tight spatial and temporal regulation of Pcdh1 expression suggests that this protocadherin plays multiple roles not only during development but also in mature tissues and organs in the mouse.

A constitutional translocation t(1;17)(p36.2;q11.2) in a neuroblastoma patient disrupts the human NBPF1 and ACCN1 genes.

The human 1p36 region is deleted in many different types of tumors, and so it probably harbors one or more tumor suppressor genes. In a Belgian neuroblastoma patient, a constitutional balanced translocation t(1;17)(p36.2;q11.2) may have led to the development of the tumor by disrupting or activating a gene. Here, we report the cloning of both translocation breakpoints and the identification of a novel gene that is disrupted by this translocation. This gene, named NBPF1 for Neuroblastoma BreakPoint Family member 1, belongs to a recently described gene family encoding highly similar proteins, the functions of which are unknown. The translocation truncates NBPF1 and gives rise to two chimeric transcripts of NBPF1 sequences fused to sequences derived from chromosome 17. On chromosome 17, the translocation disrupts one of the isoforms of ACCN1, a potential glioma tumor suppressor gene. Expression of the NBPF family in neuroblastoma cell lines is highly variable, but it is decreased in cell lines that have a deletion of chromosome 1p. More importantly, expression profiling of the NBPF1 gene showed that its expression is significantly lower in cell lines with heterozygous NBPF1 loss than in cell lines with a normal 1p chromosome. Meta-analysis of the expression of NBPF and ACCN1 in neuroblastoma tumors indicates a role for the NBPF genes and for ACCN1 in tumor aggressiveness. Additionally, DLD1 cells with inducible NBPF1 expression showed a marked decrease of clonal growth in a soft agar assay. The disruption of both NBPF1 and ACCN1 genes in this neuroblastoma patient indicates that these genes might suppress development of neuroblastoma and possibly other tumor types.

A novel gene family NBPF: intricate structure generated by gene duplications during primate evolution.

Partial and complete genome duplications occurred during evolution and resulted in the creation of new genes and gene families. We identified a novel and intricate human gene family located primarily in regions of segmental duplications on human chromosome 1. We named it NBPF, for neuroblastoma breakpoint family, because one of its members is disrupted by a chromosomal translocation in a neuroblastoma patient. The NBPF genes have a repetitive structure with high intragenic and intergenic sequence similarity in both coding and noncoding regions. These similarities might expose these genomic regions to illegitimate recombination, resulting in structural variation in the NBPF genes. The encoded proteins contain a highly conserved domain of unknown function, which we have named the NBPF repeat. In silico analysis combined with the isolation of multiple full-length cDNA clones showed that several members of this gene family are abundantly expressed in a large variety of tissues and cell lines. Strikingly, no discernable orthologues could be identified in the completed genomes of fruit fly, nematode, mouse, or rat, but sequences with low homology could be isolated from the draft canine and bovine genomes. Interestingly, this gene family shows primate-specific duplications that result in species-specific arrays of NBPF homologous sequences. Overall, this novel NBPF family reflects the continuous evolution of primate genomes that resulted in large physiological differences, and its potential role in this process is discussed.

Localization of the 17q breakpoint of a constitutional 1;17 translocation in a patient with neuroblastoma within a 25-kb segment located between the ACCN1 and TLK2 genes and near the distal breakpoints of two microdeletions in neurofibromatosis type 1 patients.

We have constructed a 1.4-Mb P1 artificial chromosome/bacterial artificial chromosome (PAC/BAC) contig spanning the 17q breakpoint of a constitutional translocation t(1;17)(p36.2;q11.2) in a patient with neuroblastoma. Three 17q breakpoint-overlapping cosmids were identified and sequenced. No coding sequences were found in the immediate proximity of the 17q breakpoint. The PAC/BAC contig covers the region between the proximally located ACCN1 gene and the distally located TLK2 gene and SCYA chemokine gene cluster. The observation that the 17q breakpoint region could not be detected in any of the screened yeast artificial chromosome libraries and the localization of the 17q breakpoint in the vicinity of the distal breakpoints of two microdeletions in patients with neurofibromatosis type 1 suggest that this chromosomal region is genetically unstable and prone to rearrangements.