A fully sequenced collection of homozygous EMS mutants for forward and reverse genetic screens in Arabidopsis thaliana.

Genetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a research model. Here, we describe the whole-genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high-confidence mutations per HEM line, over three independent mutations altering protein sequences are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental, immune and physiological traits confirmed the efficacy of the tool for identifying both null, knockdown and gain-of-function alleles. The possibility of conducting subtle repeated phenotyping and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm-browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.

The translation regulator Zar1l controls timing of meiosis in Xenopus oocytes.

Oocyte maturation and early embryo development occur in vertebrates in the near absence of transcription. Thus, sexual reproduction of vertebrates critically depends on the timely translation of mRNAs already stockpiled in the oocyte. Yet how translational activation of specific mRNAs is temporally coordinated is still incompletely understood. Here, we elucidate the function of Zar1l, a yet uncharacterized member of the Zar RNA-binding protein family, in Xenopus oocytes. Employing TRIM-Away, we demonstrate that loss of Zar1l accelerates hormone-induced meiotic resumption of Xenopus oocytes due to premature accumulation of the M-phase-promoting kinase cMos. We show that Zar1l is a constituent of a large ribonucleoparticle containing the translation repressor 4E-T and the central polyadenylation regulator CPEB1, and that it binds directly to the cMos mRNA. Partial, hormone-induced degradation of Zar1l liberates 4E-T from CPEB1, which weakens translational repression of mRNAs encoding cMos and likely additional M-phase-promoting factors. Thus, our study provides fundamental insights into the mechanisms that ensure temporally regulated translation of key cell cycle regulators during oocyte maturation, which is essential for sexual reproductivity.

A natural diversity screen in Arabidopsis thaliana reveals determinants for HopZ1a recognition in the ZAR1-ZED1 immune complex.

Pathogen pressure on hosts can lead to the evolution of genes regulating the innate immune response. By characterizing naturally occurring polymorphisms in immune receptors, we can better understand the molecular determinants of pathogen recognition. ZAR1 is an ancient Arabidopsis thaliana NLR (Nucleotide-binding [NB] Leucine-rich-repeat [LRR] Receptor) that recognizes multiple secreted effector proteins from the pathogenic bacteria Pseudomonas syringae and Xanthomonas campestris through its interaction with receptor-like cytoplasmic kinases (RLCKs). ZAR1 was first identified for its role in recognizing P. syringae effector HopZ1a, through its interaction with the RLCK ZED1. To identify additional determinants of HopZ1a recognition, we performed a computational screen for ecotypes from the 1001 Genomes project that were likely to lack HopZ1a recognition, and tested ~300 ecotypes. We identified ecotypes containing polymorphisms in ZAR1 and ZED1. Using our previously established Nicotiana benthamiana transient assay and Arabidopsis ecotypes, we tested for the effect of naturally occurring polymorphisms on ZAR1 interactions and the immune response. We identified key residues in the NB or LRR domain of ZAR1 that impact the interaction with ZED1. We demonstrate that natural diversity combined with functional assays can help define the molecular determinants and interactions necessary to regulate immune induction in response to pathogens.

Translation repression by maternal RNA binding protein Zar1 is essential for early oogenesis in zebrafish.

A large amount of maternal RNA is deposited in oocytes and is reserved for later development. Control of maternal RNA translation during oocyte maturation has been extensively investigated and its regulatory mechanisms are well documented. However, translational regulation of maternal RNA in early oogenesis is largely unexplored. In this study, we generated zebrafish zar1 mutants that result in early oocyte apoptosis and fully penetrant male development. Loss of p53 suppresses the apoptosis in zar1 mutants and restores oocyte development. zar1 immature ovaries show upregulation of proteins implicated in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). More importantly, loss of Zar1 causes marked upregulation of zona pellucida (ZP) family proteins, while overexpression of ZP proteins in oocytes causes upregulation of stress-related activating transcription factor 3 (atf3), arguing that tightly controlled translation of ZP proteins is essential for ER homeostasis during early oogenesis. Furthermore, Zar1 binds to ZP gene mRNAs and represses their translation. Together, our results indicate that regulation of translational repression and de-repression are essential for precisely controlling protein expression during early oogenesis.

Using forward genetics in Nicotiana benthamiana to uncover the immune signaling pathway mediating recognition of the Xanthomonas perforans effector XopJ4.

The immune pathway responsible for perception of the Xanthomonas perforans effector XopJ4 was identified in the plant Nicotiana benthamiana. This pathogen causes significant yield loss in commercial tomato cultivation. Genetic mapping and viral-induced gene silencing were used to identify immune signaling components of the XopJ4 perception pathway in N. benthamiana. Transient complementation assays were performed to determine the functionality of gene variants and co-immunoprecipitation assays were used to gain insight into the molecular mechanism of the pathway. Two N. benthamiana ethyl methanesulfonate (EMS) mutants deficient for XopJ4 perception were identified as having loss-of-function mutations in the gene encoding the nucleotide binding, leucine-rich repeat (NLR) protein NbZAR1. Silencing of a receptor-like cytoplasmic kinase family XII gene, subsequently named XOPJ4 IMMUNITY 2 (JIM2), blocks perception of XopJ4. This study demonstrates the feasibility of conducting mutant screens in N. benthamiana to investigate the genetic basis of the plant immune system and other processes. The identification of NbZAR1 and JIM2 as mediating XopJ4 perception in N. benthamiana supports the model of ZAR1 being involved in the perception of many different pathogen effector proteins with specificity dictated by associated receptor-like cytoplasmic kinases.

Maize ARGOS1 (ZAR1) transgenic alleles increase hybrid maize yield.

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays AR GOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer's modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer's female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance.

Disruption of Zar1 leads to arrested oogenesis by regulating polyadenylation via Cpeb1 in tilapia (Oreochromis niloticus).

Oogenesis is a complex process regulated by precise coordination of multiple factors, including maternal genes. Zygote arrest 1 (zar1) has been identified as an ovary-specific maternal gene that is vital for oocyte-to-embryo transition and oogenesis in mouse and zebrafish. However, its function in other species remains to be elucidated. In the present study, zar1 was identified with conserved C-terminal zinc finger domains in Nile tilapia. zar1 was highly expressed in the ovary and specifically expressed in phase I and II oocytes. Disruption of zar1 led to the failed transition from oogonia to phase I oocytes, with somatic cell apoptosis. Down-regulation and failed polyadenylation of figla, gdf9, bmp15 and wee2 mRNAs were observed in the ovaries of zar1-/- fish. Cpeb1, a gene essential for polyadenylation that interacts with Zar1, was down-regulated in zar1-/- fish. Moreover, decreased levels of serum estrogen and increased levels of androgen were observed in zar1-/- fish. Taken together, zar1 seems to be essential for tilapia oogenesis by regulating polyadenylation and estrogen synthesis. Our study shows that Zar1 has different molecular functions during gonadal development by the similar signaling pathway in different species.

Characterization of Two Gonadal Genes, zar1 and wt1b, in Hermaphroditic Fish Asian Seabass (Lates calcarifer).

Zygote arrest-1 (Zar1) and Wilms' tumor 1 (Wt1) play an important role in oogenesis, with the latter also involved in testicular development and gender differentiation. Here, Lczar1 and Lcwt1b were identified in Asian seabass (Lates calcarifer), a hermaphrodite fish, as the valuable model for studying sex differentiation. The cloned cDNA fragments of Lczar1 were 1192 bp, encoding 336 amino acids, and contained a zinc-binding domain, while those of Lcwt1b cDNA were 1521 bp, encoding a peptide of 423 amino acids with a Zn finger domain belonging to Wt1b family. RT-qPCR analysis showed that Lczar1 mRNA was exclusively expressed in the ovary, while Lcwt1b mRNA was majorly expressed in the gonads in a higher amount in the testis than in the ovary. In situ hybridization results showed that Lczar1 mRNA was mainly concentrated in oogonia and oocytes at early stages in the ovary, but were undetectable in the testis. Lcwt1b mRNA was localized not only in gonadal somatic cells (the testis and ovary), but also in female and male germ cells in the early developmental stages, such as those of previtellogenic oocytes, spermatogonia, spermatocytes and spermatids. These results indicated that Lczar1 and Lcwt1b possibly play roles in gonadal development. Therefore, the findings of this study will provide a basis for clarifying the mechanism of Lczar1 and Lcwt1b in regulating germ cell development and the sex reversal of Asian seabass and even other hermaphroditic species.

ZAR1: Guardian of plant kinases.

A key facet of innate immunity in plants entails the recognition of pathogen "effector" virulence proteins by host Nucleotide-Binding Leucine-Rich Repeat Receptors (NLRs). Among characterized NLRs, the broadly conserved ZAR1 NLR is particularly remarkable due to its capacity to recognize at least six distinct families of effectors from at least two bacterial genera. This expanded recognition spectrum is conferred through interactions between ZAR1 and a dynamic network of two families of Receptor-Like Cytoplasmic Kinases (RLCKs): ZED1-Related Kinases (ZRKs) and PBS1-Like Kinases (PBLs). In this review, we survey the history of functional studies on ZAR1, with an emphasis on how the ZAR1-RLCK network functions to trap diverse effectors. We discuss 1) the dynamics of the ZAR1-associated RLCK network; 2) the specificity between ZRKs and PBLs; and 3) the specificity between effectors and the RLCK network. We posit that the shared protein fold of kinases and the switch-like properties of their interactions make them ideal effector sensors, enabling ZAR1 to act as a broad spectrum guardian of host kinases.

Two plant NLR proteins confer strain-specific resistance conditioned by an effector from Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker, a devastating disease threatening the Actinidia fruit industry. In a search for non-host resistance genes against Psa, we find that the nucleotide-binding leucine-rich repeat receptor (NLR) protein ZAR1 from both Arabidopsis and Nicotiana benthamiana (Nb) recognizes HopZ5 and triggers cell death. The recognition requires ZED1 in Arabidopsis and JIM2 in Nb plants, which are members of the ZRK pseudokinases and known components of the ZAR1 resistosome. Surprisingly, Arabidopsis ZAR1 and RPM1, another NLR known to recognize HopZ5, confer disease resistance to HopZ5 in a strain-specific manner. Thus, ZAR1, but not RPM1, is solely required for resistance to P. s. maculicola ES4326 (Psm) carrying hopZ5, whereas RPM1 is primarily required for resistance to P. s. tomato DC3000 (Pst) carrying hopZ5. Furthermore, the ZAR1-mediated resistance to Psm hopZ5 in Arabidopsis is insensitive to SOBER1, which encodes a deacetylase known to suppress the RPM1-mediated resistance to Pst hopZ5. In addition, hopZ5 enhances P. syringae virulence in the absence of ZAR1 or RPM1 and that SOBER1 abolishes such virulence function. Together the study suggests that ZAR1 may be used for improving Psa resistance in Actinidia and uncovers previously unknown complexity of effector-triggered immunity and effector-triggered virulence.

Bacterial Effectors Induce Oligomerization of Immune Receptor ZAR1 In Vivo.

Plants utilize nucleotide-binding, leucine-rich repeat receptors (NLRs) to detect pathogen effectors, leading to effector-triggered immunity. The NLR ZAR1 indirectly recognizes the Xanthomonas campestris pv. campestris effector AvrAC and Pseudomonas syringae effector HopZ1a by associating with closely related receptor-like cytoplasmic kinase subfamily XII-2 (RLCK XII-2) members RKS1 and ZED1, respectively. ZAR1, RKS1, and the AvrAC-modified decoy PBL2UMP form a pentameric resistosome in vitro, and the ability of resistosome formation is required for AvrAC-triggered cell death and disease resistance. However, it remains unknown whether the effectors induce ZAR1 oligomerization in the plant cell. In this study, we show that both AvrAC and HopZ1a can induce oligomerization of ZAR1 in Arabidopsis protoplasts. Residues mediating ZAR1-ZED1 interaction are indispensable for HopZ1a-induced ZAR1 oligomerization in vivo and disease resistance. In addition, ZAR1 residues required for the assembly of ZAR1 resistosome in vitro are also essential for HopZ1a-induced ZAR1 oligomerization in vivo and disease resistance. Our study provides evidence that pathogen effectors induce ZAR1 resistosome formation in the plant cell and that the resistosome formation triggers disease resistance.

Variation Screening of Zygote Arrest 1(ZAR1) in Women with Recurrent Zygote Arrest During IVF/ICSI Programs.

Human zygote arrest during in vitro culture is rare and the etiology is unclear. The oocyte-specific gene Zar1 plays an essential role in oocyte-embryo transition, and most embryos from Zar1 knockout female mice arrest at the one-cell stage. This study investigates whether maternal ZAR1 gene variations play a role in human zygote arrest. Sequence analysis of ZAR1 was conducted for 47 women with recurrent uncleaved zygotes in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles (≥ 70% zygotes uncleaved in at least 2 cycles), 93 women from IVF/ICSI cycles with normal uncleaved rate and live birth (control subset I) and 188 women with spontaneous pregnancy and live birth (control subset II). One novel synonymous variation (c.516C>T) and one novel intron variation (c.964-55A>T) of ZAR1 were identified in the zygote arrest group but not in any of the 188 controls. However, the bioinformatics analysis revealed that neither of the mutations in ZAR1 has effect on ZAR1 protein function. Compared with control subset I, the allele frequencies of rare SNPs rs117545505 and rs17609740 were significantly different in patients with zygote arrest (P = 0.047, OR = 3.66). Allele frequencies of these two SNPs were also significantly different between the case group and control subset II (P = 0.024, OR = 3.28). In conclusion, two SNPs in ZAR1 are associated with human zygote arrest, although additional proof is needed for validation.

Genotypic Variation in Resistance Gene-Mediated Calcium Signaling and Hormonal Signaling Involved in Effector-Triggered Immunity or Disease Susceptibility in the Xanthomonas campestris pv. Campestris-Brassica napus Pathosystem.

To characterize cultivar variation in resistance gene (R-gene)-mediated calcium signaling and hormonal regulation in effector-triggered immunity (ETI) and disease susceptibility, Xanthomonas campestris pv. campestris (Xcc) was inoculated in two Brassica napus cultivars (cvs. Capitol and Mosa). At 14 days post inoculation (DPI) with Xcc, there was a necrotic lesion in cv. Mosa along with the significant accumulation of H2O2 and malondialdehyde (MDA), whereas no visual symptom was observed in cv. Capitol. The cultivar variations in the R-gene expressions were found in response to Xcc. ZAR1 is a coiled-coil-nucleotide binding site-leucine-rich repeat (CC-NB-LRR)-type R-gene that is significantly induced in cv. Capitol, whereas toll/interleukin-1 receptor-nucleotide binding site-leucine-rich repeat (TIR-NB-LRR)-type R-gene, TAO1, is significantly upregulated in cv. Mosa Xcc-inoculated plants. The defense-related gene's non-race-specific disease resistance 1 (NDR1) and mitogen-activated protein kinase 6 (MAPK6) were enhanced, whereas calcium-dependent protein kinase (CDPK5) and calcium-sensing protein 60g (CBP60g) were depressed in cv. Capitol Xcc inoculated plants, and opposite results were found in cv. Mosa. The calcium-sensing receptor (CAS), calmodulin (CaM), expression was induced in both the cultivars. However, the CAS induction rate was much higher in cv. Mosa than in cv. Capitol in response to Xcc. The phytohormone salicylic acid (SA) and jasmonic acid (JA) levels were significantly higher in cv. Capitol along with the enhanced SA receptors (NPR3 and NPR4) and JA synthesis and signaling-related gene expression (LOX2, PDF1.2), whereas the JA level was significantly lower in cv. Mosa Xcc inoculated plants. The SA synthesis and signaling-related genes (ICS1, NPR1) and SA were present at higher levels in cv. Mosa; additionally, the SA level present was much higher in the susceptible cultivar (cv. Mosa) than in the resistant cultivar (cv. Capitol) in response to Xcc. These results indicate that ZAR1 mediated the coordinated action of SA and JA synthesis and signaling to confirm ETI, whereas TAO1 enhanced the synthesis of SA through CAS and CBP60g to antagonize JA synthesis and signaling to cause disease susceptibility in the Brassica napus-Xcc pathosystem.

Genome transfer for the prevention of female infertility caused by maternal gene mutation.

Poor oocyte quality is associated with early embryo developmental arrest and infertility. Maternal gene plays crucial roles in the regulation of oocyte maturation, and its mutation is a common cause of female infertility. However, how to improve oocyte quality and develop effective therapy for maternal gene mutation remains elusive. Here, we use Zar1 as an example to assess the feasibility of genome transfer to cure maternal gene mutation-caused female infertility. We first discover that cytoplasmic deficiency primarily leads to Zar1-null embryo developmental arrest by disturbing maternal transcript degradation and minor zygotic genome activation (ZGA) during the maternal-zygotic transition. We next perform genome transfer at the oocyte (spindle transfer or polar body transfer) and zygote (early pronuclear transfer or late pronuclear transfer) stages to validate the feasibility of preventing Zar1 mutation-caused infertility. We finally demonstrate that genome transfer either at the oocyte or at the early pronuclear stage can support normal preimplantation embryo development and produce live offspring. Moreover, those pups grow to adulthood and show normal fertility. Therefore, our findings provide an effective basis of therapies for the treatment of female infertility caused by maternal gene mutation.

Immunodiversity of the Arabidopsis ZAR1 NLR Is Conveyed by Receptor-Like Cytoplasmic Kinase Sensors.

The Arabidopsis nucleotide-binding leucine-rich repeat protein ZAR1 can recognize at least six distinct families of pathogenic effector proteins to mount an effector-triggered immune response. This remarkable immunodiversity appears to be conveyed by receptor-like cytoplasmic kinase (RLCK) complexes, which associate with ZAR1 to sense several effector-induced kinase perturbations. Here we show that the recently identified ZAR1-mediated immune responses against the HopX1, HopO1, and HopBA1 effector families of Pseudomonas syringae rely on an expanded diversity of RLCK sensors. We show that individual sensors can recognize distinct effector families, thereby contributing to the expanded surveillance potential of ZAR1 and supporting its role as a guardian of the plant kinome.

The ZAR1 protein in cancer; from epigenetic silencing to functional characterisation and epigenetic therapy of tumour suppressors.

ZAR1, zygote arrest 1, is a zinc finger protein (C-terminus), which was initially identified in mouse oocytes. Later it was found that its expression is present in various human tissues e.g. lung and kidney. Interestingly, it was observed that in various tumour types the ZAR1 transcript is missing due to hypermethylation of its CpG island promoter, but not ZAR2. Since methylation of the ZAR1 promoter is described as a frequent event in tumourigenesis, ZAR1 could serve as a useful diagnostic marker in cancer screens. ZAR1 was described as a useful prognostic/diagnostic cancer marker for lung cancer, kidney cancer, melanoma and possibly liver carcinoma. Furthermore, ZAR1 was reactivated as a tumour suppressor by epigenetic therapy using CRISPR-dCas9 method. This method holds the potential to precisely target not only ZAR1 and reactivate tumour suppressors in a tailored cancer therapy. ZAR1 is highly conserved amongst vertebrates, especially its zinc finger, which is the relevant domain for its protein and RNA binding ability. ZAR1 is implicated in various cellular mechanisms including regulation of oocyte/embryo development, cell cycle control and mRNA binding, though little was known about the underlying mechanisms. ZAR1 was reported to regulate and activate translation through the binding to TCS translation control sequences in the 3'UTRs of its target mRNA the kinase WEE1. ZAR1 has a tumour suppressing function by inhibiting cell cycle progression. Here we review the current literature on ZAR1 focusing on structural, functional and epigenetic aspects. Characterising the cellular mechanisms that regulate the signalling pathways ZAR1 is involved in, could lead to a deeper understanding of tumour development and, furthermore, to new strategies in cancer treatment.

Epigenetic therapy of novel tumour suppressor ZAR1 and its cancer biomarker function.

BACKGROUND: Cancer still is one of the leading causes of death and its death toll is predicted to rise further. We identified earlier the potential tumour suppressor zygote arrest 1 (ZAR1) to play a role in lung carcinogenesis through its epigenetic inactivation. RESULTS: We are the first to report that ZAR1 is epigenetically inactivated not only in lung cancer but also across cancer types, and ZAR1 methylation occurs across its complete CpG island. ZAR1 hypermethylation significantly correlates with its expression reduction in cancers. We are also the first to report that ZAR1 methylation and expression reduction are of clinical importance as a prognostic marker for lung cancer and kidney cancer. We further established that the carboxy (C)-terminally present zinc-finger of ZAR1 is relevant for its tumour suppression function and its protein partner binding associated with the mRNA/ribosomal network. Global gene expression profiling supported ZAR1's role in cell cycle arrest and p53 signalling pathway, and we could show that ZAR1 growth suppression was in part p53 dependent. Using the CRISPR-dCas9 tools, we were able to prove that epigenetic editing and reactivation of ZAR1 is possible in cancer cell lines. CONCLUSION: ZAR1 is a novel cancer biomarker for lung and kidney, which is epigenetically silenced in various cancers by DNA hypermethylation. ZAR1 exerts its tumour suppressive function in part through p53 and through its zinc-finger domain. Epigenetic therapy can reactivate the ZAR1 tumour suppressor in cancer.

Two Arabidopsis Receptor-like Cytoplasmic Kinases SZE1 and SZE2 Associate with the ZAR1-ZED1 Complex and Are Required for Effector-Triggered Immunity.

Plants utilize intracellular nucleotide-binding leucine-rich repeat domain-containing receptors (NLRs) to recognize pathogen effectors and induce a robust defense response named effector-triggered immunity (ETI). The Arabidopsis NLR protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) forms a precomplex with HOPZ-ETI-DEFICIENT 1 (ZED1), a receptor-like cytoplasmic kinase (RLCK) XII-2 subfamily member, to recognize the Pseudomonas syringae effector HopZ1a. We previously described a dominant mutant of Arabidopsis ZED1, zed1-D, which displays temperature-sensitive autoimmunity in a ZAR1-dependent manner. Here, we report that the RLCKs SUPPRESSOR OF ZED1-D1 (SZE1) and SZE2 associate with the ZAR1-ZED1 complex and are required for the ZED1-D-activated autoimmune response and HopZ1a-triggered immunity. We show that SZE1 but not SZE2 has autophosphorylation activity, and that the N-terminal myristoylation of both SZE1 and SZE2 is critical for their plasma membrane localization and ZED1-D-activated autoimmunity. Furthermore, we demonstrate that SZE1 and SZE2 both interact with ZAR1 to form a functional complex and are required for resistance against P. syringae pv. tomato DC3000 expressing HopZ1a. We also provide evidence that SZE1 and SZE2 interact with HopZ1a and function together with ZED1 to change the intramolecular interactions of ZAR1, leading to its activation. Taken together, our results reveal SZE1 and SZE2 as critical signaling components of HopZ1a-triggered immunity.

ZAR1 knockdown promotes the differentiation of human neuroblastoma cells by suppression of MYCN expression.

Although DNA hypermethylation at non-promoter region of the Zygote arrest 1 (ZAR1) gene has been observed in many types of tumor, including neuroblastoma (NB), the role of this gene in tumor development and/or progression is unclear. One reason is that knowledge about the function of ZAR1 protein is limited. Although it has been reported that ZAR1 plays a crucial role in early embryogenesis and may act as a transcriptional repressor for some transcripts, the detailed mechanism is still elusive. In the present study, we analyzed public data of NB patients and found that higher expression levels of ZAR1 were significantly associated with a shorter survival period. Consistent with this result, ZAR1-depleted NB cells showed well-differentiated phenotypes with elongated neurites and upregulated expression of TRKA and RET, which are markers for differentiated NB. Moreover, the expression level of MYCN protein was markedly suppressed in ZAR1-depleted NB cells. MYCN-depleted cells showed similar phenotypes to ZAR1-depleted cells. The present findings indicate that ZAR1 has oncogenic effects in NB by suppressing cell differentiation via regulation of MYCN expression.

ZAR1 is a novel epigenetically inactivated tumour suppressor in lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths with 1.8 million new cases each year and poor 5-year prognosis. Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby can promote cancer development and progression. RESULTS: In this study, we analysed ZAR1 (zygote arrest 1), which has been said to be a maternal-effect gene and its expression mostly limited to certain reproductive tissues. Our study shows that ZAR1 is expressed in normal lung but inactivated by promoter methylation in lung cancer. ZAR1 is hypermethylated in primary lung cancer samples (22% small cell lung carcinoma (SCLC) and 76% non-small cell lung carcinoma (NSCLC), p < 0.001) vs. normal control lung tissue (11%). In lung cancer cell lines, ZAR1 was significantly methylated in 75% of SCLC and 83% of NSCLC vs. normal tissue (p < 0.005/0.05). In matching tumours and control tissues, we observed that NSCLC primary tumour samples exhibited a tumour-specific promoter methylation of ZAR1 in comparison to the normal control lung tissue. Demethylation treatment of various lung cancer cell lines reversed ZAR1 promoter hypermethylation and subsequently re-established ZAR1 expression. In addition, we could show the growth inhibitory potential of ZAR1 in lung cancer cell lines and cancer cell lines. Exogenous expression of ZAR1 not only inhibited colony formation but also blocked cell cycle progression of cancer cell lines. CONCLUSIONS: Our study shows for the first time the lung tumour-specific epigenetic inactivation of ZAR1 due to DNA methylation of its CpG island promoter. Furthermore, ZAR1 was characterised by the ability to block tumour growth through the inhibition of cell cycle progression in cancer cell lines. We propose that ZAR1 could serve as an epigenetically inactivated biomarker in lung cancer.