Gene Knockout Analysis Research Articles

Carbon Metabolism of a Soilborne Mn(II)-Oxidizing Escherichia coli Isolate Implicated as a Pronounced Modulator of Bacterial Mn Oxidation.

Mn(II)-oxidizing microorganisms are generally considered the primary driving forces in the biological formation of Mn oxides. However, the mechanistic elucidation of the actuation and regulation of Mn oxidation in soilborne bacteria remains elusive. Here, we performed joint multiple gene-knockout analyses and comparative morphological and physiological determinations to characterize the influence of carbon metabolism on the Mn oxide deposit amount (MnODA) and the Mn oxide formation of a soilborne bacterium, Escherichia coli MB266. Different carbon source substances exhibited significantly varied effects on the MnODA of MB266. A total of 16 carbon metabolism-related genes with significant variant expression levels under Mn supplementation conditions were knocked out in the MB266 genome accordingly, but only little effect on the MnODA of each mutant strain was accounted for. However, a simultaneous four-gene-knockout mutant (namely, MB801) showed an overall remarkable MnODA reduction and an initially delayed Mn oxide formation compared with the wild-type MB266. The assays using scanning/transmission electron microscopy verified that MB801 exhibited not only a delayed Mn-oxide aggregate processing, but also relatively smaller microspherical agglomerations, and presented flocculent deposit Mn oxides compared with normal fibrous and crystalline Mn oxides formed by MB266. Moreover, the Mn oxide aggregate formation was highly related to the intracellular ROS level. Thus, this study demonstrates that carbon metabolism acts as a pronounced modulator of MnODA in MB266, which will provide new insights into the occurrence of Mn oxidation and Mn oxide formation by soilborne bacteria in habitats where Mn(II) naturally occurs.

Evolutionarily conserved function of the even-skipped ortholog in insects revealed by gene knock-out analyses in Gryllus bimaculatus

Comparing the developmental mechanisms of segmentation among insects with different modes of embryogenesis provides insights on how the function of segmentation genes evolved. Functional analysis of eve by genetic mutants shows that the Drosophila pair-rule gene, even-skipped (eve), contributes to initial segmental patterning. However, eve orthologs tends to have diverse functions in other insects. To compare the evolutionary functional divergence of this gene, we evaluated eve function in a phylogenetically basal insect, the cricket Gryllus bimaculatus. To investigate the phenotypic effects of eve gene knock-out, we generated CRISPR/Cas9 system-mediated mutant strains of the cricket. CRISPR/Cas9 mutagenesis of multiple independent sites in the eve coding region revealed that eve null mutant embryos were defective in forming the gnathal, thoracic, and abdominal segments, consequently shortening the anterior-posterior axis. In contrast, the structures of the anterior and posterior ends (e.g., antenna, labrum, and cercus) formed normally. Hox gene expression in the gnathal, thoracic, and abdominal segments was detected in the mutant embryos. Overall, this study showed that Gryllus eve plays an important role in embryonic elongation and the formation of segmental boundaries in the gnathal to abdominal region of crickets. In the light of studies on other species, the eve function shown in Gryllus might be ancestral in insects.

Evidence for evolution and selection of drought-resistant genes based on high-throughput resequencing in weedy rice.

Weedy rice (Oryza sativa f. spontanea) is a relative of cultivated rice that propagates in paddy fields and has strong drought resistance. In this study, we used 501 rice accessions to reveal the selection mechanism of drought resistance in weedy rice through a combination of selection analysis, genome-wide association studies, gene knockout and overexpression analysis, and Ca2+ and K+ ion flux assays. The results showed that the weedy rice species investigated have gene introgression with cultivated rice, which is consistent with the hypothesis that weedy rice originated from de-domestication of cultivated rice. Regions related to tolerance have particularly diversified during de-domestication and three drought-tolerance genes were identified. Of these, Os01g0800500 was also identified using an assay of the degree of leaf withering under drought, and it was named as PAPH1, encoding a PAP family protein. The drought-resistance capacity of PAPH1-knockout lines was much lower than that of the wild type, while that of overexpression lines was much higher. Concentrations of Ca2+ and K+ were lower in the knockout lines and higher in the overexpression lines compared with those of the wild type, suggesting that PAPH1 plays important roles in coping with drought stress. Our study therefore provides new insights into the genetic mechanisms underlying adaptive tolerance to drought in wild rice and highlights potential new resistance genes for future breeding programs in cultivated rice.

Auxin Efflux Transporters OsPIN1c and OsPIN1d Function Redundantly in Regulating Rice (Oryza sativa L.) Panicle Development

The essential role of auxin in plant growth and development is well known. Pathways related to auxin synthesis, transport and signaling have been extensively studied in recent years, and the PIN-FORMED (PIN) protein family has been identified as being pivotal for polar auxin transport and distribution. However, research focused on the functional characterization of PIN proteins in rice is still lacking. In this study, we investigated the expression and function of OsPIN1c and OsPIN1d in the japonica rice variety (Nipponbare) using gene knockout and high-throughput RNA sequencing analysis. The results showed that OsPIN1c and OsPIN1d were mainly expressed in young panicles and exhibited a redundant function. Furthermore, OsPIN1c or OsPIN1d loss-of-function mutants presented a mild phenotype compared with the wild type. However, in addition to significantly decreased plant height and tiller number, panicle development was severely disrupted in double-mutant lines of OsPIN1c and OsPIN1d. Severe defects included smaller inflorescence meristem and panicle sizes, fewer primary branches, elongated bract leaves, non-degraded hair and no spikelet growth. Interestingly, ospin1cd-3, a double-mutant line with functional retention of OsPIN1d, showed milder defects than those observed in other mutants. Additionally, several critical regulators of reproductive development, such as OsPID, LAX1, OsMADS1 and OsSPL14/IPA1, were differentially expressed in ospin1c-1 ospin1d-1, supporting the hypothesis that OsPIN1c and OsPIN1d are involved in regulating panicle development. Therefore, this study provides novel insights into the auxin pathways that regulate plant reproductive development in monocots.

Genome-Scale CRISPR-Cas9 Knockout Screening in Nasopharyngeal Carcinoma for Radiosensitive and Radioresistant Genes

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), and resistance of cancer cells to radiation is one of the major causes of treatment failure. In this study, the genome-wide CRISPR/Cas9-sgRNA library virus was used for the first time to screen for the functional genes associated with radiosensitivity and radioresistance in nasopharyngeal carcinoma, which lays the foundation for new research aiming to explain the mechanisms behind the radiosensitivity of nasopharyngeal carcinoma and makes some contribution to the discovery of new therapeutic targets and the development of new drugs to increase the sensitivity of NPC cells to radiotherapy.CRISPR/Cas9 library lentivirus screening in radiation-treated nasopharyngeal carcinoma cells was combined with second-generation sequencing technology to identify specific sgRNAs. A set of samples as contrast, and early library infection was the starting control for genetic change trend analysis. Two groups were exposed to doses of 0 and 2 Gy, and cell samples were collected 7 and 14 days after irradiation. The genomic DNA of living cells was used for PCR amplification in the coding region of sgRNA, and then high-throughput sequencing analysis was performed. The radiosensitivity or radioresistance of the screened functional genes in nasopharyngeal carcinoma were further verified by colony formation and cell proliferation experiments using a cell counting kit in a stable cell line with single gene knockout and qPCR analysis in radioresistant nasopharyngeal carcinoma cells.Eleven radiosensitive and radioresistant function genes were selected based on high-throughput sequencing and bioinformatics analysis. Compared with the control group, after the single gene FBLN5, FAM3C, MUS81, or DNAJC17 knockout, the colony formation and proliferation ability of nasopharyngeal carcinoma cells C666-1 and CNE1 were significantly promoted, while after the single gene CDKN2AIP and SP1 knockout, the colony formation and proliferation ability were significantly lower (P < 0.05). Compared to the negative control cells, the expression levels of FBLN5, FAM3C, MUS81, and DNAJC17 were significantly lower in the C666-1-Rs and 5-8F-Rs cell lines (P < 0.05), while the expression levels of TOMM20, CDKN2AIP, SNX22, and SP1 were significantly higher (P < 0.05). KEGG enrichment analysis showed that the potential signaling pathway contributed to radiosensitivity or radioresistance in NPC, including the Fanconi anemia pathway and the TGF-beta signaling pathway.We found nine genes through CRISPR/Cas9-mediated genome-wide knockout involved in the radiosensitivity or radioresistance of NPC cells: four genes for radiosensitivity, FBLN5, FAM3C, MUS81, and DNAJC17; two genes for radioresistance: CDKN2AIP, SP1, while TOMM20 and SNX22 are potential radioresistant genes, and CALD1 is a potential radiosensitive gene.

A Novel Toxoplasma Inner Membrane Complex Suture-Associated Protein Regulates Suture Protein Targeting and Colocalizes with Membrane Trafficking Machinery.

ABSTRACTThe cytoskeleton of Toxoplasma gondii is composed of the inner membrane complex (IMC) and an array of underlying microtubules that provide support at the periphery of the parasite. Specific subregions of the IMC carry out distinct roles in replication, motility, and host cell invasion. Building on our previous in vivo biotinylation (BioID) experiments of the IMC, we identified here a novel protein that localizes to discrete puncta that are embedded in the parasite’s cytoskeleton along the IMC sutures. Gene knockout analysis showed that loss of the protein results in defects in cytoskeletal suture protein targeting, cytoskeletal integrity, parasite morphology, and host cell invasion. We then used deletion analyses to identify a domain in the N terminus of the protein that is critical for both localization and function. Finally, we used the protein as bait for in vivo biotinylation, which identified several other proteins that colocalize in similar spot-like patterns. These putative interactors include several proteins that are implicated in membrane trafficking and are also associated with the cytoskeleton. Together, these data reveal an unexpected link between the IMC sutures and membrane trafficking elements of the parasite and suggest that the suture puncta are likely a portal for trafficking cargo across the IMC.

The truncated TNL receptor TN2-mediated immune responses require ADR1 function.

The loss of function of exocyst subunit EXO70B1 leads to autoimmunity, which is dependent on TIR-NBS2 (TN2), a truncated intracellular nucleotide-binding and leucine-rich repeat receptor (NLR). However, how TN2 triggers plant immunity and whether typical NLRs are required in TN2-activated resistance remain unclear. Through the CRISPR/Cas9 gene editing system and knockout analysis, we found that the spontaneous cell death and enhanced resistance in exo70B1-3 were independent of the full-length NLR SOC3 and its closest homolog SOC3-LIKE 1 (SOC3-L1). Additionally, knocking out SOC3-L1 or TN2 did not suppress the chilling sensitivity conferred by chilling sensitive 1-2 (chs1-2). The ACTIVATED DISEASE RESISTANCE 1 (ADR1) family and the N REQUIREMENT GENE 1 (NRG1) family have evolved as helper NLRs for many typical NLRs. Through CRISPR/Cas9 gene editing methods, we discovered that the autoimmunity of exo70B1-3 fully relied on ADR1s, but not NRG1s, and ADR1s contributed to the upregulation of TN2 transcript levels in exo70B1-3. Furthermore, overexpression of TN2 also led to ADR1-dependent autoimmune responses. Taken together, our genetic analysis highlights that the truncated TNL protein TN2-triggered immune responses require ADR1s as helper NLRs to activate downstream signaling, revealing the importance and complexity of ADR1s in plant immunity regulation.

The chemical profile of activated secondary metabolites by overexpressing LaeA in Aspergillus niger

Although the mechanisms of regulating secondary metabolism by LaeA remains unclear, the synthesis of many secondary metabolites (SMs) in Aspergilli could be activated by LaeA mutation. In our previous sutdy, RNA-seq data has showed that the transcriptional level of many SM backbone genes could be upregulated by overexpressing LaeA. Herein, we analyzed the chemical profile of activated secondary metabolites in the variant of A. niger FGSC A1279 by overexpressing LaeA (OElaeA). 14 compounds were activated in A. niger FGSC A1279 OElaeA variant in the WATM medium. Chemical workup of organic extracts of the culture broth from the A. niger OElaeA mutant identified three pure compounds, flaviolin, orlandin and kotanin. The structures of these compounds were confirmed by HR-ESIMS, 1D/2D NMR, and computer assisted structure elucidation (CASE). Based on homologous alignment and comparison of literatures, the biosynthetic gene cluster (fla) of flaviolin was identified. The in vivo function of the backbone gene, flaA, encoding a multidomain non-reducing polyketide synthase (SAT-KS-AT-PT-ACP), was verified via gene knockout and chemical analysis. Finally, a biosynthetic model for fungal flaviolin was proposed.

Exploring gene knockout strategies to identify potential drug targets using genome-scale metabolic models

Research on new cancer drugs is performed either through gene knockout studies or phenotypic screening of drugs in cancer cell-lines. Both of these approaches are costly and time-consuming. Computational framework, e.g., genome-scale metabolic models (GSMMs), could be a good alternative to find potential drug targets. The present study aims to investigate the applicability of gene knockout strategies to be used as the finding of drug targets using GSMMs. We performed single-gene knockout studies on existing GSMMs of the NCI-60 cell-lines obtained from 9 tissue types. The metabolic genes responsible for the growth of cancerous cells were identified and then ranked based on their cellular growth reduction. The possible growth reduction mechanisms, which matches with the gene knockout results, were described. Gene ranking was used to identify potential drug targets, which reduce the growth rate of cancer cells but not of the normal cells. The gene ranking results were also compared with existing shRNA screening data. The rank-correlation results for most of the cell-lines were not satisfactory for a single-gene knockout, but it played a significant role in deciding the activity of drug against cell proliferation, whereas multiple gene knockout analysis gave better correlation results. We validated our theoretical results experimentally and showed that the drugs mitotane and myxothiazol can inhibit the growth of at least four cell-lines of NCI-60 database.

A novel biodemulsifier of Bacillus mojavensis XH1 – Oxalate decarboxylase with the potential for demulsification of oilfield emulsion

In recent years, special attention has been devoted to biodemulsifiers as a new type of environment-friendly demulsifiers. A novel biodemulsifying oxalate decarboxylase (OxdC) secreted by Bacillus mojavensis XH1 is reported in the present study. A genome-wide comparison showed that strains with high demulsification efficiencies all possess alkane degradation genes. An analysis of the differentially expressed genes and proteins induced by different substrates showed that OxdC secreted by XH1 was an effective demulsifier. Moreover, the demulsification ability was verified by prokaryotic gene expression, knockout and complementation analyses. OxdC from XH1 exhibited a strong demulsification capacity and significantly outperformed the model protein Bacillus subtilis 168 OxdC (Yvrk), which shared a high amino acid similarity but showed limited demulsification ability. Based on a comparison of the structural characteristics, the hydrophobic amino acids on the surface of OxdC were identified as a key factor driving the favorable demulsification activity of XH1. The metabolic pathways of XH1 used liquid paraffin and glucose as substrates, illustrating that hydrocarbons are necessary for biodemulsifier secretion. The present study provides new insight into the application of OxdC as an additional genetic resource in biodemulsification.

The piperazine compound ASP activates an auxin response in Arabidopsis thaliana

BackgroundAuxins play key roles in the phytohormone network. Early auxin response genes in the AUX/IAA, SAUR, and GH3 families show functional redundancy, which makes it very difficult to study the functions of individual genes based on gene knockout analysis or transgenic technology. As an alternative, chemical genetics provides a powerful approach that can be used to address questions relating to plant hormones.ResultsBy screening a small-molecule chemical library of compounds that can induce abnormal seedling and vein development, we identified and characterized a piperazine compound 1-[(4-bromophenoxy) acetyl]-4-[(4-fluorophenyl) sulfonyl] piperazine (ASP). The Arabidopsis DR5::GFP line was used to assess if the effects mentioned were correlated with the auxin response, and we accordingly verified that ASP altered the auxin-related pathway. Subsequently, we examined the regulatory roles of ASP in hypocotyl and root development, auxin distribution, and changes in gene expression. Following ASP treatment, we detected hypocotyl elongation concomitant with enhanced cell elongation. Furthermore, seedlings showed retarded primary root growth, reduced gravitropism and increased root hair development. These phenotypes were associated with an increased induction of DR5::GUS expression in the root/stem transition zone and root tips. Auxin-related mutants including tir1–1, aux1–7 and axr2–1 showed phenotypes with different root-development pattern from that of the wild type (Col-0), and were insensitive to ASP. Confocal images of propidium iodide (PI)-stained root tip cells showed no detectable damage by ASP. Furthermore, RT-qPCR analyses of two other genes, namely, Ethylene Response Factor (ERF115) and Mediator 18 (MED18), which are related to cell regeneration and damage, indicated that the ASP inhibitory effect on root growth was not attributable to toxicity. RT-qPCR analysis provided further evidence that ASP induced the expression of early auxin-response-related genes.ConclusionsASP altered the auxin response pathway and regulated Arabidopsis growth and development. These results provide a basis for dissecting specific molecular components involved in auxin-regulated developmental processes and offer new opportunities to discover novel molecular players involved in the auxin response.

Pathogenesis-related genes of entomopathogenic fungi.

All living things on Earth experience various diseases such as those caused by viruses, bacteria, and fungi. Insects are no exception to this rule, and fungi that cause disease in insects are called entomopathogenic fungi. These fungi have been developed as microbial insecticides and are used to control various pests. Generally, the mode of action of entomopathogenic fungi is divided into the attachment of conidia, germination, penetration, growth, and generation of secondary infectious conidia. In each of these steps, that entomopathogenic fungi use genes in a complex manner (specific or diverse) has been shown by gene knock-out and RNA-sequencing analysis. In this review, the information mechanism of entomopathogenic fungi was divided into six steps: (1) attachment of conidia to host, (2) germination and appressorium, (3) penetration, (4) fungal growth in hemolymph, (5) conidia production on host, and (6) transmission and dispersal. The strategy used by the fungi in each step was described at the genetic level. In addition, an approach for studying the mode of action of the fungi is presented.

Proteomic Analysis of Metastasis-Specific Biomarkers in Pancreatic Cancer: Galectin-1 Plays an Important Metastatic Role in Pancreatic Cancer

Cancer metastasis is the major cause of death in pancreatic cancer. We have established a pair of pancreatic ductal adenocarcinoma cell line, PANC1 and invasive PANC1-I5, as a model system toinvestigate the metastatic mechanism as well as potential therapeutic targets in pancreatic cancer. We used proteomic analysis based on two-dimensional differential gel electrophoresis (2D-DIGE) and matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI-TOF MS) to examine the global protein expression alterations between PANC1 and PANC1-I5. Proteomic study revealed that 88 proteins are differentially expressed between PANC1-I5 and PANC1 cells, and further functional evaluations through protein expression validation, gene knockout, migration and invasion analysis revealed that galectin-1 is one of the potential players in modulating pancreatic cancer metastasis. To conclude, we have identified numerous proteins might be associated with pancreatic cancer invasiveness in the pancreatic cancer model.

The family of amphioxus chitin synthases offers insight into the evolution of chitin formation in chordates

Chitin is a very important and widely-used biopolymer in fungi and lower metazoans, but mysteriously disappears in mammals. Recent studies reveal that at least lower vertebrates have chitin synthases (CS) and use them to synthesize endogenous chitin. Amphioxus, a basal chordate, therefore becomes critical to understand the evolution of CS, as it occupies the transitional position from invertebrates to vertebrates, and is considered as a good proxy to the chordate ancestor.Here, by exploiting multiple genome assemblies, high-depth RNA-seq data and synteny relations, we identify 11–12 CS genes for each amphioxus species. It represents the largest CS gene pool ever found in eukaryotes so far. As comparison, most metazoans have one or two CSs. Amphioxus is the only chordate that has both the very ancient type-I CS family and the more broadly distributed type-II CS family. Specifically, amphioxus has only one type-II CS but 10–11 type-I CSs, which means that amphioxus is the only metazoan with a greatly expanded type-I CS family. Further analysis suggests that the chordate ancestor have at least one type-II CS and an expanded of type-I CS family. We hypothesize that: these ancient CSs are mostly retained in amphioxus; but the whole type-I CS family was lost in urochordates and vertebrates; the type-II CS was later duplicated into two lineages in vertebrates and followed by stochastic losses, till all type-II CSs were eventually lost in birds and mammals. Finally, our expression profiling and preliminary gene knockout analysis suggest that amphioxus CSs could have highly diverse but mildly overlapping functions in various tissues and organs.Taken together, these findings not only provide insights into the evolution of chordate CSs, lay a foundation for further functional study of the chordate CSs. After all, it is mysterious that our chordate ancestor needed so many isoenzymes for chitin formation.

RNA-Seq of in planta-expressed Magnaporthe oryzae genes identifies MoSVP as a highly expressed gene required for pathogenicity at the initial stage of infection.

SummaryThe ascomycete fungus Magnaporthe oryzae is a hemibiotrophic pathogen that causes rice blast disease. Magnaporthe oryzae infects rice leaves, stems and panicles, and induces severe reductions in yield. Effector proteins secreted by M. oryzae in planta are thought to be involved its virulence activity. Here, using RNA‐sequencing (RNA‐Seq), we generated transcriptome data for M. oryzae isolate Ina168 during the initial stages of infection. We prepared samples from conidia (the inoculum) and from peeled epidermal cotyledon tissue of susceptible barley Hordeum vulgare ‘Nigrate’ at 12, 24, 36 and 48 hours post‐inoculation (hpi). We also generated a draft genome sequence of M. oryzae isolate Ina168 and used it as a reference for mapping the RNA‐Seq reads. Gene expression profiling across all stages of M. oryzae infection revealed 1728 putative secreted effector protein genes. We selected seven such genes that were strongly up‐regulated at 12 hpi and down‐regulated at 24 or 36 hpi and performed gene knockout analysis to determine their roles in pathogenicity. Knockout of MoSVP, encoding a small putative secreted protein with a hydrophobic surface binding protein A domain, resulted in a reduction in pathogenicity, suggesting that MoSVP is a novel virulence effector of M. oryzae.

Abstract 907: Understanding Cardiomyocyte Mechanosensing Utilizing CRISPR-Cas9 Based Screening Methods

Heart failure (HF) is a leading cause of premature death that affects approximately 6.5 million patients in the United States and costs over $30 billion annually. HF is a complex disease that involves genetic and environmental factors, and is characterized by gradual deterioration of cardiac pump function. A key process implicated in the development of HF is mechanotransduction—the ability of cardiomyocytes to sense and respond to mechanical forces. While mechanotransduction signaling may play an adaptive role in response to changes in mechanical load, this process can become maladaptive, and ultimately lead to pathological hypertrophy and eventual HF. Gaining insight into cardiac mechanotransduction would solidify our understanding of how cardiomyocytes sense mechanical perturbations and will provide novel targets for the prevention and management of cardiomyopathy and HF. To study perturbations in cardiomyocyte mechanosensing, we have developed a B-type natriuretic peptide (BNP) reporter assay in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), which uses bright red fluorescent protein (tdTomato) to parallel endogenous BNP expression. Our reporter has been validated as a robust, scalable, and quantitative molecular marker of sarcomere function (inside-out) and substrate stiffness (outside-in). We have also generated a custom CRISPR guide RNA (gRNA) library targeting 75 extrinsic and intrinsic components of the cardiomyocyte mechanotransduction apparatus, enabling efficient analysis of single gene knockouts in our BNP reporter and providing a novel high-throughput model for interrogating the mechanical regulation of BNP expression. These gene candidates will be further tested in an in vitro HF model to assess functional response and the potential for phenotypic correction.

Abstract 1743: Evaluating the effect of USP22 depletion on chromosome instability in colorectal cancer

Abstract Chromosome instability (CIN) is an aberrant phenotype defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost, and is observed in virtually all cancer types. Although CIN is associated with aggressive tumors, multi-drug resistance and poor patient prognosis, the genetic defects underlying CIN remain poorly understood. Preliminary data from our laboratory suggest that diminished USP22 (Ubiquitin-Specific Peptidase 22) expression induces CIN. Importantly, USP22 deletions and reduced USP22 expression are frequently observed in multiple cancer types, including colorectal cancer, suggesting diminished USP22 expression/function may be a pathogenic event underlying oncogenesis. Therefore, the present study seeks to evaluate the impact diminished USP22 expression has on CIN by determining whether karyotypically stable colorectal cancer (HCT116) and immortalized fibroblast (hTERT) cell lines can be converted into karyotypically unstable lines following the reduction of USP22 expression. Diminished USP22 expression was achieved by siRNA-based silencing or CRISPR-Cas9-mediated gene knockout (KO) and quantitative image-based single-cell analyses were employed to monitor changes in surrogate markers for CIN, including nuclear areas and chromosome numbers. USP22 silencing induced significant changes in nuclear area as well as increased frequencies of chromosomes gains and losses, which suggest USP22 is a CIN gene. Next, to assess the long-term impact diminished USP22 expression has on CIN, two HCT116 USP22-KO clones were monitored for 10 weeks and revealed dynamic changes in nuclear areas and chromosome numbers over time relative to controls. Collectively, these data identify USP22 as a novel CIN gene and indicate that USP22 deletions in cancer may have long-term impact on CIN and may thereby promote oncogenesis. Further analysis is ongoing to investigate the mechanism through which USP22 depletion induces CIN, and in particular its effect on H2B monoubiquitination. Characterizing the effect diminished USP22 expression has on CIN is the first step towards the development of therapeutic strategies targeting USP22-deficient cancers. Citation Format: Lucile M. Jeusset, Kirk J. McManus. Evaluating the effect of USP22 depletion on chromosome instability in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1743.

Gene knockout analysis reveals essentiality of estrogen receptor \u03b21 (Esr2a) for female reproduction in medaka

In vertebrates, sex steroids play crucial roles in multiple systems related to reproduction. In females, estrogens and their receptor estrogen receptor (ER or Esr) play indispensable roles in the negative sex steroid feedback regulation of pituitary gonadotropin secretion, which prevents excessive development of ovarian follicles. However, the mechanism of this feedback regulation of a gonadotropin, follicle stimulating hormone (FSH), which is essential for folliculogenesis throughout vertebrates, is poorly understood. In the present study, we generated knockouts of all subtypes of nuclear estrogen receptors in a model teleost medaka, which is suitable for the study of endocrine control and behavioral assays, and analyzed fertility, behavior and functionality of estrogen feedback in each knockout line. Among the estrogen receptors, we revealed that an estrogen receptor Esr2a plays an essential role in this feedback regulation. In addition to this, we also found that esr2a−/− females showed oviduct atresia, which causes complete infertility. Interestingly, esr2a−/− females showed apparently normal sexual behavior but without oviposition in response to male courtship. This phenotype indicates that physical readiness and motivation of sexual behavior is independently controlled.

Agouti-Related Protein 2 Is a New Player in the Teleost Stress Response System

Agouti-related protein (AgRP) is a hypothalamic regulator of food consumption in mammals. However, AgRP has also been detected in circulation, but a possible endocrine role has not been examined. Zebrafish possess two agrp genes: hypothalamically expressed agrp1, considered functionally equivalent to the single mammalian agrp, and agrp2, which is expressed in pre-optic neurons and uncharacterized pineal gland cells and whose function is not well understood. By ablation of AgRP1-expressing neurons and knockout of the agrp1 gene, we show that AgRP1 stimulates food consumption in the zebrafish larvae. Single-cell sequencing of pineal agrp2-expressing cells revealed molecular resemblance to retinal-pigment epithelium cells, and anatomic analysis shows that these cells secrete peptides, possibly into the cerebrospinal fluid. Additionally, based on AgRP2 peptide localization and gene knockout analysis, we demonstrate that pre-optic AgRP2 is a neuroendocrine regulator of the stress axis that reduces cortisol secretion. We therefore suggest that the ancestral role of AgRP was functionally partitioned in zebrafish by the two AgRPs, with AgRP1 centrally regulating food consumption and AgRP2 acting as a neuroendocrine factor regulating the stress axis.

TIR-NB-LRR immune receptor SOC3 pairs with truncated TIR-NB protein CHS1 or TN2 to monitor the homeostasis of E3 ligase SAUL1.

Intracellular nucleotide binding (NB) and leucine-rich repeat (NLR) proteins function as immune receptors to recognize effectors from pathogens. They often guard host proteins that are the direct targets of those effectors. Recent findings have revealed that a typical NLR sometimes cooperates with another atypical NLR for effector recognition. Here, by using the CRISPR/Cas9 gene editing method, knockout analysis and biochemical assays, we uncovered differential pairings of typical Toll Interleukin1 receptor (TIR) type NLR (TNL) receptor SOC3 with atypical truncated TIR-NB (TN) proteins CHS1 or TN2 to guard the homeostasis of the E3 ligase SAUL1. Overaccumulation of SAUL1 is monitored by the SOC3-TN2 pair, while SAUL1's disappearance is guarded by the SOC3-CHS1 pair. SOC3 forms a head-to-head genomic arrangement with CHS1 and TN2, indicative of transcriptional co-regulation. Such intricate cooperative interactions can probably enlarge the recognition spectrum and increase the functional flexibility of NLRs, which can partly explain the overwhelming occurrence of NLR gene clustering in higher plants.