Reverse Genetic Screen Research Articles

Protein phosphatase 2A holoenzymes regulate leucine-rich repeat kinase 2 phosphorylation and accumulation

LRRK2 is a highly phosphorylated multidomain protein and mutations in the gene encoding LRRK2 are a major genetic determinant of Parkinson's disease (PD). Dephosphorylation at LRRK2's S910/S935/S955/S973 phosphosite cluster is observed in several conditions including in sporadic PD brain, in several disease mutant forms of LRRK2 and after pharmacological LRRK2 kinase inhibition. However, the mechanism of LRRK2 dephosphorylation is poorly understood.We performed a phosphatome-wide reverse genetics screen to identify phosphatases involved in the dephosphorylation of the LRRK2 phosphosite S935. Candidate phosphatases selected from the primary screen were tested in mammalian cells, Xenopus oocytes and in vitro. Effects of PP2A on endogenous LRRK2 phosphorylation were examined via expression modulation with CRISPR/dCas9.Our screening revealed LRRK2 phosphorylation regulators linked to the PP1 and PP2A holoenzyme complexes as well as CDC25 phosphatases. We showed that dephosphorylation induced by different kinase inhibitor triggered relocalisation of phosphatases PP1 and PP2A in LRRK2 subcellular compartments in HEK-293 T cells. We also demonstrated that LRRK2 is an authentic substrate of PP2A both in vitro and in Xenopus oocytes. We singled out the PP2A holoenzyme PPP2CA:PPP2R2 as a powerful phosphoregulator of pS935-LRRK2. Furthermore, we demonstrated that this specific PP2A holoenzyme induces LRRK2 relocalization and triggers LRRK2 ubiquitination, suggesting its involvement in LRRK2 clearance. The identification of the PPP2CA:PPP2R2 complex regulating LRRK2 S910/S935/S955/S973 phosphorylation paves the way for studies refining PD therapeutic strategies that impact LRRK2 phosphorylation.

Slim shady is a novel allele of PHYTOCHROME B present in the T-DNA line SALK_015201.

Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin, rapid changes in transcript and protein abundance occur in hypocotyls, and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin‐regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we called slim shady, in an annotated insertion line in IMMUNOREGULATORY RNA‐BINDING PROTEIN (IRR). Overexpression of the IRR gene failed to rescue the slim shady phenotype and characterization of a second T‐DNA allele of IRR found that it had a wild‐type (WT) hypocotyl length. The slim shady mutant has an elevated expression of numerous genes associated with the brassinosteroid‐auxin‐phytochrome (BAP) regulatory module compared to WT, including transcription factors that regulate brassinosteroid, auxin, and phytochrome pathways. Additionally, slim shady seedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence data and genetic complementation analysis with SALK_015201C, we determined that a novel single nucleotide polymorphism in PHYTOCHROME B was responsible for the slim shady phenotype. This is predicted to induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase‐related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses with phyb‐9 confirmed that slim shady is a mutant allele of PHYB. This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin‐dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T‐DNA stocks.

Immunity onset alters plant chromatin and utilizes EDA16 to regulate oxidative homeostasis.

Perception of microbes by plants leads to dynamic reprogramming of the transcriptome, which is essential for plant health. The appropriate amplitude of this transcriptional response can be regulated at multiple levels, including chromatin. However, the mechanisms underlying the interplay between chromatin remodeling and transcription dynamics upon activation of plant immunity remain poorly understood. Here, we present evidence that activation of plant immunity by bacteria leads to nucleosome repositioning, which correlates with altered transcription. Nucleosome remodeling follows distinct patterns of nucleosome repositioning at different loci. Using a reverse genetic screen, we identify multiple chromatin remodeling ATPases with previously undescribed roles in immunity, including EMBRYO SAC DEVELOPMENT ARREST 16, EDA16. Functional characterization of the immune-inducible chromatin remodeling ATPase EDA16 revealed a mechanism to negatively regulate immunity activation and limit changes in redox homeostasis. Our transcriptomic data combined with MNase-seq data for EDA16 functional knock-out and over-expressor mutants show that EDA16 selectively regulates a defined subset of genes involved in redox signaling through nucleosome repositioning. Thus, collectively, chromatin remodeling ATPases fine-tune immune responses and provide a previously uncharacterized mechanism of immune regulation.

The Transcription Factor CoxR Negatively Regulates Alkane Hydroxylase Transcription

Both gram negative and gram positive bacteria have enzymes for metabolizing alkanes. The alpha proteobacteria Caulobacter crescentus has a medium-chain alkane hydroxylase that is a member of the CYP153 family of cytochrome P450 enzymes. The molecular mechanism controlling transcription of this hydroxylase has not been described. We carried out a reverse genetics screen and RNA-seq to find the transcription factor regulating this alkane hydroxylase. A deletion of the transcription factor, named CoxR, resulted in upregulation of both the alkane hydroxylase as well as ferredoxin and ferredoxin reductase enzymes. Gel shift assays demonstrated that CoxR bound directly to the promoter of the alkane hydroxylase. In conclusion, we have identified a novel transcription factor involved in metabolism of alkanes.

A global search for novel transcription factors impacting the Neurospora crassa circadian clock.

Eukaryotic circadian oscillators share a common circuit architecture, a negative feedback loop in which a positive element activates the transcription of a negative one that then represses the action of the former, inhibiting its own expression. While studies in mammals and insects have revealed additional transcriptional inputs modulating the expression of core clock components, this has been less characterized in the model Neurospora crassa, where the participation of other transcriptional components impacting circadian clock dynamics remains rather unexplored. Thus, we sought to identify additional transcriptional regulators modulating the N. crassa clock, following a reverse genetic screen based on luminescent circadian reporters and a collection of transcription factors (TFs) knockouts, successfully covering close to 60% of them. Besides the canonical core clock components WC-1 and -2, none of the tested transcriptional regulators proved to be essential for rhythmicity. Nevertheless, we identified a set of 23 TFs that when absent lead to discrete, but significant, changes in circadian period. While the current level of analysis does not provide mechanistic information about how these new players modulate circadian parameters, the results of this screen reveal that an important number of light and clock-regulated TFs, involved in a plethora of processes, are capable of modulating the clockworks. This partial reverse genetic clock screen also exemplifies how the N. crassa knockout collection continues to serve as an expedite platform to address broad biological questions.

Lrp5 mutant and crispant zebrafish faithfully model human osteoporosis, establishing the zebrafish as a platform for CRISPR-based functional screening of osteoporosis candidate genes

Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. (C) 2021 American Society for Bone and Mineral Research. (C) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness.

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been hindered by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying effect on CPS biosynthesis and hmv, were evaluated for their impact on CPS thickness, serum resistance, host cell association, and fitness in a murine model of disseminating pneumonia. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that hmv and CPS may serve distinct functions during pathogenesis. The integration of hmv and CPS to the metabolic status of the cell suggests that hvKp may require certain nutrients to specifically cause deep tissue infections.

Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics.

Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. We compared three DSB repair prediction algorithms – MENTHU, inDelphi, and Lindel – in identifying MMEJ-repaired, homogeneous genotypes (PreMAs) in an independent dataset of 5,885 distinct Cas9-mediated mouse embryonic stem cell DSB repair events. MENTHU correctly identified 46% of all PreMAs available, a ∼2- and ∼60-fold sensitivity increase compared to inDelphi and Lindel, respectively. In contrast, only Lindel correctly predicted predominant single-base insertions. We report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations in this large dataset. We then estimated the frequency of Cas9-targetable homogeneous frameshift-inducing DSBs in vertebrate coding regions for gene discovery using MENdel. 47 out of 54 genes (87%) contained at least one early frameshift-inducing DSB and 49 out of 54 (91%) did so when also considering Cas12a-mediated deletions. We suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.

Lrp5 Mutant and Crispant Zebrafish Faithfully Model Human Osteoporosis, Establishing the Zebrafish as a Platform for CRISPR-Based Functional Screening of Osteoporosis Candidate Genes.

Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

CIA2 and CIA2-LIKE are required for optimal photosynthesis and stress responses in Arabidopsis thaliana.

Chloroplast-to-nucleus retrograde signaling is essential for cell function, acclimation to fluctuating environmental conditions, plant growth and development. The vast majority of chloroplast proteins are nuclear-encoded, and must be imported into the organelle after synthesis in the cytoplasm. This import is essential for the development of fully functional chloroplasts. On the other hand, functional chloroplasts act as sensors of environmental changes and can trigger acclimatory responses that influence nuclear gene expression. Signaling via mobile transcription factors (TFs) has been recently recognized as a way of communication between organelles and the nucleus. In this study, we performed a targeted reverse genetic screen to identify dual-localized TFs involved in chloroplast retrograde signaling during stress responses. We found that CHLOROPLAST IMPORT APPARATUS 2 (CIA2) has a functional plastid transit peptide, and can be located both in chloroplasts and the nucleus. Further, we found that CIA2, along with its homolog CIA2-like (CIL) are involved in the regulation of Arabidopsis responses to UV-AB, high light and heat shock. Finally, our results suggest that both CIA2 and CIL are crucial for chloroplast translation. Our results contribute to a deeper understanding of signaling events in the chloroplast-nucleus cross-talk.

Modeling neurodegeneration in Caenorhabditiselegans.

ABSTRACTThe global burden of neurodegenerative diseases underscores the urgent need for innovative strategies to define new drug targets and disease-modifying factors. The nematode Caenorhabditis elegans has served as the experimental subject for multiple transformative discoveries that have redefined our understanding of biology for ∼60 years. More recently, the considerable attributes of C. elegans have been applied to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease and Huntington's disease. Transgenic nematodes with genes encoding normal and disease variants of proteins at the single- or multi-copy level under neuronal-specific promoters limits expression to select neuronal subtypes. The anatomical transparency of C. elegans affords the use of co-expressed fluorescent proteins to follow the progression of neurodegeneration as the animals age. Significantly, a completely defined connectome facilitates detailed understanding of the impact of neurodegeneration on organismal health and offers a unique capacity to accurately link cell death with behavioral dysfunction or phenotypic variation in vivo. Moreover, chemical treatments, as well as forward and reverse genetic screening, hasten the identification of modifiers that alter neurodegeneration. When combined, these chemical-genetic analyses establish critical threshold states to enhance or reduce cellular stress for dissecting associated pathways. Furthermore, C. elegans can rapidly reveal whether lifespan or healthspan factor into neurodegenerative processes. Here, we outline the methodologies employed to investigate neurodegeneration in C. elegans and highlight numerous studies that exemplify its utility as a pre-clinical intermediary to expedite and inform mammalian translational research.

Mutations disrupting neuritogenesis genes confer risk for cerebral palsy.

In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent-offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1A and CTNNB1) met genome-wide significance. We identified two novel monogenic etiologies, FBXO31 and RHOB, and showed that the RHOB mutation enhances active-state Rho effector binding while the FBXO31 mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in a Drosophila reverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.

DREAM complex suppresses DNA methylation maintenance genes and precludes DNA hypermethylation.

The DNA methyltransferases MET1 and CMT3 are known to be responsible for maintenance of DNA methylation at symmetric CG and CHG sites, respectively, in Arabidopsis thaliana. However, it is unknown how the expression of methyltransferase genes is regulated in different cell states and whether change in expression affects DNA methylation at the whole-genome level. Using a reverse genetic screen, we identified TCX5, a tesmin/TSO1-like CXC domain-containing protein, and demonstrated that it is a transcriptional repressor of genes required for maintenance of DNA methylation, which include MET1, CMT3, DDM1, KYP and VIMs. TCX5 functions redundantly with its paralogue TCX6 in repressing the expression of these genes. In the tcx5 tcx6 double mutant, expression of these genes is markedly increased, thereby leading to markedly increased DNA methylation at CHG sites and, to a lesser extent, at CG sites at the whole-genome level. Furthermore, our whole-genome DNA methylation analysis indicated that the CG and CHG methylation level is lower in differentiated quiescent cells than in dividing cells in the wild type but is comparable in the tcx5/6 mutant, suggesting that TCX5/6 are required for maintenance of the difference in DNA methylation between the two cell types. We identified TCX5/6-containing multi-subunit complexes, which are known as DREAM in other eukaryotes, and demonstrated that the Arabidopsis DREAM components function as a whole to preclude DNA hypermethylation. Given that the DREAM complexes are conserved from plants to animals, the preclusion of DNA hypermethylation by DREAM complexes may represent a conserved mechanism in eukaryotes.

A Decoy Library Uncovers U-Box E3 Ubiquitin Ligases That Regulate Flowering Time in Arabidopsis.

Targeted degradation of proteins is mediated by E3 ubiquitin ligases and is important for the execution of many biological processes. Redundancy has prevented the genetic characterization of many E3 ubiquitin ligases in plants. Here, we performed a reverse genetic screen in Arabidopsis using a library of dominant-negative U-box-type E3 ubiquitin ligases to identify their roles in flowering time and reproductive development. We identified five U-box decoy transgenic populations that have defects in flowering time or the floral development program. We used additional genetic and biochemical studies to validate PLANT U-BOX 14 (PUB14), MOS4-ASSOCIATED COMPLEX 3A (MAC3A), and MAC3B as bona fide regulators of flowering time. This work demonstrates the widespread importance of E3 ubiquitin ligases in floral reproductive development. Furthermore, it reinforces the necessity of dominant-negative strategies for uncovering previously unidentified regulators of developmental transitions in an organism with widespread genetic redundancy, and provides a basis on which to model other similar studies.

Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors.

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors perceive pathogen-derived molecules to trigger immunity. Global NLR homeostasis must be tightly controlled to ensure sufficient and timely immune output while avoiding aberrant activation, the mechanisms of which are largely unclear. In a previous reverse genetic screen, we identified two novel E3 ligases, SNIPER1 and its homolog SNIPER2, both of which broadly control the levels of NLR immune receptors in Arabidopsis. Protein levels of sensor NLRs (sNLRs) are inversely correlated with SNIPER1 amount and the interactions between SNIPER1 and sNLRs seem to be through the common nucleotide-binding (NB) domains of sNLRs. In support, SNIPER1 can ubiquitinate the NB domains of multiple sNLRs invitro. Our study thus reveals a novel process of global turnover of sNLRs by two master E3 ligases for immediate attenuation of immune output to effectively avoid autoimmunity. Such unique mechanism can be utilized in the future for engineering broad-spectrum resistance in crops to fend off pathogens that damage our food supply.

0069 Sleep in Drosophila is Regulated by the Chromatin Remodeling Factor ISWI

Abstract Introduction Sleep is commonly disrupted in patients with neurodevelopmental disorders (NDDs). Despite strong clinical associations between disrupted sleep and other NDD symptoms, we lack an understanding of how these are pathophysiologically related. Drosophila melanogaster exhibit essential characteristics of human sleep and have well-defined neural circuits underlying learning and social behaviors. This represents an ideal system to investigate the mechanistic interaction between disrupted sleep and other behavioral dysfunctions in NDDs. Methods We performed a reverse genetic RNAi-based screen targeting Drosophila homologs of human genes within NDD-associated risk loci. Pan-neuronal knockdown of risk genes was achieved using the Gal4-UAS system. Results Pan-neuronal knockdown of ISWI led to dramatic deficits in sleep and circadian arrhythmicity in the adult fly. Across species, ISWI and its homologs are ATP-dependent chromatin remodelers that regulate gene expression important for neural differentiation. We found that depleting ISWI also leads to memory and social deficits. ISWI functions during dissociable temporal windows of pre-adult development and in different circuits to establish different adult behaviors. The sleep phenotype associated with ISWI knockdown mapped to a specific population of cells. RNA-Seq of developing brains during the window important for sleep deficits revealed significant transcriptional changes in genes associated with nervous system development, suggesting ISWI acts in the development of sleep regulatory circuits. Finally, mutations in the human homologs of ISWI, SMARCA1/5, have been implicated in NDDs. Expressing either SMARCA1/5 in the setting of ISWI knockdown differentially rescued adult deficits. Conclusion Identification of ISWI provides a platform for unraveling pleiotropic behavioral effects from an NDD risk gene. Sleep, circadian rhythms, memory, and social behaviors are affected by ISWI knockdown, and map to different developmental periods and circuits. In addition, SMARCA1/5 differentially rescue adult behaviors, suggesting NDD-causing mutations in these genes may affect different behaviors. Current work aims to determine how human mutations in SMARCA1/5 affect behaviors. Support This work was supported by NIH K08 NS090461 (MSK) and T32 HL007953 (NNG), Hearst Foundation Fellowship 2018 (NNG), Burroughs Welcome Career Award for Medical Scientists, March of Dimes Basil O’Connor Scholar Award, and Sloan Research Fellowship (MSK).

The role of the bZIP transcription factor ZIP-1 in the Intracellular Pathogen Response of C. elegans

Abstract How does the nematode Caenorhabditis elegans respond to natural pathogen infections with non-professional immune cells? To answer this question, we are studying the Intracellular Pathogen Response (IPR) – a host transcriptional response common to infection with molecularly diverse natural pathogens: microsporidia and the Orsay virus. In addition to intracellular pathogens, the IPR can be induced upon exposure to heat stress and proteasome inhibition. Mutant animals with constitutive IPR activation have increased intracellular pathogen resistance and thermotolerance, indicating that the IPR confers a protective response. The IPR involves transcriptional activation of 80 genes, some of which are predicted to encode ubiquitin ligase components. One of the most highly induced IPR genes is pals-5, which serves as a robust readout for the IPR activation. Using pals-5::GFP reporters, we identified the predicted bZIP transcription factor ZIP-1 as a positive regulator of the IPR in two reverse genetic screens. Our qRT-PCR and smFISH studies demonstrated that ZIP-1 controls pals-5 mRNA expression early after proteasome blockade, but not at later time points. We further performed an RNA-Seq analysis, which revealed that ZIP-1 is required for mRNA expression of multiple IPR components. Based on the type of transcriptional regulation, we have identified three distinct types of IPR genes: a) completely ZIP-1-dependent, b) partially ZIP-1-dependent and c) ZIP-1-independent. Importantly, we found that ZIP-1 is required for the increased resistance to microsporidia infection in mutants with constitutive IPR expression, suggesting that ZIP-1-dependent genes play an important role in innate immunity.

Identification of a Transcription Factor that Represses Alkane Hydroxylase Expression

Some gram negative and gram positive bacteria have evolved enzymes for metabolizing alkanes. A medium‐chain alkane hydroxylase has been found in the model organism Caulobacter crescentus, belonging to the CYP153 family of cytochrome P450 enzymes. However, the molecular mechanisms regulating expression of this enzyme are still unknown. We carried out a reverse genetics screen and RNA‐seq to identify CoxR as a transcription factor regulating this alkane hydroxylase. A deletion of coxR resulted in upregulation of both the alkane hydroxylase as well as ferredoxin and ferredoxin reductase enzymes. EMSA assays showed that CoxR bound directly to a short region of the alkane hydroxylase promoter. In summary, we have identified a novel transcription factor involved in metabolism of alkanes.

Cancer drivers converge on NOTCH

Cancer Cancer genome–sequencing projects have emphasized the handful of genes mutated at high frequency in patients. Less attention has been directed to the hundreds of genes mutated in only a few patients—the so-called “long tail” mutations. Although rare, these mutations may nonetheless inform patient care. Loganathan et al. developed a reverse genetic CRISPR screen that allowed them to functionally assess in mice nearly 500 long tail gene mutations that occur in human head and neck squamous cell carcinoma (HNSCC). They identified 15 tumor-suppressor genes with activities that converged on the NOTCH signaling pathway. Given that NOTCH itself is mutated at high frequency in HNSCC, these results suggest that the growth of these tumors is largely driven by NOTCH inactivation. Science , this issue p. [1264][1] [1]: /lookup/doi/10.1126/science.aax0902

Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling

In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent "long tail" mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.