Forward Genetics Research Articles

Deficiency of the HGF/Met pathway leads to thyroid dysgenesis by impeding late thyroid expansion

The mechanisms of bifurcation, a key step in thyroid development, are largely unknown. Here we find three zebrafish lines from a forward genetic screening with similar thyroid dysgenesis phenotypes and identify a stop-gain mutation in hgfa and two missense mutations in met by positional cloning from these zebrafish lines. The elongation of the thyroid primordium along the pharyngeal midline was dramatically disrupted in these zebrafish lines carrying a mutation in hgfa or met. Further studies show that MAPK inhibitor U0126 could mimic thyroid dysgenesis in zebrafish, and the phenotypes are rescued by overexpression of constitutively active MEK or Snail, downstream molecules of the HGF/Met pathway, in thyrocytes. Moreover, HGF promotes thyrocyte migration, which is probably mediated by downregulation of E-cadherin expression. The delayed bifurcation of the thyroid primordium is also observed in thyroid-specific Met knockout mice. Together, our findings reveal that HGF/Met is indispensable for the bifurcation of the thyroid primordium during thyroid development mediated by downregulation of E-cadherin in thyrocytes via MAPK-snail pathway.

Spliceosomal complex components are critical for adjusting the C:N balance during high-light acclimation.

Plant acclimation to an ever-changing environment is decisive for growth, reproduction, and survival. Light availability limits biomass production on both ends of the intensity spectrum. Therefore, the adjustment of plant metabolism is central to high-light (HL) acclimation, and the accumulation of photoprotective anthocyanins is commonly observed. However, mechanisms and factors regulating the HL acclimation response are less clear. Two Arabidopsis mutants of spliceosome components exhibiting a pronounced anthocyanin overaccumulation in HL were isolated from a forward genetic screen for new factors crucial for plant acclimation. Time-resolved physiological, transcriptome, and metabolome analysis revealed a vital function of the spliceosome components for rapidly adjusting gene expression and metabolism. Deficiency of INCREASED LEVEL OF POLYPLOIDY1 (ILP1), NTC-RELATED PROTEIN1 (NTR1), and PLEIOTROPIC REGULATORY LOCUS1 (PRL1) resulted in a marked overaccumulation of carbohydrates and strongly diminished amino acid biosynthesis in HL. While not generally limited in N-assimilation, ilp1, ntr1, and prl1 showed higher glutamate levels and reduced amino acid biosynthesis in HL. The comprehensive analysis reveals a function of the spliceosome components in the conditional regulation of the carbon:nitrogen balance and the accumulation of anthocyanins during HL acclimation. The importance of gene expression, metabolic regulation, and re-direction of carbon towards anthocyanin biosynthesis for HL acclimation are discussed.

Impairment of the glial phagolysosomal system drives prion-like propagation in a Drosophila model of Huntington's disease.

Protein misfolding, aggregation, and spread through the brain are primary drivers of neurodegenerative diseases pathogenesis. Phagocytic glia are responsible for regulating the load of pathogenic protein aggregates in the brain, but emerging evidence suggests that glia may also act as vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal debris in male and female adult Drosophila expressing the gene that causes Huntington's disease (HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate burden in the brain. Finally, a forward genetic screen revealed Rab10 as a novel component of Draper-dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal degradation and acquire prion-like characteristics. Together, our findings reveal new mechanisms that enhance our understanding of the beneficial and potentially harmful effects of phagocytic glia in HD and potentially other neurodegenerative diseases.Significance Statement Deposition of amyloid aggregates is strongly associated with neurodegenerative disease progression and neuronal cell loss. Many studies point to glial cells as dynamic mediators of disease, capable of phagocytosing toxic materials, but also promoting chronic inflammation and proteopathic aggregate spread. Thus, glia have emerged as promising therapeutic targets for disease intervention. Here, we demonstrate in a Drosophila model of Huntington's disease that neuronal mHTT aggregates interfere with glial phagocytic engulfment, phagolysosomal processing, and innate immunity transcriptional responses. We also identify Rab10 as a novel modifier of prion-like transmission of mHTT aggregates. Our findings add to a growing narrative of glia as double-edged players in neurodegenerative diseases.

The plastid-localized lipoamide dehydrogenase 1 is crucial for redox homeostasis, tolerance to arsenic stress and fatty acid biosynthesis in rice.

Soil contamination with arsenic (As) can cause phytotoxicity and reduce crop yield. The mechanisms of As toxicity and tolerance are not fully understood. In this study, we used a forward genetics approach to isolate a rice mutant, ahs1, that exhibits hypersensitivity to both arsenate and arsenite. Through genomic resequencing and complementation tests, we identified OsLPD1 as the causal gene, which encodes a putative lipoamide dehydrogenase. OsLPD1 was expressed in the outer cell layer of roots, root meristem cells, and in the mesophyll and vascular tissues of leaves. Subcellular localization and immunoblot analysis demonstrated that OsLPD1 is localized in the stroma of plastids. In vitro assays showed that OsLPD1 exhibited lipoamide dehydrogenase (LPD) activity, which was strongly inhibited by arsenite, but not by arsenate. The ahs1 and OsLPD1 knockout mutants exhibited significantly reduced NADH/NAD+ and GSH/GSSG ratios, along with increased levels of reactive oxygen species and greater oxidative stress in the roots compared with wild-type (WT) plants under As treatment. Additionally, loss-of-function of OsLPD1 also resulted in decreased fatty acid concentrations in rice grain. Taken together, our finding reveals that OsLPD1 plays an important role for maintaining redox homeostasis, conferring tolerance to arsenic stress, and regulating fatty acid biosynthesis in rice.

Abstract 5849: Integrated multi-omics of hydroxychloroquine evolved cancer cells reveal survival requires transcriptional upregulation of glycolysis, exocytosis, and chromosome fidelity, but not autophagy

Abstract Repurposed drugs are undergoing a renaissance in the era of genomics, particularly in oncology. Over 2,300 FDA-approved and natural product small molecules now exist, but challenges remain in identifying which cancer patients may best benefit from such drugs. To best position repurposed drugs, mechanisms of action and resistance to the drug in a cancer cell setting must be determined. Hydroxychloroquine (HCQ) and chloroquine are repurposed drugs reported to disrupt autophagy, a molecular recycling pathway which is essential for tumor cell survival, chemotherapeutic resistance, and stem cell phenotypes. However, oncology trials with chloroquine have no biomarker or other genetic test to categorize patients who may best respond to HCQ therapy. It is unclear how tumors mechanistically evolve resistance to HCQ. We pursued a multi-omic strategy in OVCAR3 ovarian cancer and CCL218 colorectal cancer cells. Two genome-scale screens were performed. In the forward genetic screen, cell populations were passaged in 93-independent cultures for 15 drug pulse-chases with HCQ or vehicle control. Evolved cells were collected and processed for bulk RNA-seq, exome-seq, and single cell RNA-seq (scRNA-seq). In the reverse genetic screen, a pooled Brunello CRISPR-Cas9 library of 19,114 genes targeted by multiple sgRNAs was used in cells over three pulse-chases of HCQ or vehicle control treatments. HCQ evolved cells displayed remarkably few mutational differences, but substantial transcriptional differences. Bulk transcriptomes revealed multiple broad-spectrum pathways associated with resistance to HCQ, including upregulation of glycolysis, exocytosis, and chromosome condensation/segregation, as well as downregulation of translation and apoptosis. Analysis of scRNA-seq data indicated cell clusters were differentially reliant on cytoskeletal regulation, DNA repair, and metabolism. The genome-scale Cas9 screen identified remarkably few autophagy genes. However, an integrated analysis of Cas9 hits with RNA-seq upregulated genes confirmed glycolytic genes KDM3A, ENO2, and PDK3 were essential for HCQ resistance. Additional integrated hits were NEK2, involved in exocytosis, and chromosome condensation/segregation genes CDK1 and CENPA. Transcriptional plasticity was the primary mechanism by which cells evolved resistance to HCQ. Contrary to the widely presumed autophagy-related mode of cell death, the multi-omic analysis instead prioritized glycolytic metabolism, exocytosis, and chromosome condensation/segregation as most involved in cancer cell survival with HCQ. Integrated multi-omic analysis defined high-value biomarkers of HCQ sensitivity and resistance. Our analysis may serve as a model for how to better define the effects and positioning of repurposed drugs in oncology. Citation Format: Madison Clark, Silvia G. Vaena, Joe R. Delaney. Integrated multi-omics of hydroxychloroquine evolved cancer cells reveal survival requires transcriptional upregulation of glycolysis, exocytosis, and chromosome fidelity, but not autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5849.

Abstract 1998: ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature

Abstract Uveal melanoma (UM) is the leading form of intraocular cancer in adults and the second most common form of melanoma. Although rare, UM has a median survival rate of ~1 year after metastasis occurs. Metastatic UM is largely refractory to treatment and there are no effective pharmacological therapies, resulting in poor overall survival. In >90% UM cases, the oncogenic initiator proteins are constitutively active mutant GNAQ or GNA11 proteins (GNAQ/11). FR900359 (FR) is a natural compound that inhibits GNAQ/11 and has anti-proliferative effects on UM cells. To identify key oncogenic mechanisms downstream of mutant GNAQ/11, we performed a Sleeping Beauty (SB) transposon forward genetic screen to identify drivers of FR resistance. The SB screen identified ATP-binding cassette sub-family B member 1 (ABCB1) as one of the top genes upregulated by SB in FR-resistant UM cell populations. We determined that although ABCB1 does not provide immediate resistance to FR, the overexpression of ABCB1 leads to emergence of resistant colonies at a much higher rate than spontaneous resistance, validating ABCB1 as contributors to FR resistance. Unexpectedly, immunofluorescence revealed that a relatively small fraction of UM cells strongly expresses the transgenes after stable transfection. Immunoblotting confirmed that ABCB1 expression was initially modest in ABCB1-transfected populations but increased in the cells that developed resistance to FR. To examine the kinetics of FR resistance, population doubling versus time (PDVT) assays were performed in UM cells expressing ABCB1 or empty vector control treated with FR or vehicle. FR potently suppressed the proliferation of vector control cells. Drug-naïve ABCB1-transfected cells developed resistance to FR over time but accomplished fewer population doublings than vehicle-treated cells in the same time interval. Once transgenic populations emerged as resistant populations in FR, they grew similarly in drug or vehicle. Ongoing RNA-sequencing analysis from PDVT samples has identified distinct gene expression signatures associated with FR treatment and ABCB1 expression. These findings are expected to illuminate tumor cell heterogeneity amongst UM cell populations that may be related to drug resistance and metastasis. Citation Format: Sarina Dion Murray, Jesse D. Riordan, Eric R. Anderson, Michael D. Onken, Kendall J. Blumer, Adam J. Dupuy, Christopher S. Stipp. ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1998.

Genome-wide forward genetic screening to identify receptors and proteins mediating nanoparticle uptake and intracellular processing.

Understanding how cells process nanoparticles is crucial to optimize nanomedicine efficacy. However, characterizing cellular pathways is challenging, especially if non-canonical mechanisms are involved. In this Article a genome-wide forward genetic screening based on insertional mutagenesis is applied to discover receptors and proteins involved in the intracellular accumulation (uptake and intracellular processing) of silica nanoparticles. The nanoparticles are covered by a human serum corona known to target the low-density lipoprotein receptor (LDLR). By sorting cells with reduced nanoparticle accumulation and deep sequencing after each sorting, 80 enriched genes are identified. We find that, as well as LDLR, the scavenger receptor SCARB1 also mediates nanoparticle accumulation. Additionally, heparan sulfate acts as a specific nanoparticle receptor, and its role varies depending on cell and nanoparticle type. Furthermore, some of the identified targets affect nanoparticle trafficking to the lysosomes. These results show the potential of genetic screening to characterize nanoparticle pathways. Additionally, they indicate that corona-coated nanoparticles are internalized via multiple receptors.

Deciphering the genomes of motility-deficient mutants of Vibrio alginolyticus 138-2.

The motility of Vibrio species plays a pivotal role in their survival and adaptation to diverse environments and is intricately associated with pathogenicity in both humans and aquatic animals. Numerous mutant strains of Vibrio alginolyticus have been generated using UV or EMS mutagenesis to probe flagellar motility using molecular genetic approaches. Identifying these mutations promises to yield valuable insights into motility at the protein structural physiology level. In this study, we determined the complete genomic structure of 4 reference specimens of laboratory V. alginolyticus strains: a precursor strain, V. alginolyticus 138-2, two strains showing defects in the lateral flagellum (VIO5 and YM4), and one strain showing defects in the polar flagellum (YM19). Subsequently, we meticulously ascertained the specific mutation sites within the 18 motility-deficient strains related to the polar flagellum (they fall into three categories: flagellar-deficient, multi-flagellar, and chemotaxis-deficient strains) by whole genome sequencing and mapping to the complete genome of parental strains VIO5 or YM4. The mutant strains had an average of 20.6 (±12.7) mutations, most of which were randomly distributed throughout the genome. However, at least two or more different mutations in six flagellar-related genes were detected in 18 mutants specifically selected as chemotaxis-deficient mutants. Genomic analysis using a large number of mutant strains is a very effective tool to comprehensively identify genes associated with specific phenotypes using forward genetics.

The inner membrane protein YhiM links copper and CpxAR envelope stress responses in uropathogenic E. coli.

Urinary tract infection (UTI) is a ubiquitous infectious condition, and uropathogenic Escherichia coli (UPEC) is the predominant causative agent of UTI. Copper (Cu) is implicated in innate immunity, including against UPEC. Cu is a trace element utilized as a co-factor, but excess Cu is toxic due to mismetalation of non-cognate proteins. E. coli precisely regulates Cu homeostasis via efflux systems. However, Cu import mechanisms into the bacterial cell are not clear. We hypothesized that Cu import defective mutants would exhibit increased resistance to Cu. This hypothesis was tested in a forward genetic screen with transposon (Tn5) insertion mutants in UPEC strain CFT073, and we identified 32 unique Cu-resistant mutants. Transposon and defined mutants lacking yhiM, which encodes a hypothetical inner membrane protein, were more resistant to Cu than parental strain. Loss of YhiM led to decreased cellular Cu content and increased expression of copA, encoding a Cu efflux pump. The CpxAR envelope stress response system was activated in the ΔyhiM mutant as indicated by increased expression of cpxP. Transcription of yhiM was regulated by CueR and CpxR, and the CpxAR system was essential for increased Cu resistance in the ΔyhiM mutant. Importantly, activation of CpxAR system in the ΔyhiM mutant was independent of NlpE, a known activator of this system. YhiM was required for optimal fitness of UPEC in a mouse model of UTI. Our findings demonstrate that YhiM is a critical mediator of Cu homeostasis and links bacterial adaptation to Cu stress with the CpxAR-dependent envelope stress response in UPEC.IMPORTANCEUPEC is a common bacterial infection. Bacterial pathogens are exposed to host-derived Cu during infection, including UTI. Here, we describe detection of genes involved in Cu homeostasis in UPEC. A UPEC mutant lacking YhiM, a membrane protein, exhibited dramatic increase in resistance to Cu. Our study demonstrates YhiM as a nexus between Cu stress and the CpxAR-dependent envelope stress response system. Importantly, our findings establish NlpE-independent activation of CpxAR system during Cu stress in UPEC. Collectively, YhiM emerges as a critical mediator of Cu homeostasis in UPEC and highlights the interlinked nature of bacterial adaptation to survival during Cu and envelope stress.

OsGADD45a1: a multifaceted regulator of rice architecture, grain yield, and blast resistance.

The homolog gene of the Growth Arrest and DNA Damage-inducible 45 (GADD45) in rice functions in the regulation of plant architecture, grain yield, and blast resistance. The Growth Arrest and DNA Damage-inducible 45 (GADD45) family proteins, well-established stress sensors and tumor suppressors in mammals, serve as pivotal regulators of genotoxic stress responses and tumorigenesis. In contrast, the homolog and role of GADD45 in plants have remained unclear. Herein, using forward genetics, we identified an activation tagging mutant AC13 exhibited dwarf characteristics resulting from the loss-of-function of the rice GADD45α homolog, denoted as OsGADD45a1. osgadd45a1 mutants displayed reduced plant height, shortened panicle length, and decreased grain yield compared to the wild-type Kitaake. Conversely, no obvious differences in plant height, panicle length, or grain yield were observed between wild-type and OsGADD45a1 overexpression plants. OsGADD45a1 displayed relatively high expression in germinated seeds and panicles, with localization in both the nucleus and cytoplasm. RNA-sequencing analysis suggested a potential role for OsGADD45a1 in the regulation of photosynthesis, and binding partner identification indicates OsGADD45a1 interacts with OsRML1 to regulate rice growth. Intriguingly, our study unveiled a novel role for OsGADD45a1 in rice blast resistance, as osgadd45a1 mutant showed enhanced resistance to Magnaporthe oryzae, and the expression of OsGADD45a1 was diminished upon blast fungus treatment. The involvement of OsGADD45a1 in rice blast fungus resistance presents a groundbreaking finding. In summary, our results shed light on the multifaceted role of OsGADD45a1 in rice, encompassing biotic stress response and the modulation of several agricultural traits, including plant height, panicle length, and grain yield.

Forward genetic screen using a gene-breaking trap approach identifies a novel role of grin2bb-associated RNA transcript (grin2bbART) in zebrafish heart function.

LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish (Danio rerio) for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it "bigheart." Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript grin2bb associated RNA transcript (grin2bbART). Using in situ hybridization, we confirmed localized grin2bbART expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored grin2bbART expression. Morpholino-mediated antisense downregulation of grin2bbART in wild-type zebrafish embryos mimicked bigheart mutants; this suggests grin2bbART is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of grin2bbART, a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish.

Chromosomal R-loops: who R they?

R-loops, composed of DNA-RNA hybrids and displaced single-stranded DNA, are known to pose a severe threat to genome integrity. Therefore, extensive research has focused on identifying regulatory proteins involved in controlling R-loop levels. These proteins play critical roles in preventing R-loop accumulation and associated genome instability. Herein I summarize recent knowledge on R-loop regulators affecting R-loop homeostasis, involving a wide array of R-loop screening methods that have enabled their characterization, from forward genetic and siRNA-based screens to proximity labeling and machine learning. These approaches not only deepen our understanding on R-loop formation processes, but also hold promise to find new targets in R-loop dysregulation associated with human pathologies.

Mutations in KASIIB result in increased levels of palmitic acid in soybean seeds

AbstractThe plasticity of the lipid profile of soybean oil has made the soybean a crop with value in feed, food and industrial uses. Soybean oil typically consists of approximately 10% of the fully saturated palmitic acid, which has value for industrial purposes, however it is generally undesirable in food applications. Two soybean KASII (ketoacyl‐ACP synthase II) genes have been previously implicated in the control of seed palmitic acid levels. Using forward genetics, we isolated four novel alleles in the GmKASIIB gene that result in increased levels of palmitic acid, ranging from 13% to 16% of the total fatty acid content, over multiple seasons in the field. Three of the KASIIB mutations result in independent amino acid substitutions at conserved positions in the KASII protein, and one single nucleotide polymorphism is likely to cause aberrant splicing of the KASIIB transcript. These new mutations can be used in breeding applications to increase palmitic acid content and to probe the control of fatty acid biosynthesis in soybean seeds.

DTBI2: A Calibrated, Tunable Device for Administering Traumatic Brain Injury in Drosophila.

The second-generation Drosophila traumatic brain injury (TBI) device dTBI2 improves Drosophila TBI administration by providing a moderate-throughput, tunable, head-specific injury. Our updated device design improves user-friendliness, eliminates inconsistencies in injury timing, and has an updated circuit design to extend the longevity of delicate electronic components. dTBI2 improves reproducibility across users and runs, and results in more consistent post-injury phenotypes. This protocol describes the construction, calibration, and use of the dTBI2 device, which uses an Arduino-controlled piezoelectric actuator to deliver a force that compresses a fly head against a metal collar. The duration and depth of head compression is tunable, allowing calibration of injury severity. All device components are commercially available, and the entire device can be constructed in under a week for less than $1000. The dTBI2 design will enable any lab to build a highly reliable, low-cost device for Drosophila TBI, facilitating increased adoption and ease of exploration of closed-head TBI in Drosophila for forward genetic screens. We describe below the three protocols necessary for constructing a dTBI2 device. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction of the dTBI2 control device Basic Protocol 2: Construction of the piezoelectric actuator housing Basic Protocol 3: Administration of dTBI2 injuries.

Control of meiotic crossover interference by a proteolytic chaperone network.

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis.

Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium

Human induced pluripotent stem cells (hiPSCs) offer opportunities to study human biology where primary cell types are limited. CRISPR technology allows forward genetic screens using engineered Cas9-expressing cells. Here, we sought to generate a CRISPR activation (CRISPRa) hiPSC line to activate endogenous genes during pluripotency and differentiation. We first targeted catalytically inactive Cas9 fused to VP64, p65 and Rta activators (dCas9-VPR) regulated by the constitutive CAG promoter to the AAVS1 safe harbor site. These CRISPRa hiPSC lines effectively activate target genes in pluripotency, however the dCas9-VPR transgene expression is silenced after differentiation into cardiomyocytes and endothelial cells. To understand this silencing, we systematically tested different safe harbor sites and different promoters. Targeting to safe harbor sites hROSA26 and CLYBL loci also yielded hiPSCs that expressed dCas9-VPR in pluripotency but silenced during differentiation. Muscle-specific regulatory cassettes, derived from cardiac troponin T or muscle creatine kinase promoters, were also silent after differentiation when dCas9-VPR was introduced. In contrast, in cell lines where the dCas9-VPR sequence was replaced with cDNAs encoding fluorescent proteins, expression persisted during differentiation in all loci and with all promoters. Promoter DNA was hypermethylated in CRISPRa-engineered lines, and demethylation with 5-azacytidine enhanced dCas9-VPR gene expression. In summary, the dCas9-VPR cDNA is readily expressed from multiple loci during pluripotency but induces silencing in a locus- and promoter-independent manner during differentiation to mesoderm derivatives. Researchers intending to use this CRISPRa strategy during stem cell differentiation should pilot their system to ensure it remains active in their population of interest.

Discovery and characterization of anti-cancer peptides from a random peptide library.

We performed a forward genetic screen to discover peptides that specifically target breast cancer cells using a Penetratin tagged, random 15mer peptide library. We identified a group of novel peptides that specifically inhibited the proliferation and survival of breast cancer cells without affecting normal primary mammary epithelial cells or fibroblasts. The intrinsic apoptotic pathway is activated by these peptides in the face of abnormal expression of numerous cell cycle regulatory genes. Associated alterations in histone marks, nuclear structure, and levels of critical RNA binding proteins vary in a peptide specific manner. This study demonstrates a novel method for the discovery of new potential therapeutic peptides.

Pathways controlling neurotoxicity and proteostasis in mitochondrial complex I deficiency.

Neuromuscular disorders caused by dysfunction of the mitochondrial respiratory chain are common, severe and untreatable. We recovered a number of mitochondrial genes, including electron transport chain components, in a large forward genetic screen for mutations causing age-related neurodegeneration in the context of proteostasis dysfunction. We created a model of complex I deficiency in the Drosophila retina to probe the role of protein degradation abnormalities in mitochondrial encephalomyopathies. Using our genetic model, we found that complex I deficiency regulates both the ubiquitin/proteasome and autophagy/lysosome arms of the proteostasis machinery. We further performed an in vivo kinome screen to uncover new and potentially druggable mechanisms contributing to complex I related neurodegeneration and proteostasis failure. Reduction of RIOK kinases and the innate immune signaling kinase pelle prevented neurodegeneration in complex I deficiency animals. Genetically targeting oxidative stress, but not RIOK1 or pelle knockdown, normalized proteostasis markers. Our findings outline distinct pathways controlling neurodegeneration and protein degradation in complex I deficiency and introduce an experimentally facile model in which to study these debilitating and currently treatment-refractory disorders.

E3 ubiquitin ligase Herc3 deficiency leads to accumulation of subretinal microglia and retinal neurodegeneration

Activated microglia have been implicated in the pathogenesis of age-related macular degeneration (AMD), diabetic retinopathy, and other neurodegenerative and neuroinflammatory disorders, but our understanding of the mechanisms behind their activation is in infant stages. With the goal of identifying novel genes associated with microglial activation in the retina, we applied a semiquantitative fundus spot scoring scale to an unbiased, state-of-the-science mouse forward genetics pipeline. A mutation in the gene encoding the E3 ubiquitin ligase Herc3 led to prominent accumulation of fundus spots. CRISPR mutagenesis was used to generate Herc3-/- mice, which developed prominent accumulation of fundus spots and corresponding activated Iba1 + /CD16 + subretinal microglia, retinal thinning on OCT and histology, and functional deficits by Optomotory and electrophysiology. Bulk RNA sequencing identified activation of inflammatory pathways and differentially expressed genes involved in the modulation of microglial activation. Thus, despite the known expression of multiple E3 ubiquitin ligases in the retina, we identified a non-redundant role for Herc3 in retinal homeostasis. Our findings are significant given that a dysregulated ubiquitin–proteasome system (UPS) is important in prevalent retinal diseases, in which activated microglia appear to play a role. This association between Herc3 deficiency, retinal microglial activation and retinal degeneration merits further study.

The ACE inhibitor captopril inhibits ACN-1 to control dauer formation and aging.

The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in Caenorhabditis elegans, Drosophila and rodents, but its mechanism is not well defined. Here, we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril-hypersensitive mutants. We identified a missense mutation that causes a partial loss of function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNA interference promoted dauer larvae formation, suggesting that acn-1 is a daf gene. Captopril-mediated lifespan extension was abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 influence lifespan by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control.