Forward Mutagenesis Research Articles

A forward genetic screen identifies Sirtuin1 as a driver of neuroendocrine prostate cancer.

Using an unbiased forward mutagenesis screen in an autochthonous mouse model, we have investigated mechanistic determinants of aggressive prostate cancer. SIRT1 emerged as a key regulator of neuroendocrine prostate cancer differentiation and a potential target for therapeutic intervention.

SMYD5 is a regulator of the mild hypothermia response.

The mild hypothermia response (MHR) maintains organismal homeostasis during cold exposure and is thought to be critical for the neuroprotection documented with therapeutic hypothermia. To date, little is known about the transcriptional regulation of the MHR. We utilize a forward CRISPR-Cas9 mutagenesis screen to identify the histone lysine methyltransferase SMYD5 as a regulator of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia. This repression correlates with temperature-dependent levels of H3K36me3 at the SP1-locus and globally, indicating that the mammalian MHR is regulated at the level of histone modifications. We have identified 37 additional SMYD5 regulated temperature-dependent genes, suggesting a broader MHR-related role for SMYD5. Our study provides an example of how histone modifications integrate environmental cues into the genetic circuitry of mammalian cells and provides insights that may yield therapeutic avenues for neuroprotection after catastrophic events.

Loss of Sec-1 Family Domain-Containing 1 (scfd1) Causes Severe Cardiac Defects and Endoplasmic Reticulum Stress in Zebrafish.

Dilated cardiomyopathy (DCM) is a common heart muscle disorder that frequently leads to heart failure, arrhythmias, and death. While DCM is often heritable, disease-causing mutations are identified in only ~30% of cases. In a forward genetic mutagenesis screen, we identified a novel zebrafish mutant, heart and head (hahvcc43), characterized by early-onset cardiomyopathy and craniofacial defects. Linkage analysis and next-generation sequencing identified a nonsense variant in the highly conserved scfd1 gene, also known as sly1, that encodes sec1 family domain-containing 1. Sec1/Munc18 proteins, such as Scfd1, are involved in membrane fusion regulating endoplasmic reticulum (ER)/Golgi transport. CRISPR/Cas9-engineered scfd1vcc44 null mutants showed severe cardiac and craniofacial defects and embryonic lethality that recapitulated the phenotype of hahvcc43 mutants. Electron micrographs of scfd1-depleted cardiomyocytes showed reduced myofibril width and sarcomere density, as well as reticular network disorganization and fragmentation of Golgi stacks. Furthermore, quantitative PCR analysis showed upregulation of ER stress response and apoptosis markers. Both heterozygous hahvcc43 mutants and scfd1vcc44 mutants survived to adulthood, showing chamber dilation and reduced ventricular contraction. Collectively, our data implicate scfd1 loss-of-function as the genetic defect at the hahvcc43 locus and provide new insights into the role of scfd1 in cardiac development and function.

Genetic tools for the study of the mangrove killifish, Kryptolebias marmoratus, an emerging vertebrate model for phenotypic plasticity.

Kryptolebias marmoratus (Kmar), a teleost fish of the order Cyprinodontiformes, has a suite of unique phenotypes and behaviors not observed in other fishes. Many of these phenotypes are discrete and highly plastic-varying over time within an individual, and in some cases reversible. Kmar and its interfertile sister species, K. hermaphroditus, are the only known self-fertile vertebrates. This unusual sexual mode has the potential to provide unique insights into the regulation of vertebrate sexual development, and also lends itself to genetics. Kmar is easily adapted to the lab and requires little maintenance. However, its internal fertilization and small clutch size limits its experimental use. To support Kmar as a genetic model, we compared alternative husbandry techniques to maximize recovery of early cleavage-stage embryos. We find that frequent egg collection enhances yield, and that protease treatment promotes the greatest hatching success. We completed a forward mutagenesis screen and recovered several mutant lines that serve as important tools for genetics in this model. Several will serve as useful viable recessive markers for marking crosses. Importantly, the mutant kissylips lays embryos at twice the rate of wild-type. Combining frequent egg collection with the kissylips mutant background allows for a substantial enhancement of early embryo yield. These improvements were sufficient to allow experimental analysis of early development and the successful mono- and bi-allelic targeted knockout of an endogenous tyrosinase gene with CRISPR/Cas9 nucleases. Collectively, these tools will facilitate modern developmental genetics in this fascinating fish, leading to future insights into the regulation of plasticity.

Abstract 5773: A forward genetic screen identifies SIRT1 as a driver of neuroendocrine prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) is a deadly variant of prostate cancer for which no cure is yet available. Up to 20 percent of the patients with castration resistant prostate cancer (CRPC), will progress to NEPC after treatment with standard-of-care therapies, such as abiraterone and enzalutamide, that target the androgen receptor (AR) pathway. However, the molecular mechanisms that drive the emergence of NEPC are largely unknown, contributing to a lack of treatment options for patients with NEPC. We implemented a forward genetic approach to identify novel drivers of NEPC, by using the Sleeping Beauty (SB) transposon-based forward genetic mutagenesis screening system in our Pten-, Trp53-deficient mouse model that is primed to develop NEPC at a low frequency. We generated a mouse strain (NPp53-SB) in which the Nkx3.1 promoter regulates the expression of tamoxifen-inducible Cre specifically in the mouse prostate epithelium, resulting in transposition of the T2Onc2 transposable element and conditional deletion of Pten and Trp53 in mouse prostate epithelium. We observed an increased NEPC incidence in our NPp53-SB mouse model compared to control mice without the transposon T2Onc2, suggesting that the SB-mediated insertional mutagenesis events are driving the development of NEPC. To elucidate the genetic events that are driving the observed NEPC phenotype, we identified the common insertion sites (CISs) of the transposon insertions, which revealed a total of 330 CIS-associated genes. We further performed an integrative analysis using RNA-seq data from our SB cohort, a second mouse dataset and a metastatic CRPC patient cohort, which allowed us to identify the Master Regulator (MR) proteins that are associated with NEPC, as well as the upstream genomic events that modulate these MRs and the emergence of NEPC. Our integrative analysis identified the deacetylase Sirt1 as a candidate driver of NEPC. Using CRISPR activation (CRISPRa) technology in vivo we observed that SIRT1 activation induced an NEPC-like phenotype in the adenocarcinoma LNCaP cell line, while Sirt1 drug-mediated inhibition in an NEPC cell line resulted in less aggressive tumors.In summary, we have developed a Sleeping Beauty forward genetic screen in our mouse model of CRPC, which not only accelerates disease progression, but also increases the incidence of NEPC. Our integrative analysis identified a panel of CIS-associated genes that are likely to be functionally involved in progression to NEPC. In particular, Sirt1 was shown to be a promising candidate, which may shed light on mechanisms that this aggressive disease uses to evade treatments and may inform the development of novel therapies. Citation Format: Francisca Nunes de Almeida, Alessandro Vasciaveo, Min Zou, Matteo Di Bernardo, Andrea Califano, Cory Abate-Shen. A forward genetic screen identifies SIRT1 as a driver of neuroendocrine prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5773.

Molecular mechanisms of anthracycline induced cardiotoxicity: Zebrafish come into play.

Anthracyclines are among the most potent chemotherapeutics; however, cardiotoxicity significantly restricts their use. Indeed, anthracycline-induced cardiotoxicity (AIC) fares among the worst types of cardiomyopathy, and may only slowly and partially respond to standard heart failure therapies including β-blockers and ACE inhibitors. No therapy specifically designed to treat anthracycline cardiomyopathy at present, and neither is it known if any such strategy could be developed. To address this gap and to elucidate the molecular basis of AIC with a therapeutic goal in mind, zebrafish has been introduced as an in vivo vertebrate model about a decade ago. Here, we first review our current understanding of the basic molecular and biochemical mechanisms of AIC, and then the contribution of zebrafish to the AIC field. We summarize the generation of embryonic zebrafish AIC models (eAIC) and their use for chemical screening and assessment of genetic modifiers, and then the generation of adult zebrafish AIC models (aAIC) and their use for discovering genetic modifiers via forward mutagenesis screening, deciphering spatial-temporal-specific mechanisms of modifier genes, and prioritizing therapeutic compounds via chemical genetic tools. Several therapeutic target genes and related therapies have emerged, including a retinoic acid (RA)-based therapy for the early phase of AIC and an autophagy-based therapy that, for the first time, is able to reverse cardiac dysfunction in the late phase of AIC. We conclude that zebrafish is becoming an important in vivo model that would accelerate both mechanistic studies and therapeutic development of AIC.

Transcellular chaperone signaling is an intercellular stress-response distinct from the HSF-1-mediated heat shock response.

Organismal proteostasis is maintained by intercellular signaling processes including cell nonautonomous stress responses such as transcellular chaperone signaling (TCS). When TCS is activated upon tissue-specific knockdown of hsp-90 in the Caenorhabditis elegans intestine, heat-inducible hsp-70 is induced in muscle cells at the permissive temperature resulting in increased heat stress resistance and lifespan extension. However, our understanding of the molecular mechanism and signaling factors mediating transcellular activation of hsp-70 expression from one tissue to another is still in its infancy. Here, we conducted a combinatorial approach using transcriptome RNA-Seq profiling and a forward genetic mutagenesis screen to elucidate how stress signaling from the intestine to the muscle is regulated. We find that the TCS-mediated "gut-to-muscle" induction of hsp-70 expression is suppressed by HSF-1 and instead relies on transcellular-X-cross-tissue (txt) genes. We identify a key role for the PDZ-domain guanylate cyclase txt-1 and the homeobox transcription factor ceh-58 as signaling hubs in the stress receiving muscle cells to initiate hsp-70 expression and facilitate TCS-mediated heat stress resistance and lifespan extension. Our results provide a new view on cell-nonautonomous regulation of "inter-tissue" stress responses in an organism that highlight a key role for the gut. Our data suggest that the HSF-1-mediated heat shock response is switched off upon TCS activation, in favor of an intercellular stress-signaling route to safeguard survival.

Large-scale forward genetic screening of zebrafish affecting thyroid development

The thyroid follicular cells originate from the foregut endoderm and elucidating which genes and signaling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to carry an ENU-based forward mutagenesis screen aiming at identifying genes involved in the development and function of the thyroid follicular cells. ENU is an excellent chemical mutagen due to its high mutation efficiency and an indiscriminate selection of genes. A total of 1606 F2 families from 36 ENU treated founders was raised and embryos from F3 generation were collected at 5dpf to perform the whole embryo in situ hybridization with a cocktail probe of thyroid marker thyroglobulin(tg), pituitary marker thyroid stimulating hormone (tshba) to determine the mutagenic phenotype. Among the 1606 F2 families, 112 F2 mutant families with normal development stages except for thyroid dysfunction were identified and divided into three different groups according to their phenotypic characteristics. Further studies of the mutants are likely to shed more insights into the molecular basis of both the thyroid development and function in the zebrafish and vertebrate.

Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli.

A forward mutagenesis assay using the supF gene has been widely employed for the last several decades in studies addressing mutation frequencies and mutation spectra associated with various intrinsic and environmental mutagens. In this study, by using a supF shuttle vector and non-SOS-induced Escherichia coli with short-read next-generation sequencing (NGS) technology, we present an advanced method for the study of mutations, which is simple, versatile, and cost-effective. We demonstrate the performance of our newly developed assay via pilot experiments with ultraviolet (UV) irradiation, the results from which emerge more relevant than expected. The NGS data obtained from samples of the indicator E. coli grown on titer plates provides mutation frequency and spectrum data, and uncovers obscure mutations that cannot be detected by a conventional supF assay. Furthermore, a very small amount of NGS data from selection plates reveals the almost full spectrum of mutations in each specimen and offers us a novel insight into the mechanisms of mutagenesis, despite them being considered already well known. We believe that the method presented here will contribute to future opportunities for research on mutagenesis, DNA repair, and cancer.

A bacterial three-hybrid assay for forward and reverse genetic analysis of RNA-protein interactions.

This protocol describes a bacterial three-hybrid (B3H) assay, an in vivo system that reports on RNA-protein interactions and can be implemented in both forward and reverse genetic experiments. The B3H assay connects the strength of an RNA-protein interaction inside of living Escherichia coli cells to the transcription of a reporter gene (here, lacZ). We present protocols to (1) insert RNA and protein sequences into appropriate vectors for B3H experiments, (2) detect putative RNA-protein interactions with both qualitative and quantitative readouts and (3) carry out forward genetic mutagenesis screens. The B3H assay builds on a well-established bacterial two-hybrid system for genetic analyses. As a result, protein-protein interactions can be assessed in tandem with RNA interactions with a bacterial two-hybrid assay to ensure that protein variants maintain their functionality. The B3H system is a powerful complement to traditional biochemical methods for dissecting RNA-protein interaction mechanisms: RNAs and proteins of interest do not need to be purified, and their interactions can be assessed under native conditions inside of a living bacterial cell. Once cloning has been completed, an assay can be completed in under a week and a screen in 1-2 weeks.

Resistance Profile and Structural Modeling of Next-Generation ROS1 Tyrosine Kinase Inhibitors.

ROS1 fusion proteins resulting from chromosomal rearrangements of the ROS1 gene are targetable oncogenic drivers in diverse cancers. Acquired resistance to targeted inhibitors curtails clinical benefit and response durability. Entrectinib, a NTRK/ROS1/ALK targeted tyrosine kinase inhibitor (TKI), was approved for the treatment of ROS1 fusion-positive non-small cell lung cancer (NSCLC) in 2019. In addition, lorlatinib and repotrectinib are actively being explored in the setting of treatment-naïve or crizotinib-resistant ROS1 fusion driven NSCLC. Here, we employed an unbiased forward mutagenesis screen in Ba/F3 CD74-ROS1 and EZR-ROS1 cells to identify resistance liabilities to entrectinib, lorlatinib, and repotrectinib. ROS1F2004C emerged as a recurrent entrectinib resistant mutation and ROS1G2032R was discovered in entrectinib and lorlatinib-resistant clones. Cell-based and modeling data show that entrectinib is a dual type I/II mode inhibitor, and thus liable to both types of resistant mutations. Comprehensive profiling of all clinically relevant kinase domain mutations showed that ROS1L2086F is broadly resistant to all type I inhibitors, but remains sensitive to type II inhibitors. ROS1F2004C/I/V are resistant to type I inhibitors, entrectinib and crizotinib, and type II inhibitor, cabozantinib, but retain sensitivity to the type I macrocyclic inhibitors. Development of new, more selective type II ROS1 inhibitor(s) or potentially cycling type I and type II inhibitors may be one way to expand durability of ROS1-targeted agents.

Transposon Insertion Mutagenesis in Hyperthermophilic Crenarchaeon Sulfolobus islandicus.

Transposon insertion mutagenesis is a forward genetic approach that has been widely utilized for genetic characterization of bacteria and single-celled eukaryotes, and its applications are being rapidly expanded into a few archaeal model organisms for gene function analysis. Previously, we developed a Tn5-based in vivo transposon insertion mutagenesis system in the hyperthermophilic crenarchaeon S. islandicucs M.16.4 and defined the essential gene set under laboratory growth conditions. In this chapter, we will mainly focus on presenting details regarding the generation of a near-saturating transposon insertion mutant library in this crenarchaeal model. We envision that the traditional transposon-based forward mutagenesis screening paired with next generation sequencing will greatly speed up the exploration of archaeal genomic features.

Abstract LB247: A forward genetic screen to identify drivers of neuroendocrine prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) is a deadly variant of prostate cancer for which no cure is yet available. Up to 20 percent of the patients with castration resistant prostate cancer (CRPC), will progress to NEPC after treatment with standard-of-care therapies, such as abiraterone and enzalutamide, that target the androgen receptor (AR) pathway. However, the molecular mechanisms that drive the emergence of NEPC are largely unknown, contributing to a lack of treatment options for patients with NEPC. We implemented a forward genetic approach to identify novel drivers of NEPC, by using the Sleeping Beauty (SB) transposon-based forward genetic mutagenesis screening system in our Pten-, Trp53-deficient mouse model that is primed to develop NEPC at a low frequency. We generated a mouse strain (NPp53SBT2Onc) in which the Nkx3.1 promoter regulates the expression of tamoxifen-inducible Cre specifically in the mouse prostate epithelium, resulting in transposition of the T2Onc2 transposable element and conditional deletion of Pten and Trp53 in mouse prostate epithelium. We observed an increased NEPC incidence in our NPp53SBT2Onc mouse model compared to control mice without the transposon T2Onc2, suggesting that the SB-mediated insertional mutagenesis events are driving the development of NEPC. To elucidate the genetic events that are driving the observed NEPC phenotype, we identified the common insertion sites (CISs) of the transposon insertions, which revealed a total of 330 CIS-associated genes. We further performed an integrative analysis using RNA-seq data from our SB cohort, a second mouse dataset and a metastatic CRPC patient cohort, which allowed us to identify the Master Regulator (MR) proteins that are associated with NEPC, as well as the upstream genomic events that modulate these MRs and the emergence of NEPC. Altogether, our approach resulted in the identification of CIS-associated genes that are upstream of these MRs associated to NEPC and that are likely to drive this lethal subtype of prostate cancer. We will further validate and elucidate the role of these candidate genes in prostate cancer progression to NEPC, in both mouse and human using CRISPR technology. In summary, we have developed a Sleeping Beauty forward genetic screen in our mouse model of CRPC, which not only accelerates disease progression, but also increases the incidence of NEPC. Our integrative analysis identified a panel of CIS-associated genes that are likely to be functionally involved in progression to NEPC. Validation of candidate drivers resulting from our analysis will shed light on mechanisms that this aggressive disease uses to evade treatments and may inform the development of novel therapies. Citation Format: Francisca Nunes de Almeida, Alessandro Vasciaveo, Min Zou, Matteo Di Bernardo, Andrea Califano, Cory Abate-Shen. A forward genetic screen to identify drivers of neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB247.

A novel allele of SIR2 reveals a heritable intermediate state of gene silencing.

Genetic information acquires additional meaning through epigenetic regulation, the process by which genetically identical cells can exhibit heritable differences in gene expression and phenotype. Inheritance of epigenetic information is a critical step in maintaining cellular identity and organismal health. In Saccharomyces cerevisiae, one form of epigenetic regulation is the transcriptional silencing of two mating-type loci, HML and HMR, by the SIR-protein complex. To focus on the epigenetic dimension of this gene regulation, we conducted a forward mutagenesis screen to identify mutants exhibiting an epigenetic or metastable silencing defect. We utilized fluorescent reporters at HML and HMR, and screened yeast colonies for epigenetic silencing defects. We uncovered numerous independent sir1 alleles, a gene known to be required for stable epigenetic inheritance. More interestingly, we recovered a missense mutation within SIR2, which encodes a highly conserved histone deacetylase. In contrast to sir1Δ, which exhibits states that are either fully silenced or fully expressed, this sir2 allele exhibited heritable states that were either fully silenced or expressed at an intermediate level. The heritable nature of this unique silencing defect was influenced by, but not completely dependent on, changes in rDNA copy number. Therefore, this study revealed a heritable state of intermediate silencing and linked this state to a central silencing factor, Sir2.

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

Deciphering the skeletome: Contributions from mouse forward genetics ENU mutagenesis campaigns

Deciphering the skeletome: Contributions from mouse forward genetics ENU mutagenesis campaigns

Exocyst mutants suppress pollen tube growth and cell wall structural defects of hydroxyproline O-arabinosyltransferase mutants.

SummaryHYDROXYPROLINE O‐ARABINOSYLTRANSFERASEs (HPATs) initiate a post‐translational protein modification (Hyp‐Ara) found abundantly on cell wall structural proteins. In Arabidopsis thaliana, HPAT1 and HPAT3 are redundantly required for full pollen fertility. In addition to the lack of Hyp‐Ara in hpat1/3 pollen tubes (PTs), we also found broadly disrupted cell wall polymer distributions, particularly the conversion of the tip cell wall to a more shaft‐like state. Mutant PTs were slow growing and prone to rupture and morphological irregularities. In a forward mutagenesis screen for suppressors of the hpat1/3 low seed‐set phenotype, we identified a missense mutation in exo70a2, a predicted member of the vesicle‐tethering exocyst complex. The suppressed pollen had increased fertility, fewer morphological defects and partially rescued cell wall organization. A transcriptional null allele of exo70a2 also suppressed the hpat1/3 fertility phenotype, as did mutants of core exocyst complex member sec15a, indicating that reduced exocyst function bypassed the PT requirement for Hyp‐Ara. In a wild‐type background, exo70a2 reduced male transmission efficiency, lowered pollen germination frequency and slowed PT elongation. EXO70A2 also localized to the PT tip plasma membrane, consistent with a role in exocyst‐mediated secretion. To monitor the trafficking of Hyp‐Ara modified proteins, we generated an HPAT‐targeted fluorescent secretion reporter. Reporter secretion was partially dependent on EXO70A2 and was significantly increased in hpat1/3 PTs compared with the wild type, but was reduced in the suppressed exo70a2 hpat1/3 tubes.

Utricularia.

Whitewoods introduces the plant genus Utricularia.

Missense mutations in a transmembrane domain of the Komagataeibacter xylinus BcsA lead to changes in cellulose synthesis

BackgroundCellulose is synthesized by an array of bacterial species. Komagataeibacter xylinus is the best characterized as it produces copious amounts of the polymer extracellularly. Despite many advances in the past decade, the mechanisms underlying cellulose biosynthesis are not completely understood. Elucidation of these mechanisms is essential for efficient cellulose production in industrial applications.ResultsIn an effort to gain a better understanding of cellulose biosynthesis and its regulation, cellulose crystallization was investigated in K. xylinus mutants resistant to an inhibitor of cellulose I formation, pellicin. Through the use of forward genetics and site-directed mutagenesis, A449T and A449V mutations in the K. xylinus BcsA protein were found to be important for conferring high levels of pellicin resistance. Phenotypic analysis of the bcsAA449T and bcsAA449V cultures revealed that the mutations affect cellulose synthesis rates and that cellulose crystallinity is affected in wet pellicles but not dry ones.ConclusionsA449 is located in a predicted transmembrane domain of the BcsA protein suggesting that the structure of the transmembrane domain influences cellulose crystallization either by affecting the translocation of the nascent glucan chain or by allosterically altering protein-protein interactions.

How flies contribute to the discovery of human diseases and their pathogenic mechanisms

How flies contribute to the discovery of human diseases and their pathogenic mechanisms