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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Revisiting synthetic lethality of Gcn5-related N-acetyltransferase (GNAT) family mutations in Haloferax volcanii

ABSTRACT Lysine acetylation is a post-translational modification that occurs in all domains of life, highlighting its evolutionary significance. Previous genome comparison identified three Gcn5-related N-acetyltransferase (GNAT) family members as lysine acetyltransferase homologs (Pat1, Pat2, and Elp3) and two deacetylase homologs (Sir2 and HdaI) in the halophilic archaeonHaloferax volcanii, withelp3andpat2proposed as a synthetic lethal gene pair. Here, we advance these findings by performing single and double mutagenesis ofelp3with thepat1andpat2lysine acetyltransferase gene homologs. Genome sequencing and PCR screens of these strains reveal successful generation of Δelp3,Δpat1Δelp3, and Δpat2Δelp3mutant strains. Although these mutant strains exhibited a reduced growth rate compared to the parent, they remained viable. Overall, this study provides genetic evidence thatelp3andpat2, while impacting cell growth, are not a synthetic lethal gene pair as previously reported. IMPORTANCE Here, we reveal by whole-genome sequencing that the GNAT family gene homologselp3andpat2can be deleted in the sameHaloferax volcaniistrain. Beyond the targeted deletions, minimal differences between the parent and Δelp3Δpat2mutant were observed, suggesting that suppressor mutations are not responsible for our ability to generate this double mutant strain. Elp3 and Pat2, thus, may not share as close a functional relationship as implied by earlier study. Our finding is significant as Elp3 is thought to function in acetylation in tRNA modification, while Pat2 likely functions in the lysine acetylation of proteins.

Microbiology↗
DOE OSTI2023

Identification of recG genetic interactions in Escherichia coli by transposon sequencing

ABSTRACT Maintaining the integrity of the genome is of utmost importance for cell division and propagation. In Escherichia coli , the RecG protein has been implicated in processing branched recombination intermediates during DNA repair processes, but the primary cellular role(s) of RecG and the repair pathways in which it acts have been difficult to define. To gain additional insight into RecG function, we employed transposon sequencing to identify recG genetic interactions and reveal complementary or redundant functions. The strongest hits from the screen were the dam , uvrD , rnhA , radA , and rep genes. The conditional importance of these five genes in cells lacking recG was confirmed using a plasmid-based assay, revealing synthetic lethal interactions for most double deletion strains. Several of the synthetic lethal gene combinations (with uvrD, rep, and radA , but not rnhA or dam ) were suppressed by deletion of recF or recO , indicating that their genetic relationships involved roles in post-replication gap repair. Additionally, loss of the RecG/SSB interaction phenocopied a recG deletion when combined with dam , uvrD , radA , or rnhA deletions but not with rep . The results reinforce the idea of RecG as a general genome guardian. RecG has at least two functions. It plays an important role in the resolution of joint molecules behind the fork, formed during post-replication gap repair. RecG is also required to suppress genome over-replication caused by unscheduled replication initiation at R-loops, at double-strand breaks caused by dam inactivation, and during replication termination. IMPORTANCE DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing bypasses embryo lethality

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate crosslinking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect crosslinking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MPG2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not crosslink normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→ sidechain. We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Biocontainment of Genetically Engineered Algae

Algae (including eukaryotic microalgae and cyanobacteria) have been genetically engineered to convert light and carbon dioxide to many industrially and commercially relevant chemicals including biofuels, materials, and nutritional products. At industrial scale, genetically engineered algae may be cultivated outdoors in open ponds or in closed photobioreactors. In either case, industry would need to address a potential risk of the release of the engineered algae into the natural environment, resulting in potential negative impacts to the environment. Genetic biocontainment strategies are therefore under development to reduce the probability that these engineered bacteria can survive outside of the laboratory or industrial setting. These include active strategies that aim to kill the escaped cells by expression of toxic proteins, and passive strategies that use knockouts of native genes to reduce fitness outside of the controlled environment of labs and industrial cultivation systems. Several biocontainment strategies have demonstrated escape frequencies below detection limits. However, they have typically done so in carefully controlled experiments which may fail to capture mechanisms of escape that may arise in the more complex natural environment. The selection of biocontainment strategies that can effectively kill cells outside the lab, while maintaining maximum productivity inside the lab and without the need for relatively expensive chemicals will benefit from further attention.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

Approximately a quarter of the human genome consists of gene deserts, large regions devoid of genes often located adjacent to developmental genes and thought to contribute to their regulation. However, defining the regulatory functions embedded within these deserts is challenging due to their large size. Here, we explore the cis-regulatory architecture of a gene desert flanking the Shox2 gene, which encodes a transcription factor indispensable for proximal limb, craniofacial, and cardiac pacemaker development. We identify the gene desert as a regulatory hub containing more than 15 distinct enhancers recapitulating anatomical subdomains of Shox2 expression. Ablation of the gene desert leads to embryonic lethality due to Shox2 depletion in the cardiac sinus venosus, caused in part by the loss of a specific distal enhancer. The gene desert is also required for stylopod morphogenesis, mediated via distributed proximal limb enhancers. In summary, our study establishes a multi-layered role of the Shox2 gene desert in orchestrating pleiotropic developmental expression through modular arrangement and coordinated dynamics of tissue-specific enhancers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Transcriptional competition shapes proteotoxic ER stress resolution

Through dynamic activities of conserved master transcription factors (mTFs), the unfolded protein response (UPR) relieves proteostasis imbalance of the endoplasmic reticulum (ER), a condition known as ER stress. Because dysregulated UPR is lethal, the competence for fate changes of the UPR mTFs must be tightly controlled. However, the molecular mechanisms underlying regulatory dynamics of mTFs remain largely elusive. Here, we identified the abscisic acid-related regulator G-class bZIP TF2 (GBF2) and the cis-regulatory element G-box as regulatory components of the plant UPR led by the mTFs, bZIP28 and bZIP60. We demonstrate that, by competing with the mTFs at G-box, GBF2 represses UPR gene expression. Conversely, a gbf2 null mutation enhances UPR gene expression and suppresses the lethality of a bzip28 bzip60 mutant in unresolved ER stress. Here, by demonstrating that GBF2 functions as a transcriptional repressor of the UPR, we address the long-standing challenge of identifying shared signalling components for a better understanding of the dynamic nature and complexity of stress biology. Furthermore, our results identify a new layer of UPR gene regulation hinged upon an antagonistic mTFs-GFB2 competition for proteostasis and cell fate determination.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

OPEN leaf : an open‐source cloud‐based phenotyping system for tracking dynamic changes at leaf‐specific resolution in Arabidopsis

SUMMARY The first draft of the Arabidopsis genome was released more than 20 years ago and despite intensive molecular research, more than 30% of Arabidopsis genes remained uncharacterized or without an assigned function. This is in part due to gene redundancy within gene families or the essential nature of genes, where their deletion results in lethality (i.e., thedark genome). High‐throughput plant phenotyping (HTPP) offers an automated and unbiased approach to characterize subtle or transient phenotypes resulting from gene redundancy or inducible gene silencing; however, access to commercial HTPP platforms remains limited. Here we describe the design and implementation ofOPEN leaf, an open‐source phenotyping system with cloud connectivity and remote bilateral communication to facilitate data collection, sharing and processing.OPEN leaf, coupled with our SMART imaging processing pipeline was able to consistently document and quantify dynamic changes at the whole rosette level and leaf‐specific resolution when plants experienced changes in nutrient availability. Our data also demonstrate that VIS sensors remain underutilized and can be used in high‐throughput screens to identify and characterize previously unidentified phenotypes in a leaf‐specific time‐dependent manner. Moreover, the modular and open‐source design ofOPEN leafallows seamless integration of additional sensors based on users and experimental needs.

Plant Sciences↗
DOE OSTI2025

Towards engineering hybrid incompatibility in plants

The potential for gene flow between genetically modified organisms (GMOs) and non-GMO relatives poses a significant challenge to the development and regulatory approval of GMO crops (Wedger et al., 2024), for example, the spread of herbicide resistance transgenes from crops such as rice or sorghum to cross-pollinating weedy species. Addressing this concern, we developed Engineered Genetic Incompatibility (EGI) (Maselko et al., 2017), a system that establishes species-like barriers to gene flow between otherwise sexually compatible populations. EGI employs Programmable Transcriptional Activators (PTAs) to drive lethal over- and/or ectopic expression of tightly regulated genes following undesired hybridization events (Figure 1a,b). A benign mutation of the target promoter in the EGI organism protects it from ill effects of the PTA, which acts as a sentinel for the wild-type (WT) promoter sequence. Given numerous potential PTA targets, multiple mutually incompatible subpopulations are feasible (Maselko et al., 2020). EGI has been demonstrated in yeast as a proof-of-concept (Maselko et al., 2017) and in insects as a strategy for genetic biocontrol of pests (Maselko et al., 2020; Upadhyay et al., 2022). EGI in plants would provide a strategy to halt gene flow between engineered crops and their domestic and wild relatives without altering normal cultivation or propagation practices. Here, we present promising results towards the demonstration of EGI in plants and highlight technical challenges that still need to be overcome.

CRISPRa↗
DOE OSTI2024

The first intron and promoter of Arabidopsis DIACYLGLYCEROL ACYLTRANSFERASE 1 exert synergistic effects on pollen and embryo lipid accumulation

Summary Accumulation of triacylglycerols (TAGs) is crucial during various stages of plant development. In Arabidopsis , two enzymes share overlapping functions to produce TAGs, namely acyl‐CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1). Loss of function of both genes in a dgat1‐1/pdat1‐2 double mutant is gametophyte lethal. However, the key regulatory elements controlling tissue‐specific expression of either gene has not yet been identified. We transformed a dgat1‐1/dgat1‐1//PDAT1/pdat1‐2 parent with transgenic constructs containing the Arabidopsis DGAT1 promoter fused to the AtDGAT1 open reading frame either with or without the first intron. Triple homozygous plants were obtained, however, in the absence of the DGAT1 first intron anthers fail to fill with pollen, seed yield is c . 10% of wild‐type, seed oil content remains reduced (similar to dgat1‐1/dgat1‐1 ), and non‐Mendelian segregation of the PDAT1/pdat1‐2 locus occurs. Whereas plants expressing the AtDGAT1pro:AtDGAT1 transgene containing the first intron mostly recover phenotypes to wild‐type. This study establishes that a combination of the promoter and first intron of AtDGAT1 provides the proper context for temporal and tissue‐specific expression of AtDGAT1 in pollen. Furthermore, we discuss possible mechanisms of intron mediated regulation and how regulatory elements can be used as genetic tools to functionally replace TAG biosynthetic enzymes in Arabidopsis .

McGuire, Sean T.↗
DOE OSTI2023

Crystal structures of OrfX1, OrfX2 and the OrfX1–OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A–G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1–OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Complete replacement of Arabidopsis oil-producing enzymes with heterologous diacylglycerol acyltransferases

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1) share responsibility for triacylglycerol (TAG) biosynthesis, and their selectivities control TAG fatty acid (FA) compositions. For rational metabolic engineering of seed oils, replacing endogenous TAG biosynthesis with exogenous enzymes containing different substrate FA selectivities is desirable; however, the dgat1-1/pdat1-2 double mutant is pollen lethal. Here, we evaluated the ability of 3 DGAT1s, from phylogenetically diverse plants with distinct TAG assembly processes, to completely replace endogenous TAG biosynthesis in Arabidopsis ( Arabidopsis thaliana ). We transformed dgat1-1 mutant plants with expression constructs for DGAT1 s from Camelina sativa , Physaria fendleri , and castor ( Ricinus communis ). Transgene expression was properly “contextualized” by using a previously determined minimum necessary expression unit containing the promoter/5′ UTR and first intron of native AtDGAT1 ; both of these DNA elements are essential for pollen expression. Next, we crossed homozygous lines with a DGAT1/DGAT1/PDAT1/pdat1-2 parent. C. sativa and P. fendleri DGAT1s restored the FA compositions and transcriptional differences of dgat1-1 to near wild-type and rescued the dgat1-1/pdat1-2 pollen lethality. R. communis DGAT1 was active in dgat1-1 seeds but produced unique oil profiles and alterations in the expression of lipid metabolic genes; it also failed to rescue dgat1-1/pdat1-2 lethality. This study confirms that the promoter and first intron of AtDGAT1 can modulate the expression of foreign DGAT1 genes to fit the correct spatiotemporal profile necessary for completely replacing endogenous TAG biosynthesis. Furthermore, it demonstrates an additional layer of unexpected enzyme incompatibility between oilseed lineages, which may complicate bioengineering approaches that seek to replace essential genes with orthologs.

McGuire, Sean T. [Washington State Univ., Pullman,↗
DOE OSTI2024

Dynamics of ER stress-induced gene regulation in plants

Endoplasmic reticulum (ER) stress is a potentially lethal condition that is induced by the abnormal accumulation of unfolded or misfolded secretory proteins in the ER. In eukaryotes, ER stress is managed by the unfolded protein response (UPR) through a tightly regulated, yet highly dynamic, reprogramming of gene transcription. Although the core principles of the UPR are similar across eukaryotes, unique features of the plant UPR reflect the adaptability of plants to their ever-changing environments and the need to balance the demands of growth and development with the response to environmental stressors. Here, the past decades have seen notable progress in understanding the mechanisms underlying ER stress sensing and signaling transduction pathways, implicating the UPR in the effects of physiological and induced ER stress on plant growth and crop yield. Facilitated by sequencing technologies and advances in genetic and genomic resources, recent efforts have driven the discovery of transcriptional regulators and elucidated the mechanisms that mediate the dynamic and precise gene regulation in response to ER stress at the systems level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Disrupting autorepression circuitry generates “open-loop lethality” to yield escape-resistant antiviral agents

Across biological scales, gene-regulatory networks employ autorepression (negative feedback) to maintain homeostasis and minimize failure from aberrant expression. In this study, we present a proof of concept that disrupting transcriptional negative feedback dysregulates viral gene expression to therapeutically inhibit replication and confers a high evolutionary barrier to resistance. We find that nucleic-acid decoys mimicking cis-regulatory sites act as “feedback disruptors,” break homeostasis, and increase viral transcription factors to cytotoxic levels (termed “open-loop lethality”). Feedback disruptors against herpesviruses reduced viral replication >2-logs without activating innate immunity, showed sub-nM IC 50 , synergized with standard-of-care antivirals, and inhibited virus replication in mice. In contrast to approved antivirals where resistance rapidly emerged, no feedback-disruptor escape mutants evolved in long-term cultures. For SARS-CoV-2, disruption of a putative feedback circuit also generated open-loop lethality, reducing viral titers by >1-log. These results demonstrate that generating open-loop lethality, via negative-feedback disruption, may yield a class of antimicrobials with a high genetic barrier to resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Knowledge graph-aided Bayesian active learning for top- K genetic interaction discovery

In silico methods for predicting the effects of multi-gene perturbations hold great promise for advancing functional genomics, computational drug discovery, and disease modeling. However, the development of these predictive algorithms for mammalian systems has been hampered by limited datasets and high experimental costs. In this study, we present a Bayesian active learning framework designed to discover pairwise host gene knockdowns that effectively inhibit viral proliferation in an in vitro HIV-1 infection model. Our method leverages a biological knowledge graph as side information and employs a computationally efficient batch diversification approach. We evaluated this framework using a dataset of viral load measurements obtained from multi-day dual-gene depletion experiments, encompassing all possible pairwise knockdowns of over 350 host genes associated with HIV infection. We demonstrate that our framework rapidly identifies the most effective gene knockdown pairs for reducing viral load. Furthermore, we show that incorporating side information enhances performance during the early stages of active learning (low data regime), while our batch diversification strategy significantly boosts performance in later stages (high data regime). This framework is general and can be adapted to explore gene interactions in other contexts, such as synthetic lethality prediction and mapping epistatic effects across quantitative trait loci.

Computational biology and bioinformatics↗
DOE OSTI2025

Symmetric adenine methylation is an essential DNA modification in the early-diverging fungus Rhizopus microsporus

The discovery of N6-methyladenine (6mA) in eukaryotic genomes, typically found in prokaryotic DNA, has revolutionized epigenetics. Here, we show that symmetric 6mA is essential in the early diverging fungus Rhizopus microsporus, as the absence of the MT-A70 complex (MTA1c) responsible for this modification results in a lethal phenotype. 6mA is present in 70% of the genes, correlating with the presence of H3K4me3 and H2A.Z in open euchromatic regions. This modification is found predominantly in nucleosome linker regions, influencing the nucleosome positioning around the transcription start sites of highly expressed genes. Controlled downregulation of MTA1c reduces symmetric 6mA sites affecting nucleosome positioning and histone modifications, leading to altered gene expression, which is likely the cause of the severe phenotypic changes observed. Our study highlights the indispensable role of the DNA 6mA in a multicellular organism and delineates the mechanisms through which this epigenetic mark regulates gene expression in a eukaryotic genome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Determination and analysis of sex ratios in heme oxygenase 1 gene targeted mouse embryos

Abstract Heme oxygenase 1 or Hmox1 enzyme is involved in catalyzing the first and rate‐limiting step in heme breakdown reactions. Many studies have reported a partial lethality of Hmox1 knockout mice obtained from heterozygous breeding pairs. Similar results were obtained in our transgenic mice colony and a sex specific bias was observed in the favor of males in the adult mice. Hmox1 independent factors which could have caused this bias were initially analyzed and it was found that those factors were not a reason behind this anomaly. Certain studies involving gene knockout hinted toward a prenatal or neonatal lethality of female knockout mice embryos or pups, respectively. In order to check if this bias was occurring in embryonic stages, that is, either if mutant female embryos were dying or if heterozygous mothers were not carrying embryos to term, we analyzed the sex‐ratios in mid‐ and late‐gestational ages (9.5–13.5 dpc and 14.5–18.5 dpc, respectively). Our results did not indicate any significant difference in the sex ratios in embryonic stages; hence, it was concluded that females are not dying in embryonic stages. It can be speculated that these deaths were probably occurring at neonatal age. More studies are required to confirm that the lack of Hmox1 gene products is the sole reason for this female lethality.

Jain, Sidhant↗
DOE OSTI2024

Assessment of the Effect of Deleting the African Swine Fever Virus Gene R298L on Virus Replication and Virulence of the Georgia2010 Isolate

African swine fever (ASF) is a lethal disease of domestic pigs that is currently challenging swine production in large areas of Eurasia. The causative agent, ASF virus (ASFV), is a large, double-stranded and structurally complex virus. The ASFV genome encodes for more than 160 proteins; however, the functions of most of these proteins are still in the process of being characterized. The ASF gene R298L, which has previously been characterized as able to encode a functional serine protein kinase, is expressed late in the virus infection cycle and may be part of the virus particle. There is no description of the importance of the R298L gene in basic virus functions such as replication or virulence in the natural host. Based on its evolution, it is proposed that there are four different phenotypes of R298L of ASFV in nature, which may have potential implications for R298L functionality. We report here that a recombinant virus lacking the R298L gene in the Georgia 2010 isolate, ASFV-G-∆R298L, does not exhibit significant changes in its replication in primary cultures of swine macrophages. In addition, when experimentally inoculated in pigs, ASFV-G-∆R298L induced a fatal form of the disease similar to that caused by the parental virulent ASFV-G. Therefore, deletion of R298L does not significantly affect virus replication and virulence in domestic pigs of the ASFV Georgia 2010 isolate.

Virology↗
DOE OSTI2024

Multi-omics Resources for Understanding Gene Regulation in Response to ER Stress in Plants

Proteotoxic stress of the endoplasmic reticulum (ER) is a potentially lethal condition that ensues when the biosynthetic capacity of the ER is overwhelmed. A sophisticated and largely conserved signaling, known as the unfolded protein response (UPR), is designed to monitor and alleviate ER stress. In plants, the emerging picture of gene regulation by the UPR now appears to be more complex than ever before, requiring multi-omics-enabled network-level approaches to be untangled. In the past decade, with an increasing access and decreasing costs of next-generation sequencing (NGS) and high-throughput protein–DNA interaction (PDI) screening technologies, multitudes of global molecular measurements, known as omics, have been generated and analyzed by the research community to investigate the complex gene regulation of plant UPR. In this chapter, we present a comprehensive catalog of omics resources at different molecular levels (transcriptomes, protein–DNA interactomes, and networks) along with the introduction of key concepts in experimental and computational tools in data generation and analyses. Finally, this chapter will serve as a starting point for both experimentalists and bioinformaticians to explore diverse omics datasets for their biological questions in the plant UPR, with likely applications also in other species for conserved mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗