Transcriptional Reporter Research Articles

Peripubertal exposure to oxyfluorfen, a diphenyl herbicide, delays pubertal development in the male rat by antagonizing androgen receptor activity.

We recently identified the herbicide oxyfluorfen as an inhibitor of iodide uptake by the sodium iodide symporter (NIS), a key step in thyroid hormone synthesis, using in vitro assays. We also observed a suppression of serum T4 and T3 in juvenile rats exposed orally to oxyfluorfen for 4-8 days. The purpose of the present study was to further evaluate effects of an extended 31-day oral exposure using a male pubertal rat study (15 to 500 mg/kg). Oxyfluorfen delayed puberty at all doses (1.3 -3.5 days) suppressing ventral prostate at 62.5 mg/kg and above and seminal vesicle weights at 31.25 mg/kg and above with no effect on testosterone or luteinizing hormone. Serum T4 and T3 were suppressed by all doses up to 80%, with a linear increase in serum TSH. Based on delayed puberty without changes in testosterone, we hypothesized that oxyfluorfen interferes with androgen receptor (AR) function. Results from our Hershberger study, with oxyfluorfen (62.5 and 125 mg/kg) co-treated with testosterone proprionate (TP , 1 mg/kg) for 10 days showed 3 of 5 of the androgenic tissue weights were suppressed compared to TP alone indicating AR antagonism. We next confirmed this effect in an in vitro AR transcriptional activation reporter assay (0-20 μM) with 125 pM 5αDH-11-ketotestosterone and found concentration-dependent inhibition of AR luminescence activity (EC50 1.75 µM) without cytotoxicity. Here, we confirmed the endocrine disrupting effects of oxyfluorfen using both in vitro and in vivo evaluations of the thyroid hormone and AR pathways. This abstract doesn't necessarily reflect U.S. EPA policy.

Co-existence of two antibiotic-producing marine bacteria: Pseudoalteromonas piscicida reduce gene expression and production of the antibacterial compound, tropodithietic acid, in Phaeobacter sp.

Many bacteria co-exist and produce antibiotics, yet we know little about how they cope and occupy the same niche. The purpose of the present study was to determine if and how two potent antibiotic-producing marine bacteria influence the secondary metabolome of each other. We established an agar- and broth-based system allowing co-existence of a Phaeobacter species and Pseudoalteromonas piscicida that, respectively, produce tropodithietic acid (TDA) and bromoalterochromides (BACs). Co-culturing of Phaeobacter sp. strain A36a-5a on Marine Agar with P. piscicida strain B39bio caused a reduction of TDA production in the Phaeobacter colony. We constructed a transcriptional gene reporter fusion in the tdaC gene in the TDA biosynthetic pathway in Phaeobacter and demonstrated that the reduction of TDA by P. piscicida was due to the suppression of the TDA biosynthesis. A stable liquid co-cultivation system was developed, and the expression of tdaC in Phaeobacter was reduced eightfold lower (per cell) in the co-culture compared to the monoculture. Mass spectrometry imaging of co-cultured colonies revealed a reduction of TDA and indicated that BACs diffused into the Phaeobacter colony. BACs were purified from Pseudoalteromonas; however, when added as pure compounds or a mixture they did not influence TDA production. In co-culture, the metabolome was dominated by Pseudoalteromonas features indicating that production of other Phaeobacter compounds besides TDA was reduced. In conclusion, co-existence of two antibiotic-producing bacteria may be allowed by one causing reduction in the antagonistic potential of the other. The reduction (here of TDA) was not caused by degradation but by a yet uncharacterized mechanism allowing Pseudoalteromonas to reduce expression of the TDA biosynthetic pathway.IMPORTANCEThe drug potential of antimicrobial secondary metabolites has been the main driver of research into these compounds. However, in recent years, their natural role in microbial systems and microbiomes has become important to determine the assembly and development of microbiomes. Herein, we demonstrate that two potent antibiotic-producing bacteria can co-exist, and one mechanism allowing the co-existence is the specific reduction of antibiotic production in one bacterium by the other. Understanding the molecular mechanisms in complex interactions provides insights for applied uses, such as when developing TDA-producing bacteria for use as biocontrol in aquaculture.

Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in C. elegans.

Caenorhabditis elegans males undergo sex-specific tail tip morphogenesis (TTM) under the control of the DM-domain transcription factor DMD-3. To find genes regulated by DMD-3, we performed RNA-seq of laser-dissected tail tips. We identified 564 genes differentially expressed (DE) in wild-type males versus dmd-3(-) males and hermaphrodites. The transcription profile of dmd-3(-) tail tips is similar to that in hermaphrodites. For validation, we analyzed transcriptional reporters for 49 genes and found male-specific or male-biased expression for 26 genes. Only 11 DE genes overlapped with genes found in a previous RNAi screen for defective TTM. GO enrichment analysis of DE genes finds upregulation of genes within the unfolded protein response pathway and downregulation of genes involved in cuticle maintenance. Of the DE genes, 40 are transcription factors, indicating that the gene network downstream of DMD-3 is complex and potentially modular. We propose modules of genes that act together in TTM and are co-regulated by DMD-3, among them the chondroitin synthesis pathway and the hypertonic stress response.

A screen for cell envelope stress uncovers an inhibitor of prolipoprotein diacylglyceryl transferase, Lgt, in Escherichia coli

The increasing prevalence of antibiotic resistance demands the discovery of antibacterial chemical scaffolds with unique mechanisms of action. Phenotypic screening approaches, such as the use of reporters for bacterial cell stress, offer promise to identify compounds while providing strong hypotheses for follow-on mechanism of action studies. From a collection of ∼1,800 Escherichia coli GFP transcriptional reporter strains, we identified a reporter that is highly induced by cell envelope stress-pProm rcsA -GFP. After characterizing pProm rcsA -GFP induction, we assessed a collection of bioactive small molecules for reporter induction, identifying 24 compounds of interest. Spontaneous suppressors to one compound in particular, MAC-0452936, mapped to the gene encoding the essential prolipoprotein diacylglyceryl transferase, lgt. Lgt inhibition by MAC-0452936 inhibition was confirmed through genetic, phenotypic, and biochemical approaches. The oxime ester, MAC-0452936, represents a useful small molecule inhibitor of Lgt and highlights the potential of using pProm rcsA -GFP as a phenotypic screening tool.

Rapid, biochemical tagging of cellular activity history in vivo

Intracellular calcium (Ca2+) is ubiquitous to cell signaling across biology. While existing fluorescent sensors and reporters can detect activated cells with elevated Ca2+ levels, these approaches require implants to deliver light to deep tissue, precluding their noninvasive use in freely behaving animals. Here we engineered an enzyme-catalyzed approach that rapidly and biochemically tags cells with elevated Ca2+ in vivo. Ca2+-activated split-TurboID (CaST) labels activated cells within 10 min with an exogenously delivered biotin molecule. The enzymatic signal increases with Ca2+ concentration and biotin labeling time, demonstrating that CaST is a time-gated integrator of total Ca2+ activity. Furthermore, the CaST readout can be performed immediately after activity labeling, in contrast to transcriptional reporters that require hours to produce signal. These capabilities allowed us to apply CaST to tag prefrontal cortex neurons activated by psilocybin, and to correlate the CaST signal with psilocybin-induced head-twitch responses in untethered mice.

Deleterious ZNRF3 germline variants cause neurodevelopmental disorders with mirror brain phenotypes via domain-specific effects on Wnt/β-catenin signaling

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/β-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using invitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/β-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/β-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/β-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/β-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants.

Optimized reporters for multiplexed detection of transcription factor activity.

In any given cell type, dozens of transcription factors (TFs) act in concert to control the activity of the genome by binding to specific DNA sequences in regulatory elements. Despite their considerable importance in determining cell identity and their pivotal role in numerous disorders, we currently lack simple tools to directly measure the activity of many TFs in parallel. Massively parallel reporter assays (MPRAs) allow the detection of TF activities in a multiplexed fashion; however, we lack basic understanding to rationally design sensitive reporters for many TFs. Here, we use an MPRA to systematically optimize transcriptional reporters for 86 TFs and evaluate the specificity of all reporters across a wide array of TF perturbation conditions. We thus identified critical TF reporter design features and obtained highly sensitive and specific reporters for 60 TFs, many of which outperform available reporters. The resulting collection of "prime" TF reporters can be used to uncover TF regulatory networks and to illuminate signaling pathways.

SUMOylation of nuclear receptor Nor1/NR4A3 coordinates microtubule cytoskeletal dynamics and stability in neuronal cells

BackgroundNor1/NR4A3 is a member of the NR4A subfamily of nuclear receptors that play essential roles in regulating gene expression related to development, cell homeostasis and neurological functions. However, Nor1 is still considered an orphan receptor, as its natural ligand remains unclear for mediating transcriptional activation. Yet other activation signals may modulate Nor1 activity, although their precise role in the development and maintenance of the nervous system remains elusive.MethodsWe used transcriptional reporter assays, gene expression profiling, protein turnover measurement, and cell growth assays to assess the functional relevance of Nor1 and SUMO-defective variants in neuronal cells. SUMO1 and SUMO2 conjugation to Nor1 were assessed by immunoprecipitation. Tubulin stability was determined by acetylation and polymerization assays, and live-cell fluorescent microscopy.ResultsHere, we demonstrate that Nor1 undergoes SUMO1 conjugation at Lys-89 within a canonical ψKxE SUMOylation motif, contributing to the complex pattern of Nor1 SUMOylation, which also includes Lys-137. Disruption of Lys-89, thereby preventing SUMO1 conjugation, led to reduced Nor1 transcriptional competence and protein stability, as well as the downregulation of genes involved in cell growth and metabolism, such as ENO3, EN1, and CFLAR, and in microtubule cytoskeleton dynamics, including MAP2 and MAPT, which resulted in reduced survival of neuronal cells. Interestingly, Lys-89 SUMOylation was potentiated in response to nocodazole, a microtubule depolymerizing drug, although this was insufficient to rescue cells from microtubule disruption despite enhanced Nor1 gene expression. Instead, Lys-89 deSUMOylation reduced the expression of microtubule-severing genes like KATNA1, SPAST, and FIGN, and enhanced α-tubulin cellular levels, acetylation, and microfilament organization, promoting microtubule stability and resistance to nocodazole. These effects contrasted with Lys-137 SUMOylation, suggesting distinct regulatory mechanisms based on specific Nor1 input SUMOylation signals.ConclusionsOur study provides novel insights into Nor1 transcriptional signaling competence and identifies a hierarchical mechanism whereby selective Nor1 SUMOylation may govern neuronal cytoskeleton network dynamics and resistance against microtubule disturbances, a condition strongly associated with neurodegenerative diseases.

LasR regulates protease IV expression at suboptimal growth temperatures in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa causes debilitating lung infections in people with cystic fibrosis, as well as eye, burn, and wound infections in otherwise immunocompetent individuals. Many of P. aeruginosa's virulence factors are regulated by environmental changes associated with human infection, such as a change in temperature from ambient to human body temperature. One such virulence factor is protease IV (PIV). Interestingly, piv expression is higher at ambient temperatures (22-28°C) compared to human body temperature (37°C). We found that piv expression was thermoregulated at stationary phase, but not exponential phase, and that piv is thermoregulated at the level of transcription. Protein levels of known transcriptional regulators of piv, the quorum sensing regulator LasR and the gene-silencing histone nucleoid silencing proteins MvaT/MvaU, were not thermoregulated. Using a transcriptional reporter for piv, we show that LasR activates piv expression at stationary phase at 25°C but not 37°C, while MvaT/MvaU are not required for piv thermoregulation. We also identified a las box in the piv promoter, which is important for piv thermoregulation. We propose that LasR directly regulates piv at stationary phase at 25°C but has a negligible impact at 37°C. Here, we show that piv is uniquely regulated by LasR in a temperature-dependent manner. Our findings suggest that the LasRI quorum sensing regulon of P. aeruginosa may not be fully characterized and that growth at non-standard laboratory conditions such as lower temperatures could reveal previously unrecognized quorum sensing regulated genes.

Clock gene homologs lin-42 and kin-20 regulate circadian rhythms in C. elegans

Circadian rhythms are endogenous oscillations in nearly all organisms, from prokaryotes to humans, allowing them to adapt to cyclical environments for close to 24 h. Circadian rhythms are regulated by a central clock, based on a transcription-translation feedback loop. One important protein in the central loop in metazoan clocks is PERIOD, which is regulated in part by Casein kinase 1ε/δ (CK1ε/δ) phosphorylation. In the nematode Caenorhabditis elegans, period and casein kinase 1ε/δ are conserved as lin-42 and kin-20, respectively. Here, we studied the involvement of lin-42 and kin-20 in the circadian rhythms of the adult nematode using a bioluminescence-based circadian transcriptional reporter. We show that mutations of lin-42 and kin-20 generate a significantly longer endogenous period, suggesting a role for both genes in the nematode circadian clock, as in other organisms. These phenotypes can be partially rescued by overexpression of either gene under their native promoter. Both proteins are expressed in neurons and epidermal seam cells, as well as in other cells. Depletion of LIN-42 and KIN-20, specifically in neuronal cells after development, was sufficient to lengthen the period of oscillating sur-5 expression. Therefore, we conclude that LIN-42 and KIN-20 are critical regulators of the adult nematode circadian clock through neuronal cells.

Fmo induction as a tool to screen for pro-longevity drugs.

Dietary restriction (DR) and hypoxia (low oxygen) extend lifespan in Caenorhabditis elegans through the induction of a convergent downstream longevity gene, fmo-2. Flavin-containing monooxygenases (FMOs) are highly conserved xenobiotic-metabolizing enzymes with a clear role in promoting longevity in nematodes and a plausible similar role in mammals. This makes them an attractive potential target of small molecule drugs to stimulate the health-promoting effects of longevity pathways. Here, we utilize an fmo-2 fluorescent transcriptional reporter in C. elegans to screen a set of 80 compounds previously shown to improve stress resistance in mouse fibroblasts. Our data show that 19 compounds significantly induce fmo-2, and 10 of the compounds induce fmo-2 more than twofold. Interestingly, 9 of the 10 high fmo-2 inducers also extend lifespan in C. elegans. Two of these drugs, mitochondrial respiration chain complex inhibitors, interact with the hypoxia pathway to induce fmo-2, whereas two dopamine receptor type 2 (DRD2) antagonists interact with the DR pathway to induce fmo-2, indicating that dopamine signaling is involved in DR-mediated fmo-2 induction. Together, our data identify nine drugs that each (1) increase stress resistance in mouse fibroblasts, (2) induce fmo-2 in C. elegans, and (3) extend nematode lifespan, some through known longevity pathways. These results define fmo-2 induction as a viable approach to identifying and understanding mechanisms of putative longevity compounds.

Exploiting Cell-Based Assays to Accelerate Drug Development for G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are relevant targets for health and disease as they regulate various aspects of metabolism, proliferation, differentiation, and immune pathways. They are implicated in several disease areas, including cancer, diabetes, cardiovascular diseases, and mental disorders. It is worth noting that about a third of all marketed drugs target GPCRs, making them prime pharmacological targets for drug discovery. Numerous functional assays have been developed to assess GPCR activity and GPCR signaling in living cells. Here, we review the current literature of genetically encoded cell-based assays to measure GPCR activation and downstream signaling at different hierarchical levels of signaling, from the receptor to transcription, via transducers, effectors, and second messengers. Singleplex assay formats provide one data point per experimental condition. Typical examples are bioluminescence resonance energy transfer (BRET) assays and protease cleavage assays (e.g., Tango or split TEV). By contrast, multiplex assay formats allow for the parallel measurement of multiple receptors and pathways and typically use molecular barcodes as transcriptional reporters in barcoded assays. This enables the efficient identification of desired on-target and on-pathway effects as well as detrimental off-target and off-pathway effects. Multiplex assays are anticipated to accelerate drug discovery for GPCRs as they provide a comprehensive and broad identification of compound effects.

Heterogeneity of tethered agonist signaling in adhesion G protein-coupled receptors

Adhesion G protein-coupled receptor (aGPCR) signaling influences development and homeostasis in a wide range of tissues. In the current model for aGPCR signaling, ligand binding liberates a conserved sequence that acts as an intramolecular, tethered agonist (TA), yet this model has not been evaluated systematically for all aGPCRs. Here, we assessed the TA-dependent activities of all 33 aGPCRs in a suite of transcriptional reporter, G protein activation, and β-arrestin recruitment assays using a new fusion protein platform. Strikingly, only ∼50% of aGPCRs exhibited robust TA-dependent activation, and unlike other GPCR families, aGPCRs showed a notable preference for G12/13 signaling. AlphaFold2 predictions assessing TA engagement in the predicted intramolecular binding pocketaligned with the TA dependence of the cellular responses. This dataset provides a comprehensive resource to inform the investigation of all human aGPCRs and for targeting aGPCRs therapeutically.

Orfamide production in Pseudomonas protegens CHA0T promotes rhizospheric colonization and influences assemblage of the bacterial community of wheat roots in soil.

Orfamides (Ofa) are a family of closely related cyclic lipopeptides (cLPs) produced by different soil-inhabiting and plant beneficial strains of the Pseudomonas protegens subgroup. Orfamides are required for swarming motility, they mediate toxicity on oomycetes, algae, and insects, and they can act as ISR elicitors. Ofa biosynthesis depends on a non-ribosomal peptide synthetase gene cluster whose expression is tightly controlled by the post-transcriptional regulatory cascade Gac-Rsm. Although the biological properties and physiological role of Ofa have been well characterized in vitro, the role of Ofa in the fitness of the producing bacterium in its niche has not been studied so far. Here, we used an Ofa biosynthesis-deficient mutant (ΔofaABC) derived from the biocontrol model strain P. protegens CHA0T to explore the relevance of this cLP in soil for the survival of strain CHA0T, its contribution to the bacterial competitiveness for colonization of wheat roots, and its impact on the corresponding rhizobacterial community structure. To facilitate CFU counts in non-sterile soil and root samples we used chromosomally tagged versions of CHA0T and its ΔofaABC mutant with mini-Tn7 constructs carrying antibiotic resistance markers. We confirmed that the tagged Ofa− mutant does not show drop-collapse activity neither it can swarm in semi-solid medium. We also confirmed that Ofa can be exploited in vitro as a social good by the Ofa− mutant to resume swarming in the presence of CHA0T cells. We found that Ofa production did not confer any advantage for bacterial survival along a period of 5 weeks after soil inoculation. However, we observed that Ofa production was associated with a higher competitiveness for colonization of the wheat rhizosphere. Expression of the ofa biosynthetic operon, by means of a transcriptional Pofa-gfp reporter fusion, was detected in the rhizoplane of colonized wheat roots, suggesting that Ofa is produced in situ on the surface of colonized wheat roots. Finally, we observed that seed inoculation with the wild type Ofa producer strain CHA0T resulted in a differential assemblage of the bacterial community of the wheat rhizosphere with respect to that of seeds bacterized with the Ofa deficient mutant. Overall, we provide evidence that Ofa is important for the competitiveness of P. protegens CHA0T cells to colonize the surface of wheat roots and that Ofa production in the rhizosphere can impact the structure of the assembled bacterial microbiome.

A gold speciation that adds a second layer to synergistic gold-copper toxicity in Cupriavidus metallidurans.

The metal-resistant bacterium Cupriavidus metallidurans occurs in metal-rich environments. In auriferous soils, the bacterium is challenged by a mixture of copper ions and gold complexes, which exert synergistic toxicity. The previously used, self-made Au(III) solution caused a synergistic toxicity of copper and gold that was based on the inhibition of the CupA-mediated efflux of cytoplasmic Cu(I) by Au(I) in this cellular compartment. In this publication, the response of the bacterium to gold and copper was investigated by using a commercially available Au(III) solution instead of the self-made solution. The new solution was five times more toxic than the previously used one. Increased toxicity was accompanied by greater accumulation of gold atoms by the cells. The contribution of copper resistance determinants to the commercially available Au(III) solution and synergistic gold-copper toxicity was studied using single- and multiple-deletion mutants. The commercially available Au(III) solution inhibited periplasmic Cu(I) homeostasis, which is required for the allocation of copper ions to copper-dependent proteins in this compartment. The presence of the gene for the periplasmic Cu(I) and Au(I) oxidase, CopA, decreased the cellular copper and gold content. Transcriptional reporter gene fusions showed that up-regulation of gig, encoding a minor contributor to copper resistance, was strictly glutathione dependent. Glutathione was also required to resist synergistic gold-copper toxicity. The new data indicated a second layer of synergistic copper-gold toxicity caused by the commercial Au(III) solution, inhibition of the periplasmic copper homeostasis in addition to the cytoplasmic one.IMPORTANCEWhen living in auriferous soils, Cupriavidus metallidurans is not only confronted with synergistic toxicity of copper ions and gold complexes but also by different gold species. A previously used gold solution made by using aqua regia resulted in the formation of periplasmic gold nanoparticles, and the cells were protected against gold toxicity by the periplasmic Cu(I) and Au(I) oxidase CopA. To understand the role of different gold species in the environment, another Au(III) solution was commercially acquired. This compound was more toxic due to a higher accumulation of gold atoms by the cells and inhibition of periplasmic Cu(I) homeostasis. Thus, the geo-biochemical conditions might influence Au(III) speciation. The resulting Au(III) species may subsequently interact in different ways with C. metallidurans and its copper homeostasis system in the cytoplasm and periplasm. This study reveals that the geochemical conditions may decide whether bacteria are able to form gold nanoparticles or not.

Staphylococcus aureus oleate hydratase produces ligands that activate host PPARα.

Commensal gut bacteria use oleate hydratase to release a spectrum of hydroxylated fatty acids using host-derived unsaturated fatty acids. These compounds are thought to attenuate the immune response, but the underlying signaling mechanism(s) remain to be established. The pathogen Staphylococcus aureus also expresses an oleate hydratase and 10-hydroxyoctadecanoic acid (h18:0) is the most abundant oleate hydratase metabolite found at Staphylococcal skin infection sites. Here, we show h18:0 stimulates the transcription of a set of lipid metabolism genes associated with the activation of peroxisome proliferator activated receptor (PPAR) in the RAW 264.7 macrophage cell line and mouse primary bone marrow-derived macrophages. Cell-based transcriptional reporter assays show h18:0 selectively activates PPARα. Radiolabeling experiments with bone marrow-derived macrophages show [1-14C]h18:0 is not incorporated into cellular lipids, but is degraded by β-oxidation, and mass spectrometry detected shortened fragments of h18:0 released into the media. The catabolism of h18:0 was >10-fold lower in bone marrow-derived macrophages isolated from Ppara -/- knockout mice, and we recover 74-fold fewer S. aureus cells from the skin infection site of Ppara -/- knockout mice compared to wildtype mice. These data identify PPARα as a target for oleate hydratase-derived hydroxy fatty acids and support the existence of an oleate hydratase-PPARα signaling axis that functions to suppress the innate immune response to S. aureus.

Abstract 3041: The small MAF transcription factor MAFG is a potent driver of melanoma

Abstract While common mutations in potent proto-oncogenes and tumor suppressors induce melanoma formation, no recurrent mutations associated with the late stages of melanomagenesis have been identified. Instead, non-mutational mechanisms such as the deregulation of transcriptional or epigenetic programs typically promote the malignant progression and metastasis of melanoma. We have previously identified the small MAF transcription factor MAFG as a critical target of the melanoma suppressor miRNA miR-29, prompting an in-depth evaluation of the role of MAFG in melanoma. MAFG expression is elevated in melanoma compared to melanocytes and increases with tumor stage. Ectopic expression of MAFG in human melanocytes and melanoma cells enhances proliferation in vitro and promotes melanoma growth in vivo. Moreover, MAFG overexpression in BrafV600E; PtenFL/+ mice accelerated melanomagenesis, significantly shortening overall survival. Despite being a critical NRF2 dimerization partner, NRF2 was dispensable for the effects of MAFG. Moreover, MAFG overexpression in melanoma cells had no effect on the activity of NRF2 transcriptional reporters, nor did it induce transcriptional programs associated with NRF2. RNA sequencing and pathway analysis revealed MAFG-mediated effects on melanoma-associated processes such hypoxia, the PI3K/AKT pathway, and pigmentation. Indeed, MAFG induced HIF1a under normoxic conditions, enhanced AKT phosphorylation, and downregulated MITF and its target genes in the pigmentation pathway in melanoma cells. Interestingly, MAFG also potently enhanced AXL expression, suggesting that MAFG may promote melanoma, at least in part, by affecting the MITF/AXL balance that is associated with melanoma phenotype switching. We next analyzed whether MAFG is required for melanoma. Interestingly, silencing MAFG in human melanoma cells robustly inhibited growth in vitro. Moreover, the CRISPR/Cas9-mediated knockout of MAFG in an aggressive BrafV600E; PtenFL/FL model potently prevented melanoma formation. Given the mild phenotype of MAFG whole-body knockout mice, our results suggest targeting of MAFG or its downstream pathways as a viable therapeutic approach in melanoma. In conclusion, our study establishes MAFG as a potent driver of melanoma development and nominates it as a therapeutic target. Citation Format: Olga Vera, Michael Martinez, Zulaida Soto-Vargas, Xiaonan Xu, Florian Karreth. The small MAF transcription factor MAFG is a potent driver of melanoma [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 3041.

A rapid and scalable approach to build synthetic repetitive hormone-responsive promoters.

Advancement of DNA-synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high-complexity DNA sequences containing tandems of short repeats are still notoriously difficult to produce synthetically, with commercial DNA synthesis companies usually rejecting orders that exceed specific sequence complexity thresholds. To overcome this limitation, we developed a simple, single-tube reaction method that enables the generation of DNA sequences containing multiple repetitive elements. Our strategy involves commercial synthesis and PCR amplification of padded sequences that contain the repeats of interest, along with random intervening sequence stuffers that include type IIS restriction enzyme sites. GoldenBraid molecular cloning technology is then employed to remove the stuffers, rejoin the repeats together in a predefined order, and subclone the tandem(s) in a vector using a single-tube digestion-ligation reaction. In our hands, this new approach is much simpler, more versatile and efficient than previously developed solutions to this problem. As a proof of concept, two different phytohormone-responsive, synthetic, repetitive proximal promoters were generated and tested in planta in the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene-responsive promoter 10x2EBS-S10 fused to the GUS reporter gene uncovered several developmentally regulated ethylene response maxima, indicating the utility of this reporter for monitoring the involvement of ethylene in a variety of physiologically relevant processes. These encouraging results suggest that this reporter system can be leveraged to investigate the ethylene response to biotic and abiotic factors with high spatial and temporal resolution.

Interferon-induced protein with tetratricopeptide repeats 5 of black fruit bat (Pteropus alecto) displays a broad inhibition of RNA viruses.

Bats are natural host reservoirs and have adapted a unique innate immune system that permits them to host many viruses without exhibiting symptoms. Notably, bat interferon stimulated genes (ISGs) have been shown to play antiviral roles. Interferon induced protein with tetratricopeptide repeats 5 (IFIT5) is a well-characterised ISG in humans with antiviral activities against negative-sense RNA viruses via inhibiting viral transcription. Here, we aim to investigate if Pteropus alecto (pa) IFIT5 (paIFIT5) possess the ability to inhibit negative-sense RNA viruses. Initially, gene syntenic and comparative structural analyses of multiple animals highlighted a high level of similarity between Pteropus alecto and human IFIT5 proteins. Our results showed that paIFIT5 was significantly inducible by viral and dsRNA stimulation. Transient overexpression of paIFIT5 inhibited the replication of vesicular stomatitis virus (VSV). Using minireplicon and transcription reporter assays, we demonstrated the ability of paIFIT5 specifically to inhibit H17N10 polymerase activity. Mechanistically, we noticed that the antiviral potential of paIFIT5 against negative sense RNA viruses was retributed to its interaction with 5'ppp containing RNA. Taken together, these findings highlight the genetic and functional conservation of IFIT5 among mammals.

Transcription factor WOX11 modulates tolerance to cyst nematodes via adventitious lateral root formation.

The transcription factor WUSCHEL-RELATED HOMEOBOX 11 (WOX11) in Arabidopsis (Arabidopsis thaliana) initiates the formation of adventitious lateral roots upon mechanical injury in primary roots. Root-invading nematodes also induce de novo root organogenesis leading to excessive root branching, but it is not known if this symptom of disease involves mediation by WOX11 and if it benefits the plant. Here, we show with targeted transcriptional repression and reporter gene analyses in Arabidopsis that the beet cyst nematode Heterodera schachtii activates WOX11-mediated adventitious lateral rooting from primary roots close to infection sites. The activation of WOX11 in nematode-infected roots occurs downstream of jasmonic acid-dependent damage signaling via ETHYLENE RESPONSE FACTOR109, linking adventitious lateral root formation to nematode damage to host tissues. By measuring different root system components, we found that WOX11-mediated formation of adventitious lateral roots compensates for nematode-induced inhibition of primary root growth. Our observations further demonstrate that WOX11-mediated rooting reduces the impact of nematode infections on aboveground plant development and growth. Altogether, we conclude that the transcriptional regulation by WOX11 modulates root system plasticity under biotic stress, which is one of the key mechanisms underlying the tolerance of Arabidopsis to cyst nematode infections.