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bioRxiv2023

DDX3X and DDX3Y constitutively form nano-sized RNA-protein clusters that foster enzymatic activity

DEAD-box helicases, which are crucial for many aspects of RNA metabolism, often contain intrinsically disordered regions (IDRs), whose functions remain unclear. Using multiparameter confocal microscopy, we reveal that sex chromosome-encoded homologous RNA helicases, DDX3X and DDX3Y, form nano-sized RNA-protein clusters (RPCs) that foster their catalytic activities in vitro and in cells. The IDRs are critical for the formation of these RPCs. A thorough analysis of the catalytic cycle of DDX3X and DDX3Y by ensemble biochemistry and single molecule photon bursts in the confocal microscope showed that RNA release is a major step that differentiates the unwinding activities of DDX3X and DDX3Y. Our findings provide new insights that the nano-sized helicase RPCs may be the normal state of these helicases under non-stressed conditions that promote their RNA unwinding and act as nucleation points for liquid-liquid phase separation under stress. This mechanism may apply broadly among other members of the DEAD-box helicase family.

biophysics↗

bioRxiv2023

The receptor VLDLR binds East Equine Encephalitis virus through multiple distinct modes

Eastern Equine Encephalitis virus (EEEV) is an alphavirus that can cause severe diseases in infected humans. The very low-density lipoprotein receptor (VLDLR) was recently identified as a receptor of EEEV. Herein, we performed cryo-electron microscopy structural and biochemistry studies on the specific interactions between EEEV and VLDLR. Our results show that VLDLR binds EEEV at three different sites A, B and C through its membrane-distal 8 LDLR class A (LA) repeats (LA1-8). Site A is located in the cleft in between the E1-E2 heterodimers of the surface trimeric spikes. Site B is located near the connecting {beta} ribbon in between the B and A-C domains of E2 and is in proximity to site A, while site C is on the domain B of E2. LA1 and LA5 specifically recognize site A, whereas LA2, LA3 and LA6 can bind both sites A and C. The binding of VLDLR LAs to EEEV is in complex modes. The LA1-2 mediated binding plays a critical role in mediating virus entry. The mutation W132G of VLDLR impairs the binding of LA3, drives the switch of the binding modes, and significantly enhances the attachment of EEEV to the cell. The W132G variant of VLDLR could be identified in human genome and SNP sequences, implying that people with similar mutations in VLDLR may be highly susceptible to EEEV infection.

microbiology↗

bioRxiv2023

Suppression of the E. coli rnpA49 conditionally lethal phenotype via different compensatory mutations

RNase P is an essential enzyme found across all domains of life that is responsible for the 5-end maturation of precursor tRNA transcripts. Since its discovery in the 1970s, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations. In Escherichia coli, the temperature-sensitive rnpA49 mutation in the protein subunit of RNase P has arguably been one of the most well-studied and commonly used mutations for examining the enzymes activity in vivo. Here we report for the first time naturally-occurring temperature-resistant suppressor mutations of E. coli strains carrying the rnpA49 allele. We find that rnpA49 strains can partially compensate the temperature-sensitive defect via gene amplifications of either RNase P subunit (rnpA49 or rnpB) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insight into the behavior and complementation of the rnpA49 allele in vivo and provides direction for follow-up studies regarding RNase P regulation and turnover.

evolutionary biology↗

bioRxiv2023

A novel, RAS-independent role for NF1 in microtubular dynamics and damage repair dictates sensitivity to T-DM1 in HER2-positive breast cancer

Antibody-Drug Conjugates (ADC) have revolutionized the treatment of several tumors, and extensive research is being devoted to the identification of predictive biomarkers. These are particularly sought after in fields, like breast cancer, in which multiple ADCs with identical target but different payloads have been approved. NF1 is a tumor suppressor widely mutated across several cancers, best characterized as an inhibitor of RAS signaling. Additional functions have been proposed but not deeply investigated, due to its large size and complex domain structure. Whether somatic NF1 mutations can be used to guide clinical decisions is not known. Here, combining patient data, in vitro/in vivo models and protein biochemistry, we show that NF1 loss sensitizes cancer cells to T-DM1, the first approved ADC in breast cancer, through a novel, RAS-independent function on microtubular dynamics and repair. NF1 exhibits all biochemical properties of a bona fide Microtubule-Associated Protein (MAP) and specifically enhances intratubular repair, a recently discovered phenomenon whose regulation remains poorly characterized. NF1 loss results in mitotic defects and low-grade aneuploidy in cell lines and patients. Increased sensitivity to T-DM1 upon NF1 loss is confirmed in breast cancer patients analysed across institutions in Europe and USA. Our results define NF1 as a key regulatory factor for microtubular repair and the first ADC payload-associated predictive biomarker identified to date.

cancer biology↗

bioRxiv2023

A comparative analysis of planarian genomes reveals regulatory conservation in the face of rapid structural divergence

The planarian Schmidtea mediterranea can regenerate its entire body from small tissue fragments and is studied as regeneration model species. The assembly and functional analysis of planarian genomes has proven challenging due its high A/T content (70% A/T), repetitive nature, and limited transferability of routine laboratory protocols due to their divergent biochemistry. Only few and often fragmented genome assemblies are currently available, and open challenges include the provision of well-annotated chromosome-scale reference assemblies of the model species and other planarians for a comparative genome evolution perspective. Here we report a haplotype-phased, chromosome-scale genome assembly and high-quality gene annotations of the sexual S2 strain of S. mediterranea and provide putative regulatory region annotations via optimized ATAC-seq and ChIP-seq protocols. To additionally leverage sequence conservation for regulatory element annotations, we generated chromosome-scale genome assemblies and chromatin accessibility data for the three closest relatives of S. mediterranea: S. polychroa, S. nova, and S. lugubris. We find substantial divergence in protein-coding sequences and regulatory regions, yet reveal remarkable conservation in ChIP-mark bearing open chromatin regions identified as promoters and enhancers in S. mediterranea. The resulting high-confidence set of evolutionary conserved enhancers and promoters provides a valuable resource for the analysis of gene regulatory circuits and their evolution within the taxon. In addition, our four chromosome-scale genome assemblies provide a first comparative perspective on planarian genome evolution. Our analyses reveal frequent retrotransposon-associated chromosomal inversions and inter-chromosomal translocations that lead to a degradation of synteny across the genus. Interestingly, we further find independent and near-complete losses of the ancestral metazoan synteny across Schmidtea and two other flatworm groups, indicating that platyhelminth genomes largely evolve without syntenic constraints. Our work provides valuable genome resources for the planarian research community and sets a foundation for the comparative genomics of planarians. We reveal a contrast between the fast structural evolution of planarian genomes and the conservation of their regulatory elements, suggesting a unique genome evolution in flatworms where gene positioning may not be essential.

genomics↗

bioRxiv2024

PEMPS: A Phylogenetic Software Tool to Model the Evolution of Metabolic Pathways

BackgroundMetabolic pathways support the enzyme flux that converts input chemicals into energy and cellular building blocks. With a constant rate of input, steady-state flux is achieved when metabolite concentrations and reaction rates remain constant over time. Individual genes undergo mutation, while selection acts on higher level functions of the pathway, such as steady-state flux where applicable. Modeling the evolution of metabolic pathways through mechanistic sets of ordinary differential equations is a piece of the genotype-phenotype map model for interpreting genetic variation and inter-specific differences. Such models can generate distinct compensatory changes and adaptive changes from directional selection, indicating single nucleotide polymorphisms and fixed differences that could affect phenotype. If used for inference, this would ultimately enable detection of selection on metabolic pathways as well as inference of ancestral states for metabolic pathway function. ResultsA software tool for simulating the evolution of metabolic pathways based upon underlying biochemistry, phylogenetics, and evolutionary considerations is presented. The Python program, Phylogenetic Evolution of Metabolic Pathway Simulator (PEMPS), implements a mutation-selection framework to simulate the evolution of the pathway over a phylogeny by interfacing with COPASI to calculate the steady-state flux of the metabolic network, introducing mutations as alterations in parameter values according to a model, and calculating a fitness score and corresponding probability of fixation based on the change in steady-state flux value(s). Results from simulations are consistent with a priori expectations of fixation probabilities and systematic change in model parameters. ConclusionsThe PEMPS program simulates the evolution of a metabolic pathway with a mutation-selection modeling framework based on criteria like steady-state flux that is designed to work with SBML-formatted kinetic models, and Newick-formatted phylogenetic trees. The Python software is run on the Linux command line and is available at https://github.com/nmccloskey/PEMPS.

bioinformatics↗

bioRxiv2024

Poincare and SimBio: a versatile and extensible Python ecosystem for modeling systems.

Chemical Reaction Networks (CRNs) play a pivotal role in diverse fields such as systems biology, biochemistry, chemical engineering, and epidemiology. High-level modelling of CRNs enables various simulation approaches, including deterministic and stochastic methods. However, existing Python tools for CRN modelling typically wrap external C/C++ libraries for modelling and simulation, limiting their extensibility and integration with the broader Python ecosystem. In response, we developed Poincare and SimBio, two novel Python packages for the definition and simulation of dynamical systems and CRNs. Poincare serves as a foundation for dynamical system modelling, while SimBio extends this functionality to CRNs, including support for the Systems Biology Markup Language (SBML). Poincare and SimBio are developed as pure Python packages enabling users to easily extend their simulation capabilities by writing new or leveraging other Python packages. Moreover, this does not compromise the performance, as code can be Just-In-Time compiled with Numba. Our benchmark tests using curated models from the BioModels repository, demonstrate that these tools may provide a potentially superior performance advantage, compared to other existing tools. Additionally, to ensure a user-friendly experience, our packages use standard typed modern Python syntax that provides a seamless integration with Integrated Development Environments (IDEs). Python-centric approach significantly enhances code analysis, error detection, and refactoring capabilities, positioning Poincare and SimBio as valuable tools for the modelling community.

bioinformatics↗

bioRxiv2024

Optimisation of surfactin yield in Bacillus using active learning and high-throughput mass spectrometry

Integration of machine learning and high throughput measurements are essential to drive the next generation of the design-build-test-learn (DBTL) cycle in synthetic biology. Here, we report the use of active learning in combination with metabolomics for optimising production of surfactin, a complex lipopeptide resulting from a non-ribosomal assembly pathway. We designed a media optimisation algorithm that iteratively learns the yield landscape and steers the media composition toward maximal production. The algorithm led to a 160% yield increase after three DBTL runs as compared to an M9 baseline. Metabolomics data helped to elucidate the underpinning biochemistry for yield improvement and revealed Pareto-like trade-offs in production of other lipopeptides from related pathways. We found positive associations between organic acids and surfactin, suggesting a key role of central carbon metabolism, as well as system-wide anisotropies in how metabolism reacts to shifts in carbon and nitrogen levels. Our framework offers a novel data-driven approach to improve yield of biological products with complex synthesis pathways that are not amenable to traditional yield optimisation strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/576661v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@14c41ceorg.highwire.dtl.DTLVardef@1745ad2org.highwire.dtl.DTLVardef@1f6bedforg.highwire.dtl.DTLVardef@6d56c5_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Urolithin A improves Alzheimers disease cognition and restores mitophagy and lysosomal functions

BackgroundCompromised autophagy, including impaired mitophagy and lysosomal function, is thought to play a pivotal role in Alzheimers disease (AD). Urolithin A (UA) is a gut microbial metabolite of ellagic acid that stimulates mitophagy. The effects of early and/or long-term treatment, as well as more detailed mechanisms of action, are not known. MethodsWe addressed these questions in three mouse models of AD, and behavioral, electrophysiological and biochemistry assays were performed. ResultsLong-term UA treatment significantly improved learning, memory and olfactory function in different AD transgenic mice. UA also reduced A{beta} and Tau pathologies, and improved long-term potentiation. We found that UA activated autophagy/mitophagy via increasing lysosomal functions. At the cellular level, UA improved lysosomal function and normalized lysosomal cathepsins, especially targeting cathepsin Z, to restore lysosomal function in AD, indicating the important role of cathepsins in UA-induced therapeutic effects of AD. ConclusionsCollectively, our study highlights the importance of lysosomal dysfunction in AD etiology, and points to the high translational potential of UA.

neuroscience↗

bioRxiv2024

Safety, pharmacokinetics, and potential neurological interactions of ivermectin, tafenoquine and chloroquine in Rhesus Macaques

Ivermectin could be used for malaria control as treated persons are lethal to blood feeding Anopheles, resulting in reduced transmission. Tafenoquine could be used in combination with ivermectin to clear persons of liver stage Plasmodium vivax reservoir and as a prophylactic in high-risk populations. The safety of ivermectin and tafenoquine has not been evaluated. As earlier forms of 8-aminoquinolones were neurotoxic, and ivermectin is an inhibitor of the P-glycoprotein blood brain barrier transporter, there is concern that co-administration could be neurotoxic. The safety and pharmacokinetic interaction of tafenoquine, ivermectin, and chloroquine was evaluated in Rhesus macaques. No clinical, biochemistry, or hematological outcomes of concern were observed. The Cambridge Neuropsychological Test Automated Battery was employed to assess potential neurological deficits following drug administration. Some impairment was observed with tafenoquine alone and in the same monkeys with subsequent co-administrations. Co-administration of chloroquine and tafenoquine resulted in increased plasma exposure to tafenoquine. Urine concentrations of the 5,6 orthoquinone TQ metabolite were increased with co-administration of tafenoquine with ivermectin. There was an increase in ivermectin plasma exposure when co-administered with chloroquine. No interaction of tafenoquine on ivermectin was observed in vitro. Chloroquine and trace levels of ivermectin, but not tafenoquine, were observed in the cerebrospinal fluid. The 3"-O-demethyl ivermectin metabolite was observed in macaque plasma but not in urine or cerebrospinal fluid. Overall, the combination of ivermectin, tafenoquine, and chloroquine did not have clinical, neurological, or pharmacological interactions of concern in macaques, therefore this combination could be considered for evaluation in human trials.

pharmacology and toxicology↗

bioRxiv2024

Reducing stomatal density by expression of a synthetic EPF increases leaf intrinsic water use efficiency and reduces plant water use in a C4 crop

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Reducing stomatal conductance (gs) through manipulating stomatal density has been observed to translate to improved WUE in multiple C3 crop species. However, reducing gs in C3 species often reduces photosynthetic carbon gain. A different response is expected in C4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the photosynthesis (AN) relationship with intracellular CO2 concentration (ci) so that photosynthesis is CO2-saturated and reductions in gs are unlikely to impair AN. To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum, lead to reduced stomatal densities. A moderate reduction in stomatal density did not strengthen stomatal limitation to AN, improved WUE, reduced water use, and avoided loss of carbon fixation during a period of water deprivation. However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues provides a potential pathway to optimal agronomic outcomes.

plant biology↗

bioRxiv2024

Infer metabolic directions and magnitudes from moment differences of mass-weighted intensity distributions

Metabolic pathways are fundamental maps in biochemistry that detail how molecules are transformed through various reactions. The complexity of metabolic network, where a single compound can play a part in multiple pathways, poses a challenge in inferring metabolic balance changes over time or after different treatments. Isotopic labeling experiment is the standard method to infer metabolic flux, which is currently defined as the flow of a single metabolite through a given pathway over time. However, there is still no way to accurately infer the metabolic balance changes after different treatments in an experiment. This study introduces a different concept: molecular weight distribution, which is the empirical distribution of the molecular weights of all metabolites of interest. By estimating the differences of the location and scale estimates of these distributions, it becomes possible to quantitatively infer the metabolic balance changes even without requiring knowledge of the exact chemical structures of these compounds and their related pathways. This research article provides a mathematical framing for a classic biological concept.

bioinformatics↗

bioRxiv2024

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

Polysaccharides are a major class of natural polymers found abundantly across all major life forms and play a critical role as structural, metabolic, or functional components in biomolecular processes. Some polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs). Despite the fact that the GT-2 family has the maximum number of deposited sequences, the biochemistry of GT-2 family enzymes is still poorly understood due to difficulties associated with GT membrane protein expression, purification, and reconstitution in lipid carriers. Here, we chose Populus tremula x tremuloides cellulose synthase 8 (PttCesA8) and Streptococcus equisimilis hyaluronan synthase (SeHas) as putative family-2-GTs to be expressed in a wheat-germ-based cell-free expression (CFE) system as proteoliposomes. The cell-free products were obtained as reconstituted liposomes directly from CFE reactions at high yields and short processing times compared to other approaches. GT enzymes expression was confirmed using SDS-PAGE and immunoblotting, and the integration of GTs in lipid layers was observed using cryogenic electron microscopy. Both GTs tested were catalytically active when incubated with their respective substrates and cofactors. The Michalis-Menten kinetic constants, Km for PttCesA8, was 295.8 {micro}M, and SeHas was 321.51 {micro}M (toward UDP N-Acetyl Glucosamine) and 207.88 {micro}M (toward UDP Glucuronic Acid), respectively. UDP was found to actively inhibit both these GTs with apparent inhibition constants of 10.08 {micro}M and 24.38 {micro}M. Mutation of specific conserved residues in structure-deficit SeHas confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, wheat-germ-based CFE can be used to express functionally active and liposome-reconstituted family-2 GTs at high yields with relative ease to enable classical enzymology assays and will also enable more detailed structural studies in the near future.

bioengineering↗

bioRxiv2024

Charting the Cannabis plant chemical space with computational metabolomics

IntroductionThe chemical classification of Cannabis is typically confined to the cannabinoid content, whilst Cannabis encompasses diverse chemical classes that vary in abundance among all its varieties. Hence, neglecting other chemical classes within Cannabis strains results in a restricted and biased comprehension of elements that may contribute to chemical intricacy and the resultant medicinal qualities of the plant. ObjectivesThus, herein, we report a computational metabolomics study to elucidate the Cannabis metabolic map beyond the cannabinoids. MethodsMass spectrometry-based computational tools were used to mine and evaluate the methanolic leaf and flower extracts of two Cannabis cultivars: Amnesia haze (AMNH) and Royal dutch cheese (RDC). ResultsThe results revealed the presence of different chemical compound classes including cannabinoids, but extending it to flavonoids, polyketides, and phospholipids at varying distributions across the cultivar plant tissues. Therefore, the two cultivars were differentiated based on the overall chemical content of their plant tissues where AMNH was observed to be more dominant in the flavonoid content while RDC was more dominant in the lipid-like molecules. Additionally, in silico molecular docking studies in combination with biological assay studies indicated the potentially differing anti-cancer properties of the two cultivars resulting from the elucidated chemical profiles. ConclusionThese findings highlight distinctive chemical profiles beyond cannabinoids in Cannabis strains. This novel mapping of the metabolomic landscape of Cannabis provides actionable insights into plant biochemistry and justifies selecting certain varieties for medicinal use.

systems biology↗

bioRxiv2024

In-Vivo and In-Vitro Toxicity Evaluation of 2,3-Dimethylquinoxaline: an Antimicrobial Found in a Traditional Herbal Medicine

2,3-dimethylquinoxaline (DMQ) is a broad-spectrum antimicrobial phytochemical. This study aims to assess its toxicological profile. In vitro studies conducted in appropriate cell cultures, included assessment of cardiotoxicity, nephrotoxicity, and hepatotoxicity. An in vivo study was conducted in mice to determine acute oral toxicity (AOT), and subacute oral toxicity (SAOT). Acute dermal toxicity (ADT) was conducted in rats. All in-vitro toxicity studies of DMQ had negative results at concentrations [≤]100 {micro}M except for a non-significant reduction in the ATP in human hepatocellular carcinoma cell culture. The median lethal dose of DMQ was higher than 2000 mg/kg. All animals survived the scheduled necropsy and none showed any alteration in clinical signs. Biochemistry analysis revealed a significant difference between the satellite and control groups, showing an increase in platelet counts and white blood cell counts by 99.8% and 188.8%, respectively. Histology revealed enlargement of renal corpuscles; hyperplasia of testosterone-secreting cells; and dilatation of coronaries and capillaries. The present data suggests an acceptable safety profile of DMQ in rodents except for thrombocytosis, leukocytosis, and histological changes in high doses that need further investigation.

pathology↗

bioRxiv2024

Spatial control of sensory adaptation modulates mechanosensing in Pseudomonas aeruginosa

Sensory signaling pathways use adaptation to dynamically respond to changes in their environment. Here, we report the mechanism of sensory adaptation in the Pil-Chp mechanosensory system, which the important human pathogen Pseudomonas aeruginosa uses to sense mechanical stimuli during surface exploration. Using biochemistry, genetics, and cell biology, we discovered that the enzymes responsible for adaptation, a methyltransferase and a methylesterase, are segregated to opposing cell poles as P. aeruginosa explore surfaces. By coordinating the localization of both enzymes, we found that the Pil-Chp response regulators influence local receptor methylation, the molecular basis of bacterial sensory adaptation. We propose a model in which adaptation during mechanosensing spatially resets local receptor methylation, and thus Pil-Chp signaling, to modulate the pathway outputs, which are involved in P. aeruginosa virulence. Despite decades of bacterial sensory adaptation studies, our work has uncovered an unrecognized mechanism that bacteria use to achieve adaptation to sensory stimuli.

microbiology↗

bioRxiv2024

Quantitative profiling of human translation initiation reveals regulatory elements that potently affect endogenous and therapeutically modified mRNAs

mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions and identify sequences that mediate 250-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1{Psi}) selectively enhances translation by specific 5' UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissect mechanisms of human translation initiation and engineer more potent therapeutic mRNAs. HighlightsO_LIMeasurement of >30,000 human 5' UTRs reveals a 250-fold range of translation output C_LIO_LISystematic mutagenesis demonstrates the causality of short (3-6nt) regulatory elements C_LIO_LIN1-methylpseudouridine alters translation initiation in a sequence-specific manner C_LIO_LIOptimal modified 5' UTRs outperform those in the current class of mRNA vaccines C_LI

systems biology↗

bioRxiv2024

Sexually dimorphic renal expression of Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA- seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice presented normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

genetics↗