KCNH voltage-gated potassium channels regulate neuronal excitability, yet how their hallmark cyclic nucleotide-binding homology domains (CNBHDs) influence channel gating to regulate behavior remains unclear. Using an unbiased forward genetic screen in Caenorhabditis elegans, we identified a gain-of-function mutation in the EAG-subfamily KCNH channel EGL-2 that suppresses sleep. The mutation (G574E) alters a highly conserved glycine residue at the interface between CNBHDs of adjacent channel subunits. EGL-2 functions cell-autonomously in the central sleep-promoting neuron ALA. In vivo calcium imaging showed that the gain-of-function mutation suppresses sleep by inhibiting ALA neuronal activity, consistent with enhanced potassium channel activity. Genetic analysis demonstrated that the sleep-suppressing phenotype of G574E requires both an intact potassium selectivity filter and the intrinsic ligand-occupied CNBHD. A second substitution at the same glycine (G574R), which introduces a positively charged side chain in contrast to the negatively charged side chain in G574E, similarly suppresses sleep, indicating that perturbation of the CNBHD-CNBHD interface, rather than the specific amino acid change, underlies the phenotype. Moreover, mutations at the corresponding residue in the ERG-subfamily KCNH channels C. elegans UNC-103 and human hERG also produce gain-of-function effects, and electrophysiological analysis showed that hERG(G749R) shifts voltage-dependent channel activation to more negative potentials and accelerates activation kinetics. These findings identify EGL-2 as an important sleep regulator and uncover a conserved molecular mechanism by which the CNBHD-CNBHD interface restrains KCNH channel activation to regulate neuronal excitability and behavior.

Hearing loss is the most common sensory deficit impacting ~5% of the world's population. The Deafness Variation Database (DVD) is a public resource of deafness variants, containing 381,924 missense variants across 224 genes, with 303,577 classified as a variant of uncertain significance (VUS). To address the challenge of evaluating each deafness associated VUS, we evaluate a family of probabilistic frameworks to quantify the strength of computational evidence based on ACMG/AMP recommendations. First, CADD and REVEL are compared using Bayesian models parameterized using either a ClinVar 2019 dataset or labeled DVD variants. The REVEL model built using the DVD dataset demonstrates the best accuracy, sensitivity, and specificity. Incorporation of (in)tolerance to missense variation based on sorting each gene into three bins (tolerant, average, intolerant) shows that intolerant DVD genes are consistent with a higher prior probability of being pathogenic (25.7%) than average (10.7%) or tolerant (8.7%) genes. Finally, the impact of protein folding stability was incorporated into the Bayesian model and surpassed the simpler versions while also offering a biophysical rationale for the disease mechanism. The 28,866 VUSs that reach a posterior probability of pathogenicity above 98% based on the protein-folding informed Bayesian model were prioritized as likely to be pathogenic. Overall, 54,752 missense variants (14.3% of 381,924) are predicted to cause modest protein folding destabilization of greater than 1.0 kcal/mol, while 18,706 of these are prioritized VUSs (34% of 54,752). 22,237 missense variants (6.2% of 381,924) are predicted to cause more severe protein folding destabilization of at least 2 kcal/mol with 12,424 being prioritized VUSs (55.8% of 22,237). From these VUSs, we identify twelve probands where the patient's genetic diagnosis is upgraded to likely pathogenic/pathogenic. We highlight two variants that cause clear structural disruption, demonstrating the impact of biophysical characterization on variant evaluation.

This study explored the genetic links between 11 immune-mediated diseases and 13 psychiatric disorders, addressing the limited understanding of the underlying biological mechanisms linking these illnesses. Utilizing genomic structural equation modeling, we investigated whether the genetic liability for 14 immune markers (e.g., C-reactive protein [CRP], interleukin-6 [IL-6]) mediated associations between latent genomic factors of immune-mediated diseases (e.g., autoimmune) and psychiatric disorders (e.g., internalizing). While the literature extensively describes the psychiatric relevance of CRP, results indicated this is better explained by its biological precursor, IL-6. This is evidenced by highly attenuated associations of psychiatric traits with CRP when accounting for overlapping signal of IL6. Overall, the genetic liability of six immune markers significantly mediated 11 psychiatric-immune disease genetic relationships. For example, IL-6 mediated 60% of the genetic link between an internalizing factor and Crohn's disease. Identified markers often evinced mediating effects for multiple disorder clusters and many immune-psychiatric relationships were mediated by multiple markers. At the same time, highlighting their diagnostic relevance, only a subset of examined immune markers were significant mediators. These findings provide mechanistic insight by pinpointing the immune markers that may be most critical to the psychiatric-immune disease link, which notably did not include CRP.

Lysosome-rich enterocytes (LREs) are a specialized population of intestinal cells that mediate the uptake and absorption of dietary proteins in fish and neonatal mammals. Loss of LRE function causes inhibited growth and poor survival due to protein malnutrition. Previously, we reported that in zebrafish loss of Plasmolipin (pllp), an endosomal membrane protein highly expressed in LREs, impairs LRE differentiation and dietary protein absorption, resulting in marked reduction in survival rates. Raising pllp homozygous mutants surviving to adulthood and in-crossing them for multiple generations resulted in a recovery of survival and growth, even when fed a low protein diet. To uncover mechanisms underlying this phenomenon, we compared the older adapted pllp allele with genetically related wild type (WT) fish and a newly generated pllp mutant allele. Using transcriptome profiling and quantitative protein absorption assays, we found that the adapted pllp mutants exhibit upregulation of LRE endocytic components associated with a capacity for protein absorption that exceeds that of WT. This hyperactivation of LRE endocytic and absorptive activity is accompanied by transcriptional regulation of immune genes that may contribute to the enhanced survival of pllp mutants in the face of increased exposure to environmental antigens. Genetic analyses indicate that these divergent phenotypes emerge gradually over generations and can recur in an independently generated pllp allele upon inbreeding and natural selection. Overall, our study illustrates that genome-wide transcriptional changes are associated with stable improvements in intestinal function and organismal survival in response to protein malnutrition.

Bacterial cell division requires precise regulation of septal peptidoglycan (PG) synthesis by the essential SEDS-bPBP synthase FtsWI. Activation of FtsWI is thought to occur through an allosteric cascade initiated by the late-arriving divisome protein FtsN and transmitted via the FtsQ-FtsL-FtsB (FtsQLB) complex, but the molecular details of this process remain incompletely defined and differ across species. Here, using the conditional essentiality of ftsN in Pseudomonas aeruginosa, we identify substitutions in the non-enzymatic pedestal domain of FtsI that bypass the requirement for FtsN. These mutations restore cell division in ΔftsN cells, suppress dominant-negative phenotypes of activation-defective ftsL alleles, and reduce cell length in an otherwise wild-type background, demonstrating that they alter FtsWI regulation to overcome the requirement for stimulation by both FtsN and FtsQLB. Mapping these mutations onto the P. aeruginosa FtsQLBWI structure reveals that they cluster on distinct surfaces of the pedestal domain. Because some of these substitutions have been identified in clinical P. aeruginosa isolates with increased aztreonam resistance, we next asked whether these regulatory substitutions alter β-lactam susceptibility. Variants at the FtsI-FtsL interface modestly reduce susceptibility to multiple β-lactam antibiotics, whereas a variant on the opposite face of the pedestal domain produces striking FtsN-dependent hypersusceptibility to the same molecules. These findings show that the FtsI pedestal domain plays a central role in the regulation of FtsWI and reveal a previously unrecognized relationship between divisome regulation and β-lactam susceptibility. More broadly, our work identifies the pedestal domain as a regulatory hub that integrates multiple inputs controlling septal PG synthesis and highlights its potential relevance to β-lactam resistance in clinical P. aeruginosa isolates.

The mechanisms that restrict or enable latent cellular plasticity have attracted growing interest over the past decade, with important implications for cancer and regenerative therapies. However, the diversity of both pro- and anti-plasticity mechanisms remains incompletely understood. Here, we identify the THAP domain gene lin-15A as a novel factor involved in the natural rectal-to-neuronal Y-to-PDA transdifferentiation in Caenorhabditis elegans. Our genetic analyses show that lin-15A acts in parallel to previously described plasticity factors. Null mutant suggests that, rather than driving the process, LIN-15A licenses its initiation by antagonizing several chromatin-modifying complexes known to safeguard differentiated cell identities. In addition, our data show that even cells developmentally programmed to undergo transdifferentiation exhibit reprogramming barriers. Interestingly, lin-15A is not a core plasticity factor per se but acts as one specifically in the Y cell context. Together, our findings support a model in which diverse molecular activities coordinate controlled cell identity conversions: plasticity factors function as Drivers, while others like lin-15A, termed here transdifferentiation Licensers, attenuate identity safeguarding mechanisms, thereby facilitating transdifferentiation.

N6-methyladenosine (m6A), one of the most abundant chemical modifications on RNA, is installed by METTL3 and several other methyltransferases, including METTL16. Although METTL16 has been characterized in several model organisms, its function in Drosophila remains unknown. Here, we show that, unlike in mammals, Drosophila Mettl16 mutants are viable but exhibit multiple developmental and behavioral defects. Both male and female mutants are sterile, with severe gametogenesis defects. Mettl16 mutant germ cells can pass the mitotic and meiotic stages, but are defective in spermatid elongation and individualization. Mettl16-GFP localizes predominantly to the nucleus, and its absence does not impair global protein synthesis in wing disc epithelial cells. MeRIP-Seq analysis indicates that Mettl16 loss affects m6A on only a small subset of transcripts, in contrast to the broad effect of Mettl3. We further demonstrate that Mettl16 interacts with U6 snRNA and is required for its m6A modification. Accordingly, Mettl16 mutants display widespread alterations in alternative splicing. Together, the pleiotropic phenotypes of Mettl16 mutants likely stem from its role in m6A deposition and U6-dependent splicing regulation of specific target transcripts.

We present a scalable framework for computing polygenic risk scores (PRS) in high-dimensional genomic settings using the recently introduced Univariate-Guided Sparse Regression (uniLasso). UniLasso is a two-stage penalized regression procedure that leverages univariate coefficients and magnitudes to stabilize feature selection and produce sparse predictive models. Building on its theoretical and empirical advantages, we adapt uniLasso for application to the UK Biobank, a population-based repository comprising over one million genetic variants measured on hundreds of thousands of individuals from the United Kingdom. We further extend the framework to incorporate external summary statistics via uniLasso ES (external scores). These signals guide the regression toward variants with prior evidence of association by informing penalty weights and sign constraints. Both uniLasso ES and uniLasso ultimately fit multivariate models using individual-level target data; the external statistics guide, rather than replace, this fitting. Our results demonstrate that uniLasso attains predictive performance comparable to standard Lasso while selecting substantially fewer variants, yielding sparser and potentially more interpretable models. Moreover, it remains competitive with other PRS estimation methods, such as PRS-CS and lassosum2.

Rapid hair follicle cycling requires precise fate commitment and differentiation of hair follicle stem cells. While the essential role of transcription factors in establishing cell identity is well recognized, how cells control its protein synthesis to achieve tissue specificity remains unknown. RNA modifications constitute a pivotal layer of post-transcriptional regulation for protein synthesis. Among them, the N7-methylguanosine (m7G) modification has recently emerged as a critical regulator with diverse functional impacts. Here, we reveal a pivotal role of METTL1, the key enzyme for RNA m7G modification, in hair follicle cycle. Conditional knockout of Mettl1 in keratinocytes leads to severe hair follicle developmental dysplasia, impaired regeneration, and disrupted keratinocyte adhesion. Complementarily, keratinocyte-specific knock-in of Mettl1 accelerates hair regeneration. Mechanistically, beyond its effects on tRNAs, METTL1 deficiency destabilizes HOXC13 mRNA through internal m7G modification, which in turn downregulates the HOXC13/FOXN1/DSG4 signaling axis. Our findings establish METTL1-mediated internal mRNA m7G methylation as one of the essential regulatory layers of RNA modification in hair follicle morphogenesis and cycling, operating through the precise post-transcriptional control of a key transcriptional circuit to ensure structural integrity and timely regeneration.

Classic galactosemia (CG) is a potentially lethal disease that results from profound deficiency of galactose-1-P uridylyltransferase (GALT). Prior reports document elevated galactose metabolites beginning in utero in CG, raising concern that long-term complications in CG might also trace their origins to the prenatal period. However, studies document that elevated galactose metabolites in the postnatal period do not predict later developmental complications, challenging the connection. Here, we explored the relationship between elevated galactose metabolites in utero and fetal outcome using a GALT-null rat model of CG. To modify galactose metabolites in utero, we arranged rat crosses to produce GALT-null pups developing within either a GALT-null or heterozygous dam, and with or without heterozygous siblings. We reasoned that GALT activity present outside the fetus, but within the maternal-fetal compartment, might limit metabolic abnormalities in a developing GALT-null fetus -- and it did. Specifically, while galactose metabolites in GALT-null fetal pups from all crosses remained elevated over controls, the levels varied. When the dam and some siblings were heterozygous, amniotic fluid galactitol from GALT-null fetal pups at GD21 was comparable to the levels reported from second trimester human CG amniotic fluid. When the dam was GALT-null, but some siblings were heterozygous, the median galactitol level in amniotic fluid was 2.45-fold higher. When the dam and all siblings were GALT-null, the median amniotic fluid galactitol level was 5.67-fold higher. GALT-null fetal pups also showed significant growth delay in utero, but only when the dam and all siblings were GALT-null. That growth of GALT-null fetal pups was fully rescued by gestation within a heterozygous dam despite the persistence of moderately elevated galactose metabolites raises the possibility that babies with CG developing within a carrier mother might also be at least partially protected in utero from the adverse consequences of their GALT-deficiency.

In bacteria with circular chromosomes, genes near the replication origin (oriC) are replicated earlier and consequently attain higher copy numbers than genes near the terminus (ter), particularly during rapid growth. Hence, mutations altering a gene's chromosomal position can affect its expression level and be subject to selection. Two non-mutually exclusive hypotheses regarding the target of this selection have been proposed. The mean expression hypothesis (MEH) posits that the target is a gene's average expression across environments, whereas the growth-dependent expression hypothesis (GEH) proposes that the target is the growth dependence of gene expression, quantified by the expression slope-the change in expression level per unit change in growth rate. To test these hypotheses and assess their relative support, we analyze eight multi-environment protein expression datasets from three bacterial species, as well as Escherichia coli promoter strengths measured in two environments. Consistent with both MEH and GEH, we observe a significant decrease in both mean expression and expression slope from oriC to ter in six and four of the eight datasets, respectively. In regression models predicting gene position, the relative contributions of the two hypotheses differ across species. However, even when combined, the two hypotheses explain only a small fraction of chromosomal gene positioning, in part because the replication-dose effect is incompletely offset by compensatory evolution of individual promoter strengths. The positional gradients in mean and growth-dependent expression are disproportionately contributed by genes involved in translation and transcription. We conclude that patterns of bacterial chromosomal gene positioning are consistent with moderate effects of selection on both mean and growth-dependent expression.

Stationary-phase cultures contain high abundances of persisters, which are bacterial cells that are hyper-tolerant to antibiotics due to phenotypic reasons. Fluoroquinolones (FQs) are one of the most effective antibiotic classes for the treatment of non-growing bacteria, such as those found in stationary-phase cultures, and persisters in those populations have been found to survive FQ treatment by repairing the DNA damage caused by the antibiotic rather than by avoiding damage through inactivity. Interestingly, previous work demonstrated that transient growth inhibition after the conclusion of FQ treatment significantly increased persister levels from stationary-phase Escherichia coli populations if recA, a mediator of homologous recombination (HR), or uvrD, a component of nucleotide excision repair (NER), were present. Here, we sought to identify additional epistatic interaction partners in that persister recovery network by deleting DNA repair enzymes known to interact with RecA or UvrD. We found that uvrA, uvrB, and mfd also epistatically interact with recA in FQ persister recovery, whereas recB and recC are additional epistatic interaction partners of uvrD. While these results indicated that HR and NER can contribute to persister recovery, different combinations of genetic mutants suggested that the phenomenon is more nuanced than compensation for loss of one repair pathway by another. Specifically, loss of recA had farther reaching epistatic consequences than loss of other HR genes, and loss of uvrD had a greater impact on the network than any other NER gene. Delving deeper into the roles of RecA and UvrD, we found that the recombination function of RecA, via RecA(N304D), and helicase function of UvrD, via UvrD(R284A), were required for their participation in FQ persister recovery. Collectively, the data presented here deepen understanding of the roles of HR and NER machinery in the recovery FQ persisters and further emphasize the important roles of RecA and UvrD in this phenomenon.

Model organisms may help understand how p53 suppresses tumorigenesis in mammals. In Drosophila, the primary transcriptional target of p53 is the gene encoding the bZip AT-hook protein Xrp1, which is another transcription factor. We report that Xrp1 mediates multiple functions of p53 in the DNA damage response (DDR), contributing to p53-dependent gene transcription and DNA damage-induced apoptosis. In addition to this role as a p53 effector, a p53-independent role for Xrp1 in cell competition has been described. Cell competition can remove cells whose genome has been altered by DNA damage and repair. During cell competition, Xrp1 is induced by RpS12, which acts as a sensor of defective ribosome biogenesis. In irradiated discs, p53-independent RpS12-dependent Xrp1 function began as the DDR came to an end, and was even more prominent if p53 function was reduced. Such p53 inhibition resulted in persistence of DNA damage after irradiation, revealed by γH2Av accumulation. Thus, Xrp1 limited the accumulation of abnormal cells resulting from genotoxicity through both the acute, p53-dependent DDR, and also a later mechanism consistent with cell competition removing cells where DNA repair did not restore the normal genome. Both these processes might contribute to the tumor suppressor function of p53 in mammals.

In Drosophila, Arginine kinase 1 (Argk1) is involved in maintaining ATP homeostasis during bursts of activity in tissues with high and variable rates of energy turnover such as muscle. However, its role beyond stress conditions is less understood. Argk1 is the sole phosphagen kinase with dynamic expression throughout flight muscle development. Here, we show that at least one of the Argk1 isoforms localizes to mitochondria in the developing myofibers, and its function is also necessary for proper flight muscle development. Depleting Argk1 specifically in the muscles lead to low ATP level and NAD + /NADH ratio, indicative of defects in energy homeostasis, and results in animal lethality. In the wing disc-associated myoblasts, Argk1 knockdown causes a reduction in cell size without changes in cell cycle progression. Single cell RNA-sequencing (scRNA-seq) revealed that the transcriptomes of undifferentiated and differentiating Argk1-depleted myoblasts are not significantly affected compared to control. Furthermore, based on the marker expression and overall composition of scRNA-seq cell clusters, the early states of myoblasts differentiation are not severely disrupted in Argk1-depleted myoblasts. Nonetheless, Argk1 knockdown severely impacts later stages of muscle development. Remarkably, Argk1-depleted muscles completely lack spontaneous muscle contractions, which are required for proper sarcomere maturation in the formation of the indirect flight muscle. Accordingly, Argk1-depleted muscles showed defects related to sarcomere maturation, and mitochondrial morphogenesis; thus, leading to a severe reduction in muscle growth. Therefore, our data reveal an essential role for Argk1 in flight muscle development, presumably by sustaining local ATP levels to meet the energetic demand to support myofibrillogenesis, muscle growth and proper flight muscle function.