Low affinity binding site clusters confer hox specificity and regulatory robustness.

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression.

Dense and pleiotropic regulatory information in a developmental enhancer.

Changes in gene regulation underlie much of phenotypic evolution1. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations2. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

The Density of Regulatory Information Is a Major Determinant of Evolutionary Constraint on Noncoding DNA in Drosophila.

Evolutionary analyses have estimated that ∼60% of nucleotides in intergenic regions of the Drosophila melanogaster genome are functionally relevant, suggesting that regulatory information may be encoded more densely in intergenic regions than has been revealed by most functional dissections of regulatory DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene shavenbaby (svb). Most of the ∼90 kb of this large regulatory region is highly conserved in the genus Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of svb in different contexts of Drosophila development, we found that the regulatory information that drives svb expression in the abdominal pupal epidermis is organized in a different way than the elements that drive svb expression in the embryonic epidermis. While in the embryonic epidermis svb is activated by compact enhancers separated by large inactive DNA regions, svb expression in the pupal epidermis is driven by regulatory information distributed over broader regions of svb cis-regulatory DNA. In the same vein, we observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved noncoding DNA of the Drosophila genome is contained within regions of open chromatin. These results suggest that part of the evolutionary constraint on noncoding DNA of Drosophila is explained by the density of regulatory information, which may be greater than previously appreciated.

Identification of an evolutionarily conserved domain in Neurod1 favouring enteroendocrine versus goblet cell fate.

ARP/ASCL transcription factors are key determinants of cell fate specification in a wide variety of tissues, coordinating the acquisition of generic cell fates and of specific subtype identities. How these factors, recognizing highly similar DNA motifs, display specific activities, is not yet fully understood. To address this issue, we overexpressed different ARP/ASCL factors in zebrafish ascl1a-/- mutant embryos to determine which ones are able to rescue the intestinal secretory lineage. We found that Ascl1a/b, Atoh1a/b and Neurod1 factors are all able to trigger the first step of the secretory regulatory cascade but distinct secretory cells are induced by these factors. Indeed, Neurod1 rescues the enteroendocrine lineage while Ascl1a/b and Atoh1a/b rescue the goblet cells. Gain-of-function experiments with Ascl1a/Neurod1 chimeric proteins revealed that the functional divergence is encoded by a 19-aa ultra-conserved element (UCE), present in all Neurod members but absent in the other ARP/ASCL proteins. Importantly, inserting the UCE into the Ascl1a protein reverses the rescuing capacity of this Ascl1a chimeric protein that cannot rescue the goblet cells anymore but can efficiently rescue the enteroendocrine cells. This novel domain acts indeed as a goblet cell fate repressor that inhibits gfi1aa expression, known to be important for goblet cell differentiation. Deleting the UCE domain of the endogenous Neurod1 protein leads to an increase in the number of goblet cells concomitant with a reduction of the enteroendocrine cells, phenotype also observed in the neurod1 null mutant. This highlights the crucial function of the UCE domain for NeuroD1 activity in the intestine. As Gfi1 acts as a binary cell fate switch in several tissues where Neurod1 is also expressed, we can envision a similar role of the UCE in other tissues, allowing Neurod1 to repress Gfi1 to influence the balance between cell fates.

A Phylogenetic Framework to Simulate Synthetic Interspecies RNA-Seq Data.

Interspecies RNA-Seq datasets are increasingly common, and have the potential to answer new questions about the evolution of gene expression. Single-species differential expression analysis is now a well-studied problem that benefits from sound statistical methods. Extensive reviews on biological or synthetic datasets have provided the community with a clear picture on the relative performances of the available methods in various settings. However, synthetic dataset simulation tools are still missing in the interspecies gene expression context. In this work, we develop and implement a new simulation framework. This tool builds on both the RNA-Seq and the phylogenetic comparative methods literatures to generate realistic count datasets, while taking into account the phylogenetic relationships between the samples. We illustrate the usefulness of this new framework through a targeted simulation study, that reproduces the features of a recently published dataset, containing gene expression data in adult eye tissue across blind and sighted freshwater crayfish species. Using our simulated datasets, we perform a fair comparison of several approaches used for differential expression analysis. This benchmark reveals some of the strengths and weaknesses of both the classical and phylogenetic approaches for interspecies differential expression analysis, and allows for a reanalysis of the crayfish dataset. The tool has been integrated in the R package compcodeR, freely available on Bioconductor.

Transgenic expression of Rubisco accumulation factor2 and Rubisco subunits increases photosynthesis and growth in maize.

Carbon assimilation by Rubisco is often a limitation to photosynthesis and therefore plant productivity. We have previously shown that transgenic co-expression of the Rubisco large (LS) and small (SS) subunits along with an essential Rubisco accumulation factor, Raf1, leads to faster growth, increased photosynthesis, and enhanced chilling tolerance in maize (Zea mays). Maize also requires Rubisco accumulation factor2 (Raf2) for full accumulation of Rubisco. Here we have analyzed transgenic maize lines with increased expression of Raf2 or Raf2 plus LS and SS. We show that increasing Raf2 expression alone had minor effects on photosynthesis, whereas expressing Raf2 with Rubisco subunits led to increased Rubisco content, more rapid carbon assimilation, and greater plant height, most notably in plants at least 6 weeks of age. The magnitude of the effects was similar to what was observed previously for expression of Raf1 together with Rubisco subunits. Taken together, this suggests that increasing the amount of either assembly factor with Rubisco subunits can independently enhance Rubisco abundance and some aspects of plant performance. These results could also imply either synergy or a degree of functional redundancy for Raf1 and Raf2, the latter of whose precise role in Rubisco assembly is currently unknown.

Evolution of the tan locus contributed to pigment loss in Drosophila santomea: a response to Matute et al.

We have shown previously that the loss of abdominal pigmentation in D. santomea relative to its sister species D. yakuba resulted, in part, from cis-regulatory mutations at the tan locus. Matute et al. claim, based solely upon extrapolation from genetic crosses of D. santomea and D. melanogaster, a much more divergent species, that at least four X chromosome regions but not tan are responsible for pigmentation differences. Here, we provide additional evidence from introgressions of D. yakuba genes into D. santomea that support a causative role for tan in the loss of pigmentation and present analyses that contradict Matute et al.'s claims. We discuss how the choice of parental species and other factors affect the ability to identify loci responsible for species divergence, and we affirm that all of our previously reported results and conclusions stand.

Evolution of a central neural circuit underlies Drosophila mate preferences.

Courtship rituals serve to reinforce reproductive barriers between closely related species. Drosophila melanogaster and Drosophila simulans exhibit reproductive isolation, owing in part to the fact that D. melanogaster females produce 7,11-heptacosadiene, a pheromone that promotes courtship in D. melanogaster males but suppresses courtship in D. simulans males. Here we compare pheromone-processing pathways in D. melanogaster and D. simulans males to define how these sister species endow 7,11-heptacosadiene with the opposite behavioural valence to underlie species discrimination. We show that males of both species detect 7,11-heptacosadiene using homologous peripheral sensory neurons, but this signal is differentially propagated to P1 neurons, which control courtship behaviour. A change in the balance of excitation and inhibition onto courtship-promoting neurons transforms an excitatory pheromonal cue in D. melanogaster into an inhibitory cue in D. simulans. Our results reveal how species-specific pheromone responses can emerge from conservation of peripheral detection mechanisms and diversification of central circuitry, and demonstrate how flexible nodes in neural circuits can contribute to behavioural evolution.

Optimising engagement in a digital parenting intervention to prevent violence against adolescents in Tanzania: protocol for a cluster randomised factorial trial.

BACKGROUND: Violence against adolescents is a universal reality, with severe individual and societal costs. There is a critical need for scalable and effective violence prevention strategies such as parenting programmes, particularly in low- and middle-income countries where rates of maltreatment are highest. Digital interventions may be a scalable and cost-effective alternative to in-person delivery, yet maximising caregiver engagement is a substantial challenge. This trial employs a cluster randomised factorial experiment and a novel mixed-methods analytic approach to assess the effectiveness, cost-effectiveness, and feasibility of intervention components designed to optimise engagement in an open-source parenting app, ParentApp for Teens. The app is based on the evidence-based Parenting for Lifelong Health for Teens programme, developed collaboratively by academic institutions in the Global South and North, the WHO, and UNICEF. METHODS/DESIGN: Sixteen neighbourhoods, i.e., clusters, will be randomised to one of eight experimental conditions which consist of any combination of three components (Support: self-guided/moderated WhatsApp groups; App Design: sequential workshops/non-sequential modules; Digital Literacy Training: on/off). The study will be conducted in low-income communities in Tanzania, targeting socioeconomically vulnerable caregivers of adolescents aged 10 to 17 years (16 clusters, 8 conditions, 640 caregivers, 80 per condition). The primary objective of this trial is to estimate the main effects of the three components on engagement. Secondary objectives are to explore the interactions between components, the effects of the components on caregiver behavioural outcomes, moderators and mediators of programme engagement and impact, and the cost-effectiveness of components. The study will also assess enablers and barriers to engagement qualitatively via interviews with a subset of low, medium, and high engaging participants. We will combine quantitative and qualitative data to develop an optimised ParentApp for Teens delivery package. DISCUSSION: This is the first known cluster randomised factorial trial for the optimisation of engagement in a digital parenting intervention in a low- and middle-income country. Findings will be used to inform the evaluation of the optimised app in a subsequent randomised controlled trial. TRIAL REGISTRATION: Pan African Clinical Trial Registry, PACTR202210657553944. Registered 11 October 2022, https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=24051 .

CFM1, a member of the CRM-domain protein family, functions in chloroplast group II intron splicing in Setaria viridis.

The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.

Identifying modules of cooperating cancer drivers.

Identifying cooperating modules of driver alterations can provide insights into cancer etiology and advance the development of effective personalized treatments. We present Cancer Rule Set Optimization (CRSO) for inferring the combinations of alterations that cooperate to drive tumor formation in individual patients. Application to 19 TCGA cancer types revealed a mean of 11 core driver combinations per cancer, comprising 2-6 alterations per combination and accounting for a mean of 70% of samples per cancer type. CRSO is distinct from methods based on statistical co-occurrence, which we demonstrate is a suboptimal criterion for investigating driver cooperation. CRSO identified well-studied driver combinations that were not detected by other approaches and nominated novel combinations that correlate with clinical outcomes in multiple cancer types. Novel synergies were identified in NRAS-mutant melanomas that may be therapeutically relevant. Core driver combinations involving NFE2L2 mutations were identified in four cancer types, supporting the therapeutic potential of NRF2 pathway inhibition. CRSO is available at https://github.com/mikekleinsgit/CRSO/.

Natural courtship song variation caused by an intronic retroelement in an ion channel gene.

Animal species display enormous variation for innate behaviours, but little is known about how this diversity arose. Here, using an unbiased genetic approach, we map a courtship song difference between wild isolates of Drosophila simulans and Drosophila mauritiana to a 966 base pair region within the slowpoke (slo) locus, which encodes a calcium-activated potassium channel. Using the reciprocal hemizygosity test, we confirm that slo is the causal locus and resolve the causal mutation to the evolutionarily recent insertion of a retroelement in a slo intron within D. simulans. Targeted deletion of this retroelement reverts the song phenotype and alters slo splicing. Like many ion channel genes, slo is expressed widely in the nervous system and influences a variety of behaviours; slo-null males sing little song with severely disrupted features. By contrast, the natural variant of slo alters a specific component of courtship song, illustrating that regulatory evolution of a highly pleiotropic ion channel gene can cause modular changes in behaviour.

Mapping hospital data to characterize residents' educational experiences.

BACKGROUND: Experiential learning through patient care is fundamental to graduate medical education. Despite this, the actual content to which trainees are exposed in clinical practice is difficult to quantify and is poorly characterized. There remains an unmet need to define precisely how residents' patient care activities inform their educational experience.  METHODS: Using a recently-described crosswalk tool, we mapped principal ICD-10 discharge diagnosis codes to American Board of Internal Medicine (ABIM) content at four training hospitals of a single Internal Medicine (IM) Residency Program over one academic year to characterize and compare residents' clinical educational experiences. Frequencies of broad content categories and more specific condition categories were compared across sites to profile residents' aggregate inpatient clinical experiences and drive curricular change. RESULTS: There were 18,604 discharges from inpatient resident teams during the study period. The crosswalk captured > 95% of discharges at each site. Infectious Disease (ranging 17.4 to 39.5% of total discharges) and Cardiovascular Disease (15.8 to 38.2%) represented the most common content categories at each site. Several content areas (Allergy/Immunology, Dermatology, Obstetrics/Gynecology, Ophthalmology, Otolaryngology/Dental Medicine) were notably underrepresented (≤ 1% at each site). There were significant differences in the frequencies of conditions within most content categories, suggesting that residents experience distinct site-specific clinical content during their inpatient training. CONCLUSIONS: There were substantial differences in the clinical content experienced by our residents across hospital sites, prompting several important programmatic and curricular changes to enrich our residents' hospital-based educational experiences.

Setaria viridis chlorotic and seedling-lethal mutants define critical functions for chloroplast gene expression.

Deep insights into chloroplast biogenesis have been obtained by mutant analysis; however, in C4 plants a relevant mutant collection has only been developed and exploited for maize. Here, we report the initial characterization of an ethyl methyl sulfonate-induced mutant population for the C4 model Setaria viridis. Approximately 1000 M2 families were screened for the segregation of pale-green seedlings in the M3 generation, and a subset of these was identified to be deficient in post-transcriptional steps of chloroplast gene expression. Causative mutations were identified for three lines using deep sequencing-based bulked segregant analysis, and in one case confirmed by transgenic complementation. Using chloroplast RNA-sequencing and other molecular assays, we describe phenotypes of mutants deficient in PSRP7, a plastid-specific ribosomal protein, OTP86, an RNA editing factor, and cpPNP, the chloroplast isozyme of polynucleotide phosphorylase. The psrp mutant is globally defective in chloroplast translation, and has varying deficiencies in the accumulation of chloroplast-encoded proteins. The otp86 mutant, like its Arabidopsis counterpart, is specifically defective in editing of the rps14 mRNA; however, the conditional pale-green mutant phenotype contrasts with the normal growth of the Arabidopsis mutant. The pnp mutant exhibited multiple defects in 3' end maturation as well as other qualitative changes in the chloroplast RNA population. Overall, our collection opens the door to global analysis of photosynthesis and early seedling development in an emerging C4 model.

Rubisco production in maize mesophyll cells through ectopic expression of subunits and chaperones.

C4 plants, such as maize, strictly compartmentalize Rubisco to bundle sheath chloroplasts. The molecular basis for the restriction of Rubisco from the more abundant mesophyll chloroplasts is not fully understood. Mesophyll chloroplasts transcribe the Rubisco large subunit gene and, when normally quiescent transcription of the nuclear Rubisco small subunit gene family is overcome by ectopic expression, mesophyll chloroplasts still do not accumulate measurable Rubisco. Here we show that a combination of five ubiquitin promoter-driven nuclear transgenes expressed in maize leads to mesophyll accumulation of assembled Rubisco. These encode the Rubisco large and small subunits, Rubisco assembly factors 1 and 2, and the assembly factor Bundle sheath defective 2. In these plants, Rubisco large subunit accumulates in mesophyll cells, and appears to be assembled into a holoenzyme capable of binding the substrate analog CABP (carboxyarabinitol bisphosphate). Isotope discrimination assays suggest, however, that mesophyll Rubisco is not participating in carbon assimilation in these plants, most probably due to a lack of the substrate ribulose 1,5-bisphosphate and/or Rubisco activase. Overall, this work defines a minimal set of Rubisco assembly factors in planta and may help lead to methods of regulating the C4 pathway.

Distinct genetic architectures underlie divergent thorax, leg, and wing pigmentation between Drosophila elegans and D. gunungcola.

Pigmentation divergence between Drosophila species has emerged as a model trait for studying the genetic basis of phenotypic evolution, with genetic changes contributing to pigmentation differences often mapping to genes in the pigment synthesis pathway and their regulators. These studies of Drosophila pigmentation have tended to focus on pigmentation changes in one body part for a particular pair of species, but changes in pigmentation are often observed in multiple body parts between the same pair of species. The similarities and differences of genetic changes responsible for divergent pigmentation in different body parts of the same species thus remain largely unknown. Here we compare the genetic basis of pigmentation divergence between Drosophila elegans and D. gunungcola in the wing, legs, and thorax. Prior work has shown that regions of the genome containing the pigmentation genes yellow and ebony influence the size of divergent male-specific wing spots between these two species. We find that these same two regions of the genome underlie differences in leg and thorax pigmentation; however, divergent alleles in these regions show differences in allelic dominance and epistasis among the three body parts. These complex patterns of inheritance can be explained by a model of evolution involving tissue-specific changes in the expression of Yellow and Ebony between D. elegans and D. gunungcola.

Valsartan nano-filaments alter mitochondrial energetics and promote faster healing in diabetic rat wounds.

Chronic wounds are a common and debilitating condition associated with aging populations that impact more than 6.5 million patients in the United States. We have previously demonstrated the efficacy of daily topical 1% valsartan in treating wounds in diabetic mouse and pig models. Despite these promising results, there remains a need to develop an extended-release formulation that would reduce patient burden by decreasing the frequency of daily applications. Here, we used nanotechnology to self-assemble valsartan amphiphiles into a filamentous structure (val-filaments) that would serve as a scaffold in wound beds and allow for steady, localised and tunable release of valsartan amphiphiles over 24 days. Two topical treatments of this peptide-based hydrogel on full-thickness wounds in Zucker Diabetic Fatty rats resulted in faster rates of wound closure. By day 23, all val-filament treated wounds were completely closed, as compared to one wound closed in the placebo group. Mechanistically, we observed enrichment of proteins involved in cell adhesion and energetics pathways, downregulation of Tgf-ß signalling pathway mediators (pSmad2, pSmad3 and Smad4) and increased mitochondrial metabolic pathway intermediates. This study demonstrates the successful synthesis of a sustained-release valsartan filament hydrogel, its impact on mitochondrial energetics and efficacy in treating diabetic wounds.

Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures.

Chloroplast transcription requires numerous quality control steps to generate the complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we systematically mapped transcript ends by developing a protocol called Terminome-seq. Using Arabidopsis thaliana as a model, we catalogued >215 primary 5' ends corresponding to transcription start sites (TSS), as well as 1628 processed 5' ends and 1299 3' ends. While most termini were found in intergenic regions, numerous abundant termini were also found within coding regions and introns, including several major TSS at unexpected locations. A consistent feature was the clustering of both 5' and 3' ends, contrasting with the prevailing description of discrete 5' termini, suggesting an imprecision of the transcription and/or RNA processing machinery. Numerous termini correlated with the extremities of small RNA footprints or predicted stem-loop structures, in agreement with the model of passive RNA protection. Terminome-seq was also implemented for pnp1-1, a mutant lacking the processing enzyme polynucleotide phosphorylase. Nearly 2000 termini were altered in pnp1-1, revealing a dominant role in shaping the transcriptome. In summary, Terminome-seq permits precise delineation of the roles and regulation of the many factors involved in organellar transcriptome quality control.

Accurate and sensitive quantification of protein-DNA binding affinity.

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Selective Capture and Recovery of Monoclonal Antibodies by Self-Assembling Supramolecular Polymers of High Affinity for Protein Binding.

The separation and purification of therapeutic proteins from their biological resources pose a great limitation for industrial manufacturing of biologics in an efficient and cost-effective manner. We report here a supramolecular polymeric system that can undergo multiple reversible processes for efficient capture, precipitation, and recovery of monoclonal antibodies (mAbs). These supramolecular polymers, namely immunofibers (IFs), are formed by coassembly of a mAb-binding peptide amphiphile with a rationally designed filler molecule of varying stoichiometric ratios. Under the optimized conditions, IFs can specifically capture mAbs with a precipitation yield greater than 99%, leading to an overall mAb recovery yield of 94%. We also demonstrated the feasibility of capturing and recovering two mAbs from clarified cell culture harvest. These results showcase the promising potential of peptide-based supramolecular polymers as reversible affinity precipitants for mAb purification.