Biology subjects

Abraham, E.

Publications and source records attributed to Abraham, E..

16 recordsLinked to original sources

bioRxiv2026

Functionally dark genes and the transcriptomic landscape of sporulation in a model mushroom-forming fungus

Spores are the primary means of fungal reproduction, contributing to genetic diversity, colonization, and adaptation. Although spore formation is a pivotal part of the fungal life cycle, its genetic underpinnings remain poorly known. In this study, we characterize transcriptomic changes from late meiosis to early basidiospore formation in the mushroom-forming fungus Coprinopsis cinerea, decipher several cellular processes, and identify novel genes involved in this process. We identify distinct trajectories of gene expression, each of which display different functional signals, corresponding to meiotic and morphogenetic processes and transitions between these. Our analyses identify diverse arrays of fungal cell wall modifying carbohydrate-active enzymes, ferritins, a putative catechol-melanin synthesis pathway, as well as components of the mitotic/meiotic apparatus. We present twelve highly conserved genes with roles specific to sexual sporulation in both budding yeast and C. cinerea, indicating deep conservation of the gene networks driving sexual spore formation. Reverse genetics identified three conserved but functionally poorly characterized genes conferring sporeless and spore-poor phenotypes that result from postmeiotic developmental arrests stemming from spore inflation and nuclear migration problems. Overall, this study provides novel insight into basidiomycete spore formation and highlights the cornucopia of novel functions encoded by functionally dark genes.

microbiology↗

bioRxiv2026

Coordinated regulation of Barrier-to-Autointegration Factor (BAF) phosphorylation by protein phosphatases PP2A and PP4

Phosphorylation is crucial for the regulation of Barrier-to-Autointegration Factor (BAF), a conserved nuclear envelope (NE)-associated protein that, among its diverse functions, binds chromatin and mediates its anchoring to the NE. In interphase, cycles of BAF phosphorylation, which inhibits chromatin binding, and dephosphorylation, which restores it, are essential for the dynamic anchoring of chromatin to the NE. During mitosis, BAF becomes highly phosphorylated and dissociates from chromatin, except for a small centromeric pool (cenBAF). Upon mitotic exit, BAF is dephosphorylated and re-associates with chromosomes. Two Ser/Thr protein phosphatases, PP2A and PP4, are known to dephosphorylate BAF, but their specific roles in regulating BAF phosphorylation are not yet fully understood. Here, we show that PP2A is the main phosphatase driving BAF dephosphorylation in interphase, whereas PP4 plays a minor role. During mitosis, both PP2A and PP4 localize to centromeres; however, only PP4 directly interacts with cenBAF, suggesting that it maintains this fraction in an unphosphorylated state. Notably, PP4s centromeric localization depends on PP2A. At mitotic exit, PP2A dephosphorylates the large pool of mitotically phosphorylated BAF, while PP4 controls the timing of its re-association with chromatin. Together, these findings reveal a coordinated interplay between PP2A and PP4 in controlling BAF dephosphorylation and function.

Molecular Biology↗

bioRxiv2026

Rapid in vitro synthesis of DNA templates via Sidewinder for polyadenylated Hantavirus mRNA vaccine candidates

As the recent COVID-19 pandemic illustrated, zoonotic viruses and other pathogens pose a credible threat to public health. Recent advancements in vaccine technology, particularly mRNA vaccines, provide key tools for an effective and swift public health response. Although mRNA vaccines can be developed more quickly than traditional vaccines, fast and accurate construction of DNA templates for these vaccines remains a critical bottleneck. Using our novel DNA assembly technology, Sidewinder, we rapidly designed and built multiple mRNA vaccine candidates to guard against a potential outbreak of Hantavirus (ANDV). We successfully constructed the DNA templates from oligo pools and produced the mRNA for three vaccine candidates in just 2 days after delivery of the synthetic DNA oligos.

biochemistry↗

bioRxiv2026

A multifunctional polyketide synthase in nematodes produces divergent families of signaling molecules that control different developmental arrests

To improve their chances of reproductive success, nematodes not only must arrest their development in response to adverse growth conditions but also must quickly recover if conditions improve. A polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid assembly line that is expressed in the canal-associated neurons (CANs) of Caenorhabditis elegans promotes recovery from starvation-induced larval arrest. Here, we show that in the predatory nematode Pristionchus pacificus this assembly line produces a suite of secondary metabolites, including a family of hybrid polyketide-nonribosomal peptides known as the nemamides, the related nemaketides, and a family of ascarylose-modified polyketides named ascarenes. Depending on the starter unit that is loaded onto the PKS, the assembly line can produce dramatically different downstream products. Whereas the nemamides promote recovery from starvation-induced larval arrest, the ascarenes inhibit development of the dauer larval stage and promote recovery. This dichotomy suggests that the PKS-NRPS megasynthetase serves as a signaling hub in the CANs, controlling multiple developmental events. The PKS-NRPS assembly line is highly conserved across many nematode species, and identification of these chemical signals will help to elucidate the signaling pathways that control development in the worm and lead to novel anthelmintics.

biochemistry↗

bioRxiv2026

One-pot parallel Sidewinder construction from oligo pools

Reliable and cost-effective de novo DNA production at scale has become increasingly important to studying and engineering biology. Double-stranded DNA is constructed from short synthetic single-stranded DNA oligonucleotides synthesized either individually or as a pool. Oligonucleotide pools offer substantially reduced costs at the sacrifice of yield and individual oligo isolation, making efficient multiplexed DNA construction from pools a complex engineering challenge, yet one which promises to reduce cost, labor, and turnaround time if solved. Here we introduce Oligo Pool Sidewinder assembly, which enables one-pot, parallel assembly of hundreds of DNA fragments simultaneously into dozens of defined constructs with high fidelity from oligo pools. We pair a computational workflow for string-based bespoke oligo design with a set of construction rules for highly multiplexed assembly. We demonstrate recovery of individual sequences from the pool by construct-specific amplification with misconnection rates as low as 1 in 10. This high fidelity for connection enables hundreds of oligos to co-assemble in a single reaction. Further, we show universal amplification of libraries of defined target sequences in a single PCR and extend the approach through in vitro hierarchical assembly to construct a large 12.5-kilobase synthetic linear DNA product.

molecular biology↗

bioRxiv2025

YAP levels regulate anteroposterior elongation of hESC-derived gastruloids

Human embryonic stem cells (hESCs) can self-organize into anteroposterior patterned three-dimensional structures, characterized by polarized expression of CDX2 and GATA6, known as human 3D gastruloids (h3D-gastruloids). This patterning emerges through hESC-intrinsic mechanisms that remain poorly understood. Here, we demonstrate that the formation and elongation of h3D-gastruloids is modulated by the activity levels of the Hippo pathway effector YAP. Using complementary chemical and genetic perturbations, we show that elevated nuclear YAP activity inhibits gastruloid elongation, whereas YAP inhibition enhances axial elongation relative to controls. Single-cell RNA sequencing (scRNA-seq) analyses reveal that YAP activation disrupts the establishment of distinct anterior GATA6 and posterior CDX2 poles. Conversely, YAP inhibition promotes clearer segregation of these domains, facilitating symmetry breaking and elongation. Finally, we developed a high-throughput platform for h3D-gastruloid generation, enabling the production of over 500 gastruloids per well. Together, our findings uncover a role for YAP in orchestrating the three-dimensional organization of human gastruloids.

developmental biology↗

bioRxiv2025

Functional characterization of a Con7-related transcription factor in Coprinopsis cinerea indicates evolutionary conservation of morphogenetic roles

Fruiting bodies of mushroom-forming fungi (Agaricomycetes) exhibit the highest degree of multicellular complexity in fungi, yet the molecular underpinnings of their developmental programs remain incompletely understood. Here, we characterize gcd1, a gene encoding a transcription factor in the Con7 subfamily of C2H2-type zinc finger proteins. This subfamily has previously been implicated in pathogenic morphogenesis in Ascomycota, but their role in Agaricomycetes has not previously been addressed. In Coprinopsis cinerea, CRISPR/Cas9-mediated deletion of gcd1 resulted in strains with severely impaired fruiting body morphogenesis, with malformed cap, stipe, and gill tissues. Gcd1 deletion strains lacked universal veil, resembling species with open (gymnocarpous) development. We find that GCD1/Con7 homologs are widely distributed in most Dikarya species and are mostly encoded by a single gene in each species genome. Transcriptome analyses identified several misregulated genes in the {Delta}gcd1 mutant, which pinpoint potential mechanisms underlying its developmental defects as well as provided insights into the morphogenesis of mushroom fruiting bodies. These findings establish GCD1 as a key regulator of multicellular development in C. cinerea and broaden the known functions of Con7-like transcription factors to include fruiting body morphogenesis in Agaricomycetes. Overall, our results and the morphogenetic role of Con7-like transcription factors of Ascomycota suggest functional conservation over half a billion years of evolution.

microbiology↗

bioRxiv2025

YAP1 and QSER1 are Key Modulators of Embryonic Signaling Pathways in the Mammalian Epiblast

YAP1 signaling is essential for development but its specific roles in early embryogenesis remain poorly understood. To shed light on this, we analyzed YAP1s role in regulating the pluripotency of the mammalian epiblast, using scRNAseq approaches. Conditional deletion of Yap1 in the mouse epiblast (Sox2-Cre) altered the expression of signaling genes, including Nodal, Wnt3, and Fgf8. Accordingly, Yap1 loss led to enhanced differentiation of the epiblast toward primitive streak lineages, as evidenced by the upregulation of T/Brachyury and Eomes genes. Furthermore, a proximity labeling assay in human pluripotent stem cells, followed by biochemical assays and molecular modeling predictions, revealed that YAP1 cooperates with QSER1 protein to regulate lineage genes. Our analysis shows that YAP1:TEAD4 enhancers recruit QSER1 to prevent RNA Polymerase II recruitment. Accordingly, QSER1 depletion, similar to YAP1, increases NODAL gene expression and leads to hyperactive NODAL signaling in human 2D-gastruloids. Overall, our findings define a role of YAP1 in the epiblast in vivo and uncovered an interplay with QSER1 controlling the activity of developmental signaling pathways in pluripotent cells.

developmental biology↗

bioRxiv2024

A new regulator of sporulation sheds light on spore morphogenesis and ballistospory in mushroom-forming fungi

Sporulation is the most widespread means of reproduction and dispersal in fungi. In the Basidiomycota, sexual spores are produced on specialised cells known as basidia, from which they are discharged forcibly by a powered process called ballistospory, the highest known acceleration in nature. However, the genetics of sporulation, in particular postmeiotic events related to spore morphogenesis and ballistospory, remain poorly known. Here, we explore the genetics of these processes, based on a new, highly conserved transcription factor, Sporulation-Related Regulator 1 (SRR1), and its putative downstream regulatory network. Reverse genetics of Srr1 in the model mushroom Coprinopsis cinerea and commercially produced oyster mushroom indicated a conserved role of Srr1 in sporulation across Agaricomycetes. RNA-Seq analysis and motif-based inference of a hypothetical SRR1 gene regulatory network allowed delimiting putative targets regulated by SRR1 in a direct and indirect manner. Using this network and comparative genomics, we identified genes associated with ballistospory, including a putative SRR1-target chitinase, which was found to be required for normal spore production and morphology. Overall, our study offers new insights into the genetic mechanisms governing postmeiotic spore morphogenesis and ballistospory in the Agaricomycetes.

microbiology↗

bioRxiv2024

Retracing the horizontal transfer of a novel innate immune factor in Drosophila

Immune systems are among the most dynamically evolving traits across the tree of life, and long-lived macroparasites play an outsized role in shaping animal immunity. Even without adaptive immunity, insects have evolved potent innate immune strategies to neutralize such enemies, including nematodes and parasitoid wasps. One such strategy relies on endosymbioses between insects and toxin-expressing bacteria. Here, we use genome editing in Drosophila melanogaster to retrace the evolution of two of such toxins -- cytolethal distending toxin B (cdtB) and apoptosis inducing protein of 56kDa (aip56) -- that were horizontally transferred from bacteriophages to insects. We found that a cdtB::aip56 fusion gene (fusionB), which is conserved in Drosophila ananassae subgroup species, dramatically promoted fly survival and suppressed wasp development when expressed in D. melanogaster immune tissues. FusionB, a functional nuclease, was secreted into the host hemolymph where it targeted the parasitoid embryos serosal tissue and is to our knowledge the first humoral anti-parasitoid toxin in Drosophila. When expressed ubiquitously, fusionB slowed development in late stage fly larvae and eventually killed flies, pointing to the salience of regulatory constraint in preventing autoimmunity. Our findings demonstrate how horizontal gene transfer, in the right regulatory context, can instantly provide new and potent innate immune modules in animals.

evolutionary biology↗

bioRxiv2024

Distinct components of nucleoside-modified messenger RNA vaccines cooperate to instruct efficient germinal center responses

Nucleoside-modified mRNA vaccines elicit protective antibodies through their ability to promote T follicular helper (Tfh) cells. The lipid nanoparticle (LNP) component of mRNA vaccines possesses inherent adjuvant activity. However, to what extent the nucleoside-modified mRNA can be sensed and contribute to Tfh cell responses remains largely undefined. Herein, we deconvoluted the signals induced by LNP and mRNA that instruct dendritic cells (DCs) to promote Tfh cell differentiation. We demonstrated that the nucleoside-modified mRNA drives the production of type I interferons that act on DCs to induce their maturation and the induction of Th1-biased Tfh responses. Conversely, LNP favors the acquisition of a Tfh cell-inducing program in DCs, a stronger Th2 polarization in Tfh cells, and allows for rapid mRNA translation by DCs within the draining lymph node. Our work unravels distinct adjuvant features of mRNA and LNP necessary for the induction of Tfh cells, with implications for vaccine design.

immunology↗

bioRxiv2024

Single-cell RNA sequencing of mutant whole mouse embryos: from the epiblast to the end of gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula. SUMMARYWe establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

developmental biology↗

bioRxiv2024

Chemical genetics in C. elegans identifies anticancer mycotoxins chaetocin and chetomin as potent inducers of a nuclear metal homeostasis response

C. elegans numr-1/2 (nuclear-localized metal-responsive) is an identical gene pair encoding a nuclear protein previously shown to be activated by cadmium and disruption of the integrator RNA metabolism complex. We took a chemical genetic approach to further characterize regulation of this novel metal response by screening 41,716 compounds and extracts for numr-1p::GFP activation. The most potent activator was chaetocin, a fungal 3,6-epidithiodiketopiperazine (ETP) with promising anticancer activity. Chaetocin activates numr-1/2 strongly in the alimentary canal but is distinct from metal exposure because it represses canonical cadmium-responsive metallothionine genes. Chaetocin has diverse targets in cancer cells including thioredoxin reductase, histone lysine methyltransferase, and acetyltransferase p300/CBP; further work is needed to identify the mechanism in C. elegans as genetic disruption and RNAi screening of homologs did not induce numr-1/2 in the alimentary canal and chaetocin did not affect markers of integrator dysfunction. We demonstrate that disulfides in chaetocin and chetomin, a dimeric ETP analog, are required to induce numr-1/2. ETP monomer gliotoxin, despite possessing a disulfide linkage, had almost no effect on numr-1/2, suggesting a dimer requirement. Chetomin inhibits C. elegans growth at low micromolar levels and loss of numr-1/2 increases sensitivity; C. elegans and Chaetomiaceae fungi inhabit similar environments raising the possibility that numr-1/2 functions as a defense mechanism. There is no direct ortholog of numr-1/2 in humans, but RNAseq suggests that chaetocin affects expression of cellular processes linked to stress response and metal homeostasis in colorectal cancer cells. Our results reveal interactions between metal response gene regulation and ETPs and identify a potential mechanism of resistance to this versatile class of preclinical compounds.

pharmacology and toxicology↗

bioRxiv2023

Restoration of Cone Circuit Functionality in the Regenerating Adult Zebrafish Retina

Unlike humans, teleosts like zebrafish exhibit robust retinal regeneration after injury from endogenous stem cells. However, understanding the functional recovery of the regenerated retina remains a challenge. In particular, it is unclear if regenerating cone-photoreceptors can regain physiological function and integrate correctly into post-synaptic circuits. To bridge this gap, we employed two-photon calcium imaging of living retina, focusing on photoreceptor responses before and after an intense light-induced lesion in the adult zebrafish retina. To assess the functional recovery of cones and their downstream circuits, we exploited the colour opponency in adult zebrafish cones. We find that UV cones exhibit an intrinsic Off-response to short-wavelength blue light, but an On-response to longer-wavelength green light, which depends on feedback signals from outer retinal circuits. Accordingly, we examined the presence and quality of Off-versus On-responses, to assess the functional recovery of cones and their correct integration into outer retinal circuits. We find that regenerated UV cones regain both Off-responses to short-wavelength and On-responses to long-wavelength within 3 months after light lesion. Hence, physiological circuit functionality is restored in regenerated cone photoreceptors, suggesting that inducing endogenous regeneration is a promising strategy for human retinal repair. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/566441v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@c9f038org.highwire.dtl.DTLVardef@1ba255borg.highwire.dtl.DTLVardef@1ea7f60org.highwire.dtl.DTLVardef@50adf3_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Protein phosphatase 4 is required for Centrobin function in DNA damage repair

Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein Phosphatase 4 (PP4) plays a pivotal role in coordinating the cellular response to DNA damage. Meanwhile, Centrobin (Ctb), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with Ctb in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and Ctb and shed light on the importance of two specific motifs in Ctb, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells lacking PP4 or Ctb and cells harboring FRVP and SQ mutations in Ctb, which result in similarly abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms and interrelationships of PP4 and Ctb in the regulation of HR repair.

cell biology↗

bioRxiv2021

YAP1 Regulates the Self-organized Fate Patterning of hESCs-Derived Gastruloids

During gastrulation, the coordinated activity of BMP, WNT and NODAL signaling pathways guide the differentiation of the pluripotent epiblast into the three germinal layers. Recent studies underline the role of the Hippo-effector YAP1 regulating WNT and NODAL signaling pathways and repressing mesoendodermal differentiation in human embryonic stem cells (hESCs). However, the contribution of YAP1 to the cell-fate patterning decisions that transform the epiblast in a three-germ layer gastrula remains unknown. We address this question by analyzing micropatterned 2D-gastruloids derived from hESCs, in the presence and absence of YAP1. Our findings show that YAP1 is necessary for gastrulation. YAP1 KO-gastruloids display reduced ectoderm layer and enlarged mesoderm and endoderm layers, compared to WT. Furthermore, YAP1 regulates the self-organized patterning of the hESCs, as the discrete position of the three germ layers is altered in the YAP1 KO-gastruloids. Our epigenome (single-nuclei ATACseq) and transcriptome (RNA-seq) analysis revealed that YAP1 directly represses the chromatin accessibility and transcription of key genes in the NODAL pathway, including the NODAL and FOXH1 genes. In WT gastruloids, a gradient of NODAL: SMAD2.3 signaling from the periphery to the center of the colony regulates the exit of pluripotency toward endoderm, mesoderm and ectoderm, respectively. Hence, in the absence of YAP1, a hyperactive NODAL signaling retains SMAD2.3 in the nuclei impeding the self-organized differentiation of hESCs. Accordingly, the partial inhibition of NODAL signaling is sufficient to rescue the differentiation and pattern -defective phenotypes of the YAP1 KO gastruloids. Our work revealed that YAP1 is a master regulator of NODAL signaling, essential to instruct germ layer fate patterning in human gastruloids.

developmental biology↗