Biology subjects

Sims, J.

Publications and source records attributed to Sims, J..

14 recordsLinked to original sources

bioRxiv2026

Preclinical Characterization of Hippo pathway inhibition: Insights from Pharmacological and Genetic Studies

The Hippo pathway is an evolutionarily conserved regulator of growth, regeneration, and organ homeostasis, and while its dysregulation is well established in cancer, the effects of inhibiting this pathway on normal tissues are less understood. Here we have systematically investigated the impact of Hippo pathway inhibition by comparing pharmacologic perturbation using a covalent small-molecule TEAD inhibitor (TEADi CMPD1, also known as GNE-8025) with genetic suppression of YAP/TAZ. We identified three key target organs that consistently emerged upon TEAD inhibition: the kidney, as well as the pancreas, and thymus. Across models, both perturbations led to comparable disease phenotypes in these organs, including tubular degeneration in the kidney, acinar atrophy in the pancreas, and lymphoid depletion in the thymus. However, the extent of damage was more pronounced in mice treated with the small-molecule inhibitor, highlighting potential dose and compound specific effects while remaining broadly consistent with the phenotypes observed upon genetic ablation of YAP/TAZ. This highlights the key role of evaluating both genetic and pharmacological perturbations to characterize the phenotypes and potential toxicities when modulating novel targets in oncology. To further investigate the mechanisms underlying pan-TEAD inhibition and kidney related adverse effects, we further characterized this class effect through a comprehensive transcriptomic analysis of the kidney to map the pathways involved in renal response. SignificanceUnderstanding on target toxicities is critical for the safe clinical development of TEAD inhibitors. Here, by integrating pharmacologic TEAD inhibition with genetic suppression by developing a mouse model that characterizes systemic, inducible knockdown of YAP/TAZ, we provide a systematic framework to define the Hippo pathway liabilities in vivo. We identify kidney, pancreas, and thymus as conserved target organs with concomitant phenotypes across both genetic and pharmacological methods, establishing these as pathway driven effects. Importantly, we uncover dose dependent and partially irreversible injury, particularly in kidney and pancreas, alongside mechanistic insight linking TEAD inhibition to aldosterone signaling disruption in kidney. These findings highlight the importance of strategies to identify monitorable, manageable adverse effect to guide clinical translation of TEAD targeting strategies.

cancer biology↗

bioRxiv2024

Massively parallel jumping assay decodes Alu retrotransposition activity

The human genome contains millions of retrotransposons, several of which could become active due to somatic mutations having phenotypic consequences, including disease. However, it is not thoroughly understood how nucleotide changes in retrotransposons affect their jumping activity. Here, we developed a novel massively parallel jumping assay (MPJA) that can test the jumping potential of thousands of transposons en masse. We generated nucleotide variant library of selected four Alu retrotransposons containing 165,087 different haplotypes and tested them for their jumping ability using MPJA. We found 66,821 unique jumping haplotypes, allowing us to pinpoint domains and variants vital for transposition. Mapping these variants to the Alu-RNA secondary structure revealed stem-loop features that contribute to jumping potential. Combined, our work provides a novel high-throughput assay that assesses the ability of retrotransposons to jump and identifies nucleotide changes that have the potential to reactivate them in the human genome.

genomics↗

bioRxiv2023

MPRAbase: A Massively Parallel Reporter Assay Database

Massively parallel reporter assays (MPRAs) represent a set of high-throughput technologies that measure the functional effects of thousands of sequences/variants on gene regulatory activity. There are several different variations of MPRA technology and they are used for numerous applications, including regulatory element discovery, variant effect measurement, saturation mutagenesis, synthetic regulatory element generation or characterization of evolutionary gene regulatory differences. Despite their many designs and uses, there is no comprehensive database that incorporates the results of these experiments. To address this, we developed MPRAbase, a manually curated database that currently harbors 129 experiments, encompassing 17,718,677 elements tested across 35 cell types and 4 organisms. The MPRAbase web interface (http://www.mprabase.com) serves as a centralized user-friendly repository to download existing MPRA data for independent analysis and is designed with the ability to allow researchers to share their published data for rapid dissemination to the community.

genomics↗

bioRxiv2023

A TOPBP1 Allele Causing Male Infertility Uncouples XY Silencing Dynamics From Sex Body Formation

Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. Topbp1B5/B5 males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted including phosphorylation and localization of the RNA:DNA helicase Senataxin. Topbp1B5/B5 spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.

cell biology↗

bioRxiv2022

Vascular fungi alter the apoplastic purinergic signaling in plants by deregulating the homeostasis of extracellular ATP and its metabolite adenosine

Purinergic signaling activated by extracellular nucleotides and their derivative nucleosides trigger sophisticated signaling networks. The outcome of these pathways determine the capacity of the organism to survive under challenging conditions. Both extracellular ATP (eATP) and Adenosine (eAdo) act as secondary messengers in mammals, essential for immunosuppressive responses. Despite the clear role of eATP as a plant damage-associated molecular pattern, the function of its nucleoside, eAdo, and of the eAdo/eATP balance in plant stress response remain to be fully elucidated. This is particularly relevant in the context of plant/microbe interaction, where the intruder manipulates the extracellular matrix. Here, we identify Ado as a main elicitor secreted by the vascular fungus Fusarium oxysporum. We show that eAdo modulates the plants susceptibility to fungal colonization by altering eATP-mediated physiological immune responses, such as apoplastic pH and calcium homeostasis. Our work indicates that plant pathogens actively imbalance the eAdo/eATP levels as a virulence mechanism. One Sentence SummaryThe apoplastic Adenosine/ATP balance is a messenger for plant defense and can be manipulated by the fungal pathogen F. oxysporum.

plant biology↗

bioRxiv2022

The cohesin subunit RAD21.2 functions as a recombination silencer of ribosomal DNA arrays

Introductory ParagraphIn many species, including Arabidopsis, heterochromatin often comprises repetitive DNA elements, such as arrays of ribosomal DNA (rDNA). Repetitive regions pose a risk in meiosis since recombination between them can lead to gross genomic rearrangements. However, meiotic recombination at rDNA arrays and other heterochromatic repeat regions is blocked by not well understood mechanisms. Here, we have identified RAD21.2, an -kleisin subunit of cohesin, as a repressor of meiotic recombination at the rDNA regions in Arabidopsis. We show that RAD21.2 co-localizes with heterochromatic factors and is specifically enriched at rDNA repeats, which are devoid of the meiosis specific -kleisin REC8, needed for recombination. Knocking down RAD21.2, we find that REC8 moves into the nucleolus organizing regions (NORs), where we see an increase of RAD51 recombinase foci numbers. Concomitantly, we find extensive rearrangements of the NORs and the offspring of these plants have large variation in rDNA copy numbers demonstrating that RAD21.2 is necessary for transgenerational genome stability. One-Sentence SummaryThe cohesin component RAD21.2 represses meiotic recombination and by that contributes to genome stability over generations.

cell biology↗

bioRxiv2021

Phosphoproteomics of ATR Signaling in Prophase I of Mouse Meiosis

During mammalian meiosis, the ATR kinase plays crucial roles in the coordination of DNA repair, meiotic sex chromosome inactivation and checkpoint signaling. Despite the importance of ATR in meiosis, the meiotic ATR signaling network remains largely unknown. Here we defined ATR signaling during prophase I in mice. Quantitative analysis of phosphoproteomes obtained after genetic ablation of the ATR-activating 9-1-1 complex or chemical inhibition of ATR revealed over 12,000 phosphorylation sites, of which 863 phosphorylation sites were dependent on both 9-1-1 and ATR. ATR and 9-1-1-dependent signaling was enriched for S/T-Q and S/T-X-X-K motifs and included proteins involved in DNA damage signaling, DNA repair, and piRNA and mRNA metabolism. We find that ATR targets the RNA processing factors SETX and RANBP3 and regulate their localization to the sex body. Overall, our analysis establishes a comprehensive map of ATR signaling in spermatocytes and highlights potential meiotic-specific actions of ATR during prophase I.

developmental biology↗

bioRxiv2021

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here we report that testis-specific RAD1 disruption resulted in impaired DSB repair, germ cell depletion and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss also caused defects in homolog synapsis, ATR signaling and meiotic sex chromosome inactivation. Comprehensive testis phosphoproteomics revealed that RAD1 and ATR coordinately regulate numerous proteins involved in DSB repair, meiotic silencing, synaptonemal complex formation, and cohesion. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

genetics↗

bioRxiv2021

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis. Author summarySARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis.

genetics↗

bioRxiv2020

Sequencing and analysis of Arabidopsis thaliana NOR2 reveal its distinct organization and tissue-specific expression of rRNA ribosomal variants

Despite vast differences between organisms, some characteristics of their genomes are conserved, such as the nucleolus organizing region (NOR). The NOR is constituted of multiple, highly repetitive rDNA genes, encoding the catalytic ribosomal core RNAs which are transcribed from 45S rDNA units. Their precise sequence information and organization remained uncharacterized. We used a combination of long- and short-read sequencing technologies to assemble contigs of the Arabidopsis NOR2 rDNA domain providing a first map. We identified several expressed rRNA gene variants which are integrated into translating ribosomes in a tissue-specific manner. These findings support the concept of tissue specific ribosome subpopulations that differ in their rRNA composition and provide the higher order organization of NOR2.

plant biology↗

bioRxiv2020

Optimization and scaling of patient-derived brain organoids uncovers deep phenotypes of disease

Cerebral organoids provide unparalleled access to human brain development in vitro. However, variability induced by current culture methodologies precludes using organoids as robust disease models. To address this, we developed an automated Organoid Culture and Assay (ORCA) system to support longitudinal unbiased phenotyping of organoids at scale across multiple patient lines. We then characterized organoid variability using novel machine learning methods and found that the contribution of donor, clone, and batch is significant and remarkably consistent over gene expression, morphology, and cell-type composition. Next, we performed multi-factorial protocol optimization, producing a directed forebrain protocol compatible with 96-well culture that exhibits low variability while preserving tissue complexity. Finally, we used ORCA to study tuberous sclerosis, a disease with known genetics but poorly representative animal models. For the first time, we report highly reproducible early morphological and molecular signatures of disease in heterozygous TSC+/- forebrain organoids, demonstrating the benefit of a scaled organoid system for phenotype discovery in human disease models.

neuroscience↗

bioRxiv2020

Genetic elucidation of complex biochemical traits mediating maize innate immunity

Specialized metabolites constitute key layers of immunity underlying crop resistance; however, challenges in resolving complex pathways limit our understanding of their functions and applications. In maize (Zea mays) the inducible accumulation of acidic terpenoids is increasingly considered as a defense regulating disease resistance. To understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies, and targeted mutagenesis. We now define ten genes in three zealexin (Zx) gene clusters comprised of four sesquiterpene synthases and six cytochrome P450s that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants blocked in the production of {beta}-macrocarpene exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate-promiscuity creating a biosynthetic hourglass pathway utilizing diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes underlying disease resistance demonstrates a predominant maize defense pathway and informs innovative strategies for transferring chemical immunity between crops.

plant biology↗

bioRxiv2019

CpG-creating Mutations are Costly in Many Human Viruses

Mutations can occur throughout the virus genome and may be beneficial or deleterious. We are interested in mutations that yield a C next to a G, producing CpG sites. CpG sites are rare in eukaryotic and viral genomes. For the eukaryotes, it is thought that CpG sites are rare because they are prone to mutation when methylated. In viruses, we know less about why CpG sites are rare. A previous study in HIV suggested that CpG-creating transition mutations are more costly that similar non-CpG-creating mutations. To determine if this is the case in other viruses, we analyzed the allele frequencies of CpG-creating and non-CpG-creating mutations across various strains, subtypes, and genes of viruses using existing data obtained from Genbank, HIV Databases, and Virus Pathogen Resource. Our results suggest that CpG sites are costly for most viruses. By understanding the cost of CpG sites, we can obtain further insights into the evolution and adaptation of viruses.

evolutionary biology↗

bioRxiv2019

Meiotic DNA repair in the nucleolus employs a non-homologous end joining mechanism

Ribosomal RNA genes are arranged in large arrays with hundreds of rDNA units in tandem. These highly repetitive DNA elements pose a risk to genome stability since they can undergo non-allelic exchanges. During meiosis DNA double strand breaks (DSBs) are induced as part of the regular program to generate gametes. Meiotic DSBs initiate homologous recombination (HR) which subsequently ensures genetic exchange and chromosome disjunction. In Arabidopsis thaliana we demonstrate that all 45S rDNA arrays become transcriptionally active and are recruited into the nucleolus early in meiosis. This shields the rDNA from acquiring canonical meiotic chromatin modifications, meiotic cohesin and meiosis-specific DSBs. DNA breaks within the rDNA arrays are repaired in a RAD51-independent, but LIG4-dependent manner, establishing that it is non-homologous end joining (NHEJ) that maintains rDNA integrity during meiosis. Utilizing ectopically integrated rDNA repeats we validate our findings and demonstrate that the rDNA constitutes a HR-refractory genome environment.

molecular biology↗