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1. Idiosyncratic and dose-dependent epistasis drives variation in tomato fruit size

   https://doi.org/10.1126/science.adi5222  

   Aguirre, Lyndsey; Hendelman, Anat; Hutton, Samuel F; McCandlish, David M; Lippman, Zachary B (October 2023, Science)

   Epistasis between genes is traditionally studied with mutations that eliminate protein activity, but most natural genetic variation is in cis-regulatory DNA and influences gene expression and function quantitatively. In this study, we used natural and engineered cis-regulatory alleles in a plant stem-cell circuit to systematically evaluate epistatic relationships controlling tomato fruit size. Combining a promoter allelic series with two other loci, we collected over 30,000 phenotypic data points from 46 genotypes to quantify how allele strength transforms epistasis. We revealed a saturating dose-dependent relationship but also allele-specific idiosyncratic interactions, including between alleles driving a step change in fruit size during domestication. Our approach and findings expose an underexplored dimension of epistasis, in which cis-regulatory allelic diversity within gene regulatory networks elicits nonlinear, unpredictable interactions that shape phenotypes. 

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2. Enabling the study of gene function in gymnosperms: Virus‐induced gene silencing in Ephedra tweedieana

   https://doi.org/10.1002/aps3.70027  

   Garcia, Anthony_G K; Bùi, Jo Trang; Michael, Todd P; Ickert‐Bond, Stefanie M; Di_Stilio, Verónica S (December 2025, Applications in Plant Sciences)

   Abstract PremiseAs the sister clade to angiosperms, extant gymnosperms are crucial for reconstructing ancestral gene regulatory networks in seed plants. This highlights the need for model systems representing each of their distinct lineages. However, tools to quickly and effectively investigate gene function in gymnosperms are still limited due to the challenges of long life cycles and large genome sizes. Species within the xerophytic genusEphedra(Gnetales) have comparatively smaller genomes and shrubby growth habits with shorter life spans, making them better suited for greenhouse cultivation and laboratory experiments. MethodsWe implement virus‐induced gene silencing (VIGS) to manipulate gene expression inEphedra tweedieanaviaAgrobacterium‐mediated vacuum infiltration of tobacco rattle virus (TRV1 and TRV2) into seedlings. ResultsTreatment resulted in highly efficient gene silencing of theE. tweedieana PHYTOENE DESATURASE(PDS) orthologEtwPDS. The expected photobleaching phenotype was observed as early as two weeks, and lasted at least five months in stems, shoot tips, leaves, axillary meristems, and lateral branches of treated plants. DiscussionWe report on virus‐induced targeted gene silencing ofPDSin a Gnetales representative to further enable functional studies of the genetic mechanisms underpinning adaptations in gymnosperms, an important and underrepresented lineage of seed plants. 

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3. Discovery of target genes and pathways at GWAS loci by pooled single-cell CRISPR screens

   https://doi.org/10.1126/science.adh7699  

   Morris, John A.; Caragine, Christina; Daniloski, Zharko; Domingo, Júlia; Barry, Timothy; Lu, Lu; Davis, Kyrie; Ziosi, Marcello; Glinos, Dafni A.; Hao, Stephanie; et al (May 2023, Science)

   INTRODUCTION Genome-wide association studies (GWASs) have identified thousands of human genetic variants associated with diverse diseases and traits, and most of these variants map to noncoding loci with unknown target genes and function. Current approaches to understand which GWAS loci harbor causal variants and to map these noncoding regulators to target genes suffer from low throughput. With newer multiancestry GWASs from individuals of diverse ancestries, there is a pressing and growing need to scale experimental assays to connect GWAS variants with molecular mechanisms. Here, we combined biobank-scale GWASs, massively parallel CRISPR screens, and single-cell sequencing to discover target genes of noncoding variants for blood trait loci with systematic targeting and inhibition of noncoding GWAS loci with single-cell sequencing (STING-seq). RATIONALE Blood traits are highly polygenic, and GWASs have identified thousands of noncoding loci that map to candidate cis -regulatory elements (CREs). By combining CRE-silencing CRISPR perturbations and single-cell readouts, we targeted hundreds of GWAS loci in a single assay, revealing target genes in cis and in trans . For select CREs that regulate target genes, we performed direct variant insertion. Although silencing the CRE can identify the target gene, direct variant insertion can identify magnitude and direction of effect on gene expression for the GWAS variant. In select cases in which the target gene was a transcription factor or microRNA, we also investigated the gene-regulatory networks altered upon CRE perturbation and how these networks differ across blood cell types. RESULTS We inhibited candidate CREs from fine-mapped blood trait GWAS variants (from ~750,000 individual of diverse ancestries) in human erythroid progenitors. In total, we targeted 543 variants (254 loci) mapping to candidate CREs, generating multimodal single-cell data including transcriptome, direct CRISPR gRNA capture, and cell surface proteins. We identified target genes in cis (within 500 kb) for 134 CREs. In most cases, we found that the target gene was the closest gene and that specific enhancer-associated biochemical hallmarks (H3K27ac and accessible chromatin) are essential for CRE function. Using multiple perturbations at the same locus, we were able to distinguished between causal variants from noncausal variants in linkage disequilibrium. For a subset of validated CREs, we also inserted specific GWAS variants using base-editing STING-seq (beeSTING-seq) and quantified the effect size and direction of GWAS variants on gene expression. Given our transcriptome-wide data, we examined dosage effects in cis and trans in cases in which the cis target is a transcription factor or microRNA. We found that trans target genes are also enriched for GWAS loci, and identified gene clusters within trans gene networks with distinct biological functions and expression patterns in primary human blood cells. CONCLUSION In this work, we investigated noncoding GWAS variants at scale, identifying target genes in single cells. These methods can help to address the variant-to-function challenges that are a barrier for translation of GWAS findings (e.g., drug targets for diseases with a genetic basis) and greatly expand our ability to understand mechanisms underlying GWAS loci. Identifying causal variants and their target genes with STING-seq. Uncovering causal variants and their target genes or function are a major challenge for GWASs. STING-seq combines perturbation of noncoding loci with multimodal single-cell sequencing to profile hundreds of GWAS loci in parallel. This approach can identify target genes in cis and trans , measure dosage effects, and decipher gene-regulatory networks. 

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4. A Variational Inference Approach to Single-Cell Gene Regulatory Network Inference using Probabilistic Matrix Factorization

   https://doi.org/10.1101/2022.09.09.507305  

   Mahmood, Omar; Skok Gibbs, Claudia; Bonneau, Richard; Cho, Kyunghyun (March 2023, ICML 2023)

   Inferring gene regulatory networks (GRNs) from single-cell gene expression datasets is a challenging task. Existing methods are often designed heuristically for specific datasets and lack the flexibility to incorporate additional information or compare against other algorithms. Further, current GRN inference methods do not provide uncertainty estimates with respect to the interactions that they predict, making inferred networks challenging to interpret. To overcome these challenges, we introduce Probabilistic Matrix Factorization for Gene Regulatory Network inference (PMF-GRN). PMF-GRN uses single-cell gene expression data to learn latent factors representing transcription factor activity as well as regulatory relationships between transcription factors and their target genes. This approach incorporates available experimental evidence into prior distributions over latent factors and scales well to single-cell gene expression datasets. By utilizing variational inference, we facilitate hyperparameter search for principled model selection and direct comparison to other generative models. To assess the accuracy of our method, we evaluate PMF-GRN using the model organisms Saccharomyces cerevisiae and Bacillus subtilis, benchmarking against database-derived gold standard interactions. We discover that, on average, PMF-GRN infers GRNs more accurately than current state-of-the-art single-cell GRN inference methods. Moreover, our PMF-GRN approach offers well-calibrated uncertainty estimates, as it performs gene regulatory network (GRN) inference in a probabilistic setting. These estimates are valuable for validation purposes, particularly when validated interactions are limited or a gold standard is incomplete. 

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5. Local chromatin context regulates the genetic requirements of the heterochromatin spreading reaction

   https://doi.org/10.1371/journal.pgen.1010201  

   Greenstein, R. A.; Ng, Henry; Barrales, Ramon R.; Tan, Catherine; Braun, Sigurd; Al-Sady, Bassem (May 2022, PLOS Genetics)

   van Steensel, Bas (Ed.)

   Heterochromatin spreading, the expansion of repressive chromatin structure from sequence-specific nucleation sites, is critical for stable gene silencing. Spreading re-establishes gene-poor constitutive heterochromatin across cell cycles but can also invade gene-rich euchromatin de novo to steer cell fate decisions. How chromatin context (i.e. euchromatic, heterochromatic) or different nucleation pathways influence heterochromatin spreading remains poorly understood. Previously, we developed a single-cell sensor in fission yeast that can separately record heterochromatic gene silencing at nucleation sequences and distal sites. Here we couple our quantitative assay to a genetic screen to identify genes encoding nuclear factors linked to the regulation of heterochromatin nucleation and the distal spreading of gene silencing. We find that mechanisms underlying gene silencing distal to a nucleation site differ by chromatin context. For example, Clr6 histone deacetylase complexes containing the Fkh2 transcription factor are specifically required for heterochromatin spreading at constitutive sites. Fkh2 recruits Clr6 to nucleation-distal chromatin sites in such contexts. In addition, we find that a number of chromatin remodeling complexes antagonize nucleation-distal gene silencing. Our results separate the regulation of heterochromatic gene silencing at nucleation versus distal sites and show that it is controlled by context-dependent mechanisms. The results of our genetic analysis constitute a broad community resource that will support further analysis of the mechanisms underlying the spread of epigenetic silencing along chromatin. 

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