Biology subjects

Cohen, B. A.

Publications and source records attributed to Cohen, B. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Features affecting Cas9-Induced Editing Efficiency and Patterns in Tomato: Evidence from a Large CRISPR Dataset

CRISPR/Cas9 is a cornerstone of genome editing, yet the determinants of editing efficiency and DNA Double Strand Break (DSB) repair outcomes remain poorly understood, particularly in plants. To address this gap, we generated a dataset of 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts and quantified editing efficiencies together with ATAC-seq-derived chromatin accessibility and transcriptional states in the same cellular system. Editing efficiency was consistently higher at open chromatin sites and modestly elevated in promoters and introns relative to exons, whereas transcriptional activity did not measurably influence editing outcomes. Additionally, we identified a local genomic effect resulting in less variable editing between sgRNAs targeting the same compared to different genes. A distinct subset of sgRNAs achieved nearly complete editing, producing long deletions with extended microhomology tracts. These repair footprints closely parallel those observed for high-efficiency guides in human datasets, implicating conserved sequence-driven biases and a predominant role for microhomology-mediated end joining at these sites. Yet, widely used human-trained prediction models failed to rank sgRNA performance in plants, underscoring the limits of cross-species generalization. This dataset defines how chromatin accessibility, genomic context, and intrinsic sequence characteristics shape Cas9 activity in plants, and provides a resource for improving guide design and advancing mechanistic understanding of plant DNA repair.

plant biology↗

bioRxiv2025

Systematic evaluation of metatranscriptomic differential gene expression in silico, in vitro, and in vivo enables elucidation of inter-species cross-feeding

Metatranscriptomic (MTX) sequencing quantifies gene expression from the collective genomes of microbial communities (microbiomes), enabling assessment of functional activity rather than functional potential. While differential expression testing is essential for RNA-sequencing analysis, current metatranscriptomic approaches have only been benchmarked on simulated data, resulting in a lack of standard practices for analysis of real datasets. Here, we use mock communities (defined mixtures of microbial cells with known properties) to quantitatively assess robustness and susceptibility of current approaches to various confounders including organisms low relative abundance, differential abundance, low prevalence, global transcriptional output changes, and compositional effects. We show that no current method is robust to all confounders and method performance on simulated data does not generalize to real datasets. We then apply the same approaches to MTX datasets generated from gnotobiotic mice colonized with defined consortia of human bacterial strains and show that the method nominated by the mock community comparisons successfully inferred cross-feeding dynamics that were subsequently validated in vitro. Finally, using metagenome-assembled genomes from a human clinical study, we leverage genome-level sequencing depth and detection of genes to exclude low information samples on a per-organism basis to overcome confounding low prevalence and enhance differential expression inference.

genomics↗

bioRxiv2025

Long-range massively parallel reporter assay reveals rules of distal enhancer-promoter interactions

Long-range activation is an essential property of enhancers, yet the features determining long-range enhancer activities have not been systematically investigated due to a lack of high-throughput methods to measure long-range enhancer activities efficiently. To address this gap, we present a long-range massively parallel reporter assay (long-range MPRA), an assay allowing the measurement of hundreds of enhancers at multiple distances from a genome-integrated promoter. The long-range MPRA assay features two independent landing pads, allowing modular control over the genome-integrated promoter and enhancer libraries. We showcased the capability of long-range MPRA by testing over 300 K562 enhancers, as well as a set of enhancer combinations, at distances up to 100 kb. We found that enhancers long-range activities are primarily determined by their intrinsic strength, with strong enhancers retaining more activity over long distances, while weak enhancers rapidly lose activity. Additionally, we found that GATA1-bound enhancers are more resistant to distance-dependent loss of activity, suggesting that TF binding also modulates long-range function. Finally, testing long-range enhancer activities with three different promoters (HBE, HBG and GAPDH) revealed that long-range enhancer-promoter interactions rely on not only enhancer properties but also promoter responsiveness.

systems biology↗

bioRxiv2024

Two non-coding variants associated with isolated orofacial cleft promote binding of transcriptional repressors FOXE1 or ETS2 and reduce expression of IRF6

Oral facial cleft (OFC) is a multifactorial disorder that can present as a cleft lip with or without cleft palate (CL/P) or a cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) at the 1q32/IRF6 locus and many other loci where, like IRF6, the presumed OFC-relevant gene is expressed in embryonic oral epithelium. To identify the functional subset of SNPs at eight such loci we conducted a massively parallel reporter assay in a cell line derived from fetal oral epithelium, revealing SNPs with allele-specific effects on enhancer activity. We filtered these against chromatin-mark evidence of enhancers in relevant cell types or tissues, and then tested a subset in traditional reporter assays, yielding six candidates for functional SNPs in five loci (1q32/IRF6, 3q28/TP63, 6p24.3/TFAP2A, 20q12/MAFB, and 9q22.33/FOXE1). We further tested two SNPs near IRF6 and one near FOXE1 by engineering the genome of induced pluripotent stem cells, differentiating the cells into embryonic oral epithelium, and measuring expression of IRF6 or FOXE1 and binding of transcription factors; the results strongly supported their candidacy. Conditional analyses of a meta-analysis of GWAS suggest that the two functional SNPs near IRF6 account for the majority of risk for CL/P associated with variation at this locus. This study connects genetic variation associated with orofacial cleft to mechanisms of pathogenesis.

genetics↗

bioRxiv2024

Mutational scanning of CRX classifies clinical variants and reveals biochemical properties of the transcriptional effector domain

Cone-Rod Homeobox, encoded by CRX, is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Structurally, CRX comprises an ordered DNA-binding homeodomain and an intrinsically disordered transcriptional effector domain. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity. We calculated a statistical confidence for each activity score derived from multiple independent measurements of each variant marked by unique sequence barcodes, curating a high-confidence list of nearly 2,000 variants with significantly altered transcriptional activity compared to wild-type CRX. We evaluated the performance of the DMS assay as a clinical variant classification tool using gold-standard classified human variants from ClinVar, and determined that activity scores could be used to identify pathogenic variants with high specificity. That this performance could be achieved using a synthetic reporter assay in a foreign cell type, even for a highly cell type-specific TF like CRX, suggests that this approach shows promise for DMS of other TFs that function in cell types that are not easily accessible. Per-position average activity scores closely aligned to a predicted structure of the ordered homeodomain and demonstrated position-specific residue requirements. The intrinsically disordered transcriptional effector domain, by contrast, displayed a qualitatively different pattern of substitution effects, following compositional constraints without specific residue position requirements in the peptide chain. The observed compositional constraints of the effector domain were consistent with the acidic exposure model of transcriptional activation. Together, the results of the CRX DMS identify molecular features of the CRX effector domain and demonstrate clinical utility for variant classification.

genomics↗

bioRxiv2023

Active learning of enhancer and silencer regulatory grammar in photoreceptors

Cis-regulatory DNA elements (CRE) are composed of transcription factor (TF) binding sites that direct cell type-specific gene expression. Deep learning is an emerging strategy to model CREs, but the genome offers too few training examples to learn the complex interactions between TF binding sites that govern CRE activities. We address this limitation using active learning to iteratively train models that predict enhancer and silencer activities in the developing mouse retina. Active learning doubled the performance of models trained on genomic data, resulting in models that accurately distinguish between enhancers and silencers composed of the same TF binding sites. The ability of these models to discriminate between functionally non-equivalent binding sites establishes active learning as an effective strategy for modeling regulatory DNA. One sentence summaryModels of regulatory DNA learn critical interactions between transcription factor binding sites with active machine learning.

genomics↗

bioRxiv2023

Pathogenic variants in CRX have distinct cis-regulatory effects on enhancers and silencers in photoreceptors

Dozens of variants in the photoreceptor-specific transcription factor (TF) CRX are linked with human blinding diseases that vary in their severity and age of onset. It is unclear how different variants in this single TF alter its function in ways that lead to a range of phenotypes. We examined the effects of human disease-causing variants on CRX cis-regulatory function by deploying massively parallel reporter assays (MPRAs) in live mouse retinas carrying knock-ins of two variants, one in the DNA binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). The degree of reporter gene dysregulation caused by the variants corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, while p.E168d2 has stronger effects on silencers. Cis-regulatory elements (CREs) near cone photoreceptor genes are enriched for silencers that are de-repressed in the presence of p.E168d2. Chromatin environments of CRX-bound loci were partially predictive of episomal MPRA activity, and silencers were notably enriched among distal elements whose accessibility increases later in retinal development. We identified a set of potentially pleiotropic regulatory elements that convert from silencers to enhancers in retinas that lack a functional CRX effector domain. Our findings show that phenotypically distinct variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to misregulation of similar sets of enhancers, while having a qualitatively different impact on silencers.

genomics↗

bioRxiv2023

Transcription factor interactions explain the context-dependent activity of CRX binding sites

The effects of transcription factor binding sites (TFBSs) on the activity of a cis-regulatory element (CRE) depend on the local sequence context. In rod photoreceptors, binding sites for the transcription factor (TF) Cone-rod homeobox (CRX) occur in both enhancers and silencers, but the sequence context that determines whether CRX binding sites contribute to activation or repression of transcription is not understood. To investigate the context-dependent activity of CRX sites, we fit neural network-based models to the activities of synthetic CREs composed of photoreceptor TFBSs. The models revealed that CRX binding sites consistently make positive, independent contributions to CRE activity, while negative homotypic interactions between sites cause CREs composed of multiple CRX sites to function as silencers. The effects of negative homotypic interactions can be overcome by the presence of other TFBSs that either interact cooperatively with CRX sites or make independent positive contributions to activity. The context-dependent activity of CRX sites is thus determined by the balance between positive heterotypic interactions, independent contributions of TFBSs, and negative homotypic interactions. Our findings explain observed patterns of activity among genomic CRX-bound enhancers and silencers, and suggest that enhancers may require diverse TFBSs to overcome negative homotypic interactions between TFBSs.

systems biology↗

bioRxiv2022

Massively parallel characterization of insulator activity across the genome

Insulators are cis-regulatory sequences (CRSs) that can block enhancers from activating target promoters or act as barriers to block the spread of heterochromatin. Their name derives from their ability to insulate transgenes from genomic position effects, an important function in gene therapy and biotechnology applications that require high levels of sustained transgene expression. In theory, flanking transgenes with insulators protects them from position effects, but in practice, efforts to insulate transgenes meet with mixed success because the contextual requirements for insulator function in the genome are not well understood. A key question is whether insulators are modular elements that can function anywhere in the genome or whether they are adapted to function only in certain genomic locations. To distinguish between these two possibilities we developed MPIRE (Massively Parallel Integrated Regulatory Elements) and used it to measure the effects of three insulators (A2, cHS4, ALOXE3) and their mutants at thousands of locations across the genome. Our results show that each insulator functions in only a small number of genomic locations, and that insulator function depends on the sequence motifs that comprise each insulator. All three insulators can block enhancers in the genome, but specificity arises because each insulator blocks enhancers that are bound by different sets of transcription factors. All three insulators can block enhancers in the genome, but only ALOXE3 can act as a heterochromatin barrier. We conclude that insulator function is highly context dependent and that MPIRE is a robust and systematic method for revealing the context dependencies of insulators and other cis-regulatory elements across the genome.

genomics↗

bioRxiv2022

Transcription factor fluctuations underlie cell-to-cell variability in a signaling pathway response

Stochastic differences among clonal cells can initiate cell fate decisions in development or cause cell-to-cell differences in the responses to drugs or extracellular ligands. We hypothesize that some of this phenotypic variability is caused by stochastic fluctuations in the activities of transcription factors. We tested this hypothesis in NIH3T3-CG cells using the response to Hedgehog signaling as a model cellular response. Here we present evidence for the existence of distinct fast and slow responding substates of NIH3T3-CG cells. These two substates have distinct expression profiles, and fluctuations in the activity of the Prrx1 transcription factor (TF) underlie some of the differences in expression and responsiveness between fast and slow cells. We speculate that similar variability in other TFs may underlie other phenotypic differences among genetically identical cells.

genomics↗

bioRxiv2022

Effect of genomic and cellular environments on gene expression noise

Individual cells from isogenic populations often display large cell-to-cell differences in gene expression. This "noise" in expression derives from several sources, including the genomic and cellular environment in which a gene resides. Large-scale maps of genomic environments have revealed the effects of epigenetic modifications and transcription factor occupancy on mean expression levels, but leveraging such maps to explain expression noise will require new methods to assay how expression noise changes at locations across the genome. To address this gap, we present Single-cell Analysis of Reporter Gene Expression Noise and Transcriptome (SARGENT), a method that simultaneously measures the noisiness of reporter genes integrated throughout the genome and the global mRNA profiles of individual reporter-gene-containing cells. Using SARGENT, we performed the first comprehensive genome-wide survey of how genomic locations impact gene expression noise. We found that the mean and noise of expression correlate with different histone modifications. We quantified the intrinsic and extrinsic components of reporter gene noise and, using the associated mRNA profiles, assigned the extrinsic component to differences between the CD24+ "stem-like" sub-state and the more "differentiated" sub-state. SARGENT also reveals the effects of transgene integrations on endogenous gene expression, which will help guide the search for "safe-harbor" loci. Taken together, we show that SARGENT is a powerful tool to measure both the mean and noise of gene expression at locations across the genome, and that the data generated by SARGENT reveals important insights into the regulation of gene expression noise genome-wide.

genomics↗

bioRxiv2022

A quantitative metric of pioneer activity reveals that HNF4A has stronger in vivo pioneer activity than FOXA1

We and others have suggested that pioneer activity-a transcription factors (TFs) ability to bind and open inaccessible loci-is not a qualitative trait limited to a select class of pioneer TFs. We hypothesize that most TFs display pioneering activity that depends on the TF concentration and the motif content at their target loci. Here we present a quantitative measure of pioneer activity that captures the relative difference in a TFs ability to bind accessible versus inaccessible DNA. The metric is based on experiments that use CUT&Tag to measure binding of doxycycline (dox) inducible TFs. For each location across the genome we determine a "dox50," the concentration of dox required for a TF to reach half-maximal occupancy. We propose that the ratio of a TFs average dox50 between ATAC-seq labeled inaccessible and accessible binding sites, its {Delta}dox50, is a measure of its pioneer activity. We measured {Delta}dox50s for the endodermal TFs FOXA1 and HNF4A and show that HNF4A has a smaller {Delta}dox50 than FOXA1, suggesting that HNF4A has stronger pioneer activity than FOXA1. We further show that FOXA1 binding sites with more copies of its motif have a lower {Delta}dox50, suggesting that strong motif content may compensate for weak pioneer activity. The quantitative analysis of binding suggests different modes of binding for FOXA1, including an anti-cooperative mode of binding at certain accessible loci. Our results suggest that {Delta}dox50s, or other similar measures that assess the difference in TF affinity for inaccessible and accessible DNA, are reasonable measures of pioneer activity.

genomics↗

bioRxiv2022

Evolution of transposable element-derived enhancer activity

Many transposable elements (TEs) contain transcription factor binding sites and are implicated as potential regulatory elements. However, TEs are rarely functionally tested for regulatory activity, which in turn limits our understanding of how TE regulatory activity has evolved. We systematically tested the human LTR18A subfamily for regulatory activity using massively parallel reporter assay (MPRA) and found AP-1 and C/EBP-related binding motifs as drivers of enhancer activity. Functional analysis of evolutionarily reconstructed ancestral sequences revealed that LTR18A elements have generally lost regulatory activity over time through sequence changes, with the largest effects occurring due to mutations in the AP-1 and C/EBP motifs. We observed that the two motifs are conserved at higher rates than expected based on neutral evolution. Finally, we identified LTR18A elements as potential enhancers in the human genome, primarily in epithelial cells. Together, our results provide a model for the origin, evolution, and co-option of TE-derived regulatory elements.

genetics↗

bioRxiv2021

The origins and functional effects of postzygotic mutations throughout the human lifespan

Postzygotic mutations (PZMs) begin to accrue in the human genome immediately after fertilization, but how and when PZMs affect development and lifetime health remains unclear. To study the origins and functional consequences of PZMs, we generated a multi-tissue atlas of PZMs from 948 donors using the final major release of the Genotype-Tissue Expression (GTEx) project. Nearly half the variation in mutation burden among tissue samples can be explained by measured technical and biological effects, while 9% can be attributed to donor-specific effects. Through phylogenetic reconstruction of PZMs, we find that their type and predicted functional impact varies during prenatal development, across tissues, and the germ cell lifecycle. Remarkably, a class of prenatal mutations was predicted to be more deleterious than any other category of genetic variation investigated and under positive selection as strong as somatic mutations in cancers. In total, the data indicate that PZMs can contribute to phenotypic variation throughout the human lifespan, and, to better understand the relationship between genotype and phenotype, we must broaden the long-held assumption of one genome per individual to multiple, dynamic genomes per individual. One-Sentence SummaryThe predicted rates, functional effects and selection pressure of postzygotic mutations vary through the human lifecycle.

genomics↗

bioRxiv2021

A single-cell massively parallel reporter assay detects cell type specific cis-regulatory activity

Massively parallel reporter gene assays are key tools in regulatory genomics, but cannot be used to identify cell-type specific regulatory elements without performing assays serially across different cell types. To address this problem, we developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell-types simultaneously. We assayed a library of core promoters in a mixture of HEK293 and K562 cells and showed that scMPRA is a reproducible, highly parallel, single-cell reporter gene assay that detects cell-type specific cis-regulatory activity. We then measured a library of promoter variants across multiple cell types in ex vivo mouse retinas and showed that subtle genetic variants can produce cell-type specific effects on cis-regulatory activity. We anticipate that scMPRA will be widely applicable for studying the role of CRSs across diverse cell types.

genomics↗

bioRxiv2021

A Test of the Pioneer Factor Hypothesis

The Pioneer Factor Hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (nonPFs) that activate batteries of silent genes. We tested the PFH by expressing the endodermal PF FoxA1 and nonPF Hnf4a in K562 lymphoblast cells. While co-expression of FoxA1 and Hnf4a activated a burst of endoderm-specific gene expression, we found no evidence for functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and "pioneered" for each other, although FoxA1 required fewer copies of its motif to bind at inaccessible sites. A subset of targets required both TFs, but the mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results we propose an alternative to the PFH where "pioneer activity" depends not on the existence of discrete TF subclasses, but on TF binding affinity and genomic context.

genomics↗

bioRxiv2021

A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of genetic variants in vivo

The function of regulatory elements is highly dependent on the cellular context, and thus for understanding the function of elements associated with psychiatric diseases these would ideally be studied in neurons in a living brain. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3'UTR MPRA library with genomic elements containing variants from ASD patients, we developed a method to achieve reproducible measurements of element effects in vivo in a cell type-specific manner, using excitatory cortical neurons and striatal medium spiny neurons as test cases. This new technique should enable robust, functional annotation of genetic elements in the cellular contexts most relevant to psychiatric disease.

genetics↗

bioRxiv2021

Modular effects of gene promoters and chromatin environments on noise in gene expression.

Genetically identical cells growing in the same environment can have large differences in gene expression. Both locally acting cis-regulatory sequences (CRS) and the regional properties of chromosomal environments influence the noisiness of a genes expression. Whether or not local CRS and regional chromosomal environments act independently on noise, or whether they interact in complex ways is unknown. To address this question, we measured the expression mean and noise of reporter genes driven by different CRS at multiple chromosomal locations. While a strong power law relationship between mean expression and noise explains ~60% of noise for diverse promoters across chromosomal locations, modeling the residual mean-independent noise suggests that chromosomal environments have strong effects on expression noise by influencing how quickly genes transition from their inactive states to their active states and that the effects of local CRS and regional chromatin on noise are largely independent. Our results support a modular genome in which regional chromatin modifies the inherent relationship between the mean and noise of expression regardless of the identity of the promoter sequence.

systems biology↗