Biology subjects

Beer, M. A.

Publications and source records attributed to Beer, M. A..

At least 19 recordsLinked to original sources

bioRxiv2026

SARAF represses the mild hypothermia response through the regulation of JUN

The mild hypothermia response (MHR) is a conserved mammalian cytoprotective program activated upon exposure to mild hypothermia (32 degrees C) that contributes to the neuroprotective effects of therapeutic hypothermia following hypoxic injury. Although rapid changes in intracellular calcium occur upon cooling, the mechanisms linking calcium dynamics to the activation of core MHR factors such as SP1 and RBM3 remain incompletely defined. In this study, we used siRNA-mediated knockdown (KD) of candidate regulators in conjunction with novel mild hypothermia indicator (MHI) reporters to identify upstream modulators of MHR-associated transcription. We identify SARAF, a negative regulator of store-operated calcium entry (SOCE), as a repressor of both SP1 and RBM3 under normothermic conditions. SARAF depletion is associated with increased intracellular calcium release and enhanced SP1- and RBM3-linked transcriptional outputs. We identify JUN as an important downstream factor mediating SARAF depletion-dependent de-repression of the MHR and demonstrate that it undergoes activation rapidly upon cooling. Finally, SARAF depletion conferred significant cytoprotection against hypoxia-induced early apoptosis. Collectively, these findings establish SARAF as an upstream regulator of MHR-associated transcription and provide a functional link between cold-induced intracellular calcium dynamics and the induction of core MHR effectors.

molecular biology↗

bioRxiv2026

Reprogramming of neuronal genome function and phenotype by astrocytes

Heterotypic cell-cell interactions are critical to governing cellular physiology, disease progression, and responses to the environment and pharmacologic interventions. For example, neurons and astrocytes engage in intricate interactions that are essential for brain development and function1-3. However, the transformation of these extracellular signals into epigenomic regulation that governs cell function is poorly understood. Here, we report that weeks of co-culture between human induced pluripotent stem cell (hiPSC)-derived neurons and mouse cortical astrocytes extensively reprograms gene expression and the chromatin accessibility landscape in neurons, affecting thousands of genes and putative gene regulatory elements (REs), including many transcription factors (TFs). These genes are enriched for functions implicated in neuronal differentiation and maturation, and tend to be impacted in schizophrenia, and autosomal dominant Alzheimers disease. Through complementary CRISPR interference and activation screens, we recapitulated hundreds of astrocyte-induced transcriptional and chromatin remodeling events in mono-cultured neurons at both promoters and distal regulatory elements (REs) of TF genes. We discovered functional REs for [~]50 astrocyte-responsive TF genes, providing a map of gene regulatory network control. Astrocyte-responsive TF genes fall into groups that exert independent or counter-balancing transcriptional effects, highlighting the complex coordination of the neuronal response to astrocytes. Functional effects of specific TFs, including POU3F2 and TFAP2E, on neurite morphology and neuronal electrophysiology are consistent with transcriptional effects, demonstrating the capacity of direct epigenetic control to mimic heterotypic cellular signals. This work illuminates the regulation of neurodevelopment-and disease-relevant gene modules by neuron-astrocyte interactions, and provides a blueprint for applying modern functional genomics to uncover the links between cell microenvironment and epigenomic programming. HighlightsO_LINeuronal gene expression and chromatin accessibility landscape are profoundly remodeled by astrocytes over weeks of co-culture C_LIO_LIAstrocyte-responsive neuronal gene modules and neuron-responsive astrocytic gene modules are enriched for genes associated with schizophrenia and familial Alzheimers Disease C_LIO_LISingle-cell CRISPR interference and activation screens of astrocyte-responsive gene regulatory elements identified dozens of functional regulatory elements of TF genes in neurons C_LIO_LISingle-cell CRISPR interference and activation screens of >200 astrocyte-responsive TF genes uncovered discrete functional clusters that promote neuronal maturity or stemness C_LIO_LIAstrocyte-responsive TF genes reprogram neuronal electrophysiology and neurite morphology C_LI

genomics↗

bioRxiv2025

Perturb-seq reveals distinct responses to pluripotency regulator dosages underlying the control of self-renewal and differentiation

Precise regulation of transcription factor (TF) expression is critical for maintaining cell identity, but studies on how graded expression levels affect cellular phenotypes are limited. To address this gap, we employed human embryonic stem cells (hESCs) as a dynamic model to study gene dosage effects and systematically titrated key TFs NANOG and OCT4 expression using CRISPR interference (CRISPRi). We then profiled transcriptomic changes in hESCs under self-renewal and differentiation conditions using single-cell RNA-seq (scRNA-seq). Quantitative modeling of these Perturb-seq datasets uncovers distinct response patterns for different types of genes, including a striking non-monotonic response of lineage-specific genes during differentiation, indicating that mild perturbations of hESC TFs promote differentiation while strong perturbations compromise it. These discoveries suggest that fine-tuning the dosage of stem cell TFs can enhance differentiation efficiency and underscore the importance of characterizing TF function across a gradient of expression levels.

developmental biology↗

bioRxiv2024

An Expanded Registry of Candidate cis-Regulatory Elements for Studying Transcriptional Regulation

Mammalian genomes contain millions of regulatory elements that control the complex patterns of gene expression. Previously, The ENCODE consortium mapped biochemical signals across many cell types and tissues and integrated these data to develop a Registry of 0.9 million human and 300 thousand mouse candidate cis-Regulatory Elements (cCREs) annotated with potential functions1. We have expanded the Registry to include 2.35 million human and 927 thousand mouse cCREs, leveraging new ENCODE datasets and enhanced computational methods. This expanded Registry covers hundreds of unique cell and tissue types, providing a comprehensive understanding of gene regulation. Functional characterization data from assays like STARR-seq, MPRA, CRISPR perturbation, and transgenic mouse assays now cover over 90% of human cCREs, revealing complex regulatory functions. We identified thousands of novel silencer cCREs and demonstrated their dual enhancer/silencer roles in different cellular contexts. Integrating the Registry with other ENCODE annotations facilitates genetic variation interpretation and trait-associated gene identification, exemplified by discovering KLF1 as a novel causal gene for red blood cell traits. This expanded Registry is a valuable resource for studying the regulatory genome and its impact on health and disease.

genomics↗

bioRxiv2023

An encyclopedia of enhancer-gene regulatory interactions in the human genome

Identifying transcriptional enhancers and their target genes is essential for understanding gene regulation and the impact of human genetic variation on disease1-6. Here we create and evaluate a resource of >13 million enhancer-gene regulatory interactions across 352 cell types and tissues, by integrating predictive models, measurements of chromatin state and 3D contacts, and large-scale genetic perturbations generated by the ENCODE Consortium7. We first create a systematic benchmarking pipeline to compare predictive models, assembling a dataset of 10,411 element-gene pairs measured in CRISPR perturbation experiments, >30,000 fine-mapped eQTLs, and 569 fine-mapped GWAS variants linked to a likely causal gene. Using this framework, we develop a new predictive model, ENCODE-rE2G, that achieves state-of-the-art performance across multiple prediction tasks, demonstrating a strategy involving iterative perturbations and supervised machine learning to build increasingly accurate predictive models of enhancer regulation. Using the ENCODE-rE2G model, we build an encyclopedia of enhancer-gene regulatory interactions in the human genome, which reveals global properties of enhancer networks, identifies differences in the functions of genes that have more or less complex regulatory landscapes, and improves analyses to link noncoding variants to target genes and cell types for common, complex diseases. By interpreting the model, we find evidence that, beyond enhancer activity and 3D enhancer-promoter contacts, additional features guide enhancer-promoter communication including promoter class and enhancer-enhancer synergy. Altogether, these genome-wide maps of enhancer-gene regulatory interactions, benchmarking software, predictive models, and insights about enhancer function provide a valuable resource for future studies of gene regulation and human genetics.

genomics↗

bioRxiv2023

Gapped-kmer sequence modeling robustly identifies regulatory vocabularies and distal enhancers conserved between evolutionarily distant mammals

Gene regulatory elements drive many complex biological phenomena such as fetal development, and their mutations are linked to a multitude of common human diseases. The phenotypic impacts of regulatory variants are often tested using their conserved orthologous counterparts in model organisms such as mice. However, mapping human enhancers to conserved elements in mice remains a challenge, due to both rapid evolution of enhancers and limitations of current computational methods to detect conserved regulatory sequences. To improve upon existing computational methods and to better understand the sources of this apparent regulatory divergence, we comprehensively measured the evolutionary dynamics of distal enhancers across 45 matched human/mouse cell/tissue pairs from more than 1,000 DNase-seq experiments. Using this expansive dataset, we show that while cell-specific regulatory vocabulary is conserved, enhancers evolve more rapidly than other genomic elements such as promoters and CTCF binding sites. We observed surprisingly high levels of cell-specific variability in enhancer conservation rates, in part explainable by tissue specific transposable element activity. To improve orthologous enhancer mapping, we developed an improved genome alignment algorithm using gapped-kmer sequence features, and using the matched cell/tissue pairs, we show that this novel computational method, gkm-align, discovers 23,660 novel human/mouse conserved enhancers missed by standard alignment algorithms.

genomics↗

bioRxiv2023

The ENCODE4 long-read RNA-seq collection reveals distinct classes of transcript structure diversity

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3 end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of 200,000 full-length transcripts, 40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3 processing are deployed across human tissues, with nearly half of multitranscript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

genomics↗

bioRxiv2023

Parallel genome-scale CRISPR screens distinguish pluripotency and self-renewal

Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.

developmental biology↗

bioRxiv2023

Dynamic network-guided CRISPRi screen reveals CTCF loop-constrained nonlinear enhancer-gene regulatory activity in cell state transitions

Comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Our network modeling revealed that nonlinear enhancer-gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Utilizing hESC definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core endoderm lineage-specifying transcription factors, and many enhancers had strong effects mid-transition but weak effects post-transition. Through integrating enhancer activity measurements and three-dimensional enhancer-promoter interaction information, we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.

genetics↗

bioRxiv2023

Early transcriptional responses of human nasal epithelial cells to infection with Influenza A and SARS-CoV-2 virus differ and are influenced by physiological temperature

Influenza A (IAV) and SARS-CoV-2 (SCV2) viruses represent an ongoing threat to public health. Both viruses target the respiratory tract, which consists of a gradient of cell types, receptor expression, and temperature. Environmental temperature has been an un-derstudied contributor to infection susceptibility and understanding its impact on host responses to infection could help uncover new insights into severe disease risk factors. As the nasal passageways are the initial site of respiratory virus infection, in this study we investigated the effect of temperature on host responses in human nasal epithelial cells (hNECs) utilizing IAV and SCV2 in vitro infection models. We demonstrate that temperature affects SCV2, but not IAV, viral replicative fitness and that SCV2 infected cultures are slower to mount an infection-induced response, likely due to suppression by the virus. Additionally, we show that that temperature not only changes the basal transcriptomic landscape of epithelial cells, but that it also impacts the response to infection. The induction of interferon and other innate immune responses were not drastically affected by temperature, suggesting that while the baseline antiviral response at different temperatures remains consistent, there may be metabolic or signaling changes that affect how well the cultures are able to adapt to new pressures such as infection. Finally, we show that hNECs respond differently to IAV and SCV2 infection in ways that give insight into how the virus is able to manipulate the cell to allow for replication and release. Taken together, these data give new insight into the innate immune response to respiratory infections and can assist in identifying new treatment strategies for respiratory infections.

microbiology↗

bioRxiv2022

Multi-center integrated analysis of non-coding CRISPR screens

The ENCODE Consortiums efforts to annotate non-coding, cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes which play a major role in health and disease. Pooled, non-coding CRISPR screens are a promising approach for systematically investigating gene regulatory mechanisms. Here, the ENCODE Functional Characterization Centers report 109 screens comprising 346,970 individual perturbations across 13.3Mb of the genome, using a variety of methods, readouts, and statistical analyses. Across 332 functionally confirmed CRE-gene links, we identify principles for screening endogenous, non-coding elements for causal regulatory mechanisms. Nearly all CREs show strong evidence of open chromatin, and targeting accessibility peak summits is a critical component of our proposed sgRNA design rules. We provide experimental guidelines to accurately detect CREs with variable, often low, transcriptional effects. We discover a previously undescribed DNA strand-bias for CRISPRi in transcribed regions with implications for screen design and analysis. Benchmarking five screen analysis tools, we find CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity sgRNAs. Together, we provide an accessible data resource, predesigned sgRNAs targeting 3,275,697 ENCODE SCREEN candidate CREs, and screening guidelines to accelerate functional characterization of the non-coding genome.

genomics↗

bioRxiv2022

Stronger sexual dimorphism in fruit flies may be favored when congeners are present and females actively search for mates

Why are some species sexually dimorphic while other closely related species are not? When the degree of sexual dimorphism varies within a genus, an integrative phylogenetic approach may help reveal underlying patterns favoring the evolution of dimorphism. While all female flies in genus Strauzia - a genus of true fruit flies - share a multiply-banded wing pattern, males of four species have patterns wherein bands have "coalesced" into a continuous dark streak across much of the wing. We find that the origin of coalesced male wing patterns and pronounced differences in male wing shape correlate with the presumed origin of host plant sharing in this genus. A survey of North American Tephritidae finds just three other genera with specialist species that share host plants. Each has one or more congeners with wing patterns unusual for that genus, and just one genus, Eutreta, has those unusual wing patterns only in the male sex. Eutreta is also the only other genus among this subset wherein, like Strauzia, males hold territories while females search for mates. Sharing the same hosts may result in reproductive character displacement, and when coupled with a biology wherein females actively search for males, may specifically favor sexually dimorphic wing patterns.

evolutionary biology↗

bioRxiv2021

Sequence-based correction of barcode bias in massively parallel reporter assays

Massively parallel reporter assays (MPRA) are a high-throughput method for evaluating in vitro activities of thousands of candidate cis-regulatory elements (CREs). In these assays, candidate sequences are cloned upstream or downstream of a reporter gene tagged by unique DNA sequences. However, tag sequences may themselves affect reporter gene expression and lead to major potential biases in the measured cis-regulatory activity. Here, we present a sequence-based method for correcting tag sequence-specific effects and demonstrate that our method can significantly reduce this source of variation, and improve the identification of functional regulatory variants by MPRAs. We also show that our model captures sequence features associated with post-transcriptional regulation of mRNA. Thus, this new method helps to not only improve detection of regulatory signals in MPRA experiments but also to design better MPRA protocols.

bioinformatics↗

bioRxiv2020

Loop extrusion model predicts CTCF interaction specificity

Three-dimensional chromatin looping interactions play an important role in constraining enhancer-promoter interactions and mediating transcriptional gene regulation. CTCF is thought to play a critical role in the formation of these loops, but the specificity of which CTCF binding events form loops and which do not is difficult to predict. Loops often have convergent CTCF binding site motif orientation, but this constraint alone is only weakly predictive of genome-wide interaction data. Here we present an easily interpretable and simple mathematical model of CTCF mediated loop formation which is consistent with Cohesin extrusion and can predict ChIA-PET CTCF looping interaction measurements with high accuracy. Competition between overlapping loops is a critical determinant of loop specificity. We show that this model is consistent with observed chromatin interaction frequency changes induced by CTCF binding site deletion, inversion, and mutation, and is also consistent with observed constraints on validated enhancer-promoter interactions.

genomics↗

bioRxiv2020

Host shifting and host sharing in a genus of specialist flies diversifying alongside their sunflower hosts

Congeneric parasites are unlikely to specialize on the same tissues of the same host species, likely because of strong multifarious selection against niche overlap. Exceptions where multiple congeneric species overlap on the same tissues may therefore reveal important insights into the ecological factors underlying the origins and maintenance of diversity. Larvae of sunflower maggot flies in genus Strauzia feed on the pith of plants in the family Asteraceae. Although Strauzia tend to be host specialists, some species overlap in their host use. To resolve the origins of host sharing among these specialist flies, we used reduced representation genomic sequencing to infer the first multi-locus phylogeny of genus Strauzia. Our results show that Helianthus tuberosus and Helianthus grosseserratus each host three different fly species, and that the flies co-occurring on a host are not one anothers closest relatives. Though this pattern implies that host sharing is most likely the result of host shifts, these may not be host shifts in the conventional sense of an insect moving onto an entirely new plant. Many hosts of Strauzia belong to a young (1-2 MYA) clade of perennial sunflowers noted for their frequent introgression and hybrid speciation events. In at least one case, flies may have converged upon a host after their respective ancestral host plants hybridized to form a new sunflower species (H. tuberosus). Broadly, we suggest that rapid and recent adaptive introgression and speciation in this group of plants may have instigated the diversification of their phytophagous fly associates, including the convergence of >1 species onto the same shared host plants.

evolutionary biology↗

bioRxiv2018

Local epigenomic state cannot discriminate interacting and non-interacting enhancer-promoter pairs with high accuracy

We report an overfitting issue in recent machine learning formulations of the enhancer-promoter interaction problem arising from the fact that many enhancer-promoter pairs share features. Cross- fold validation schemes which do not correctly separate these feature sharing enhancer-promoter pairs into one test set report high accuracy, which is actually due to overfitting. Cross-fold validation schemes which properly segregate pairs with shared features show markedly reduced ability to predict enhancer-promoter interactions from epigenomic state. Parameter scans with multiple models indicate that local epigenomic features of individual pairs of enhancers and promoters cannot distinguish those pairs that interact from those which do with high accuracy, suggesting that additional information is required to predict enhancer-promoter interactions.

genomics↗

bioRxiv2018

Parkinson-associated SNCA enhancer variants revealed by open chromatin in mouse dopamine neurons

The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD) and modulation of risk by common variation in PD has been well established through GWAS. Anticipating that a fraction of PD-associated genetic variation mediates their effects within this neuronal population, we acquired open chromatin signatures of purified embryonic mouse MB DA neurons. Correlation with >2,300 putative enhancers assayed in mice reveals enrichment for MB cis-regulatory elements (CRE), data reinforced by transgenic analyses of six additional sequences in zebrafish and mice. One CRE, within intron 4 of the familial PD gene SNCA, directs reporter expression in catecholaminergic neurons of transgenic mice and zebrafish. Sequencing of this CRE in 986 PD patients and 992 controls reveals two common variants associated with elevated PD risk. To assess potential mechanisms of action, we screened >20,000 DNA interacting proteins and identify a subset whose binding is impacted by these enhancer variants. Additional genotyping across the SNCA locus identifies a single PD-associated haplotype, containing the minor alleles of both of the aforementioned PD-risk variants. Our work posits a model for how common variation at SNCA may modulate PD risk and highlights the value of cell context-dependent guided searches for functional non-coding variation.

genetics↗

bioRxiv2018

Quantifying the unquantifiable: why Hymenoptera -- not Coleoptera -- is the most speciose animal order

BackgroundWe challenge the oft-repeated claim that the beetles (Coleoptera) are the most species-rich order of animals. Instead, we assert that another order of insects, the Hymenoptera, are more speciose, due in large part to the massively diverse but relatively poorly known parasitoid wasps. The idea that the beetles have more species than other orders is primarily based on their respective collection histories and the relative availability of taxonomic resources, which both disfavor parasitoid wasps. Though it is unreasonable to directly compare numbers of described species in each order, the ecology of parasitic wasps - specifically, their intimate interactions with their hosts - allows for estimation of relative richness. We present a simple logical model that shows how the specialization of many parasitic wasps on their hosts suggests few scenarios in which there would be more beetle species than parasitic wasp species. We couple this model with an accounting of what we call the \"genus-specific parasitoid-host ratio\" from four well-studied genera of insect hosts, a metric by which to generate extremely conservative estimates of the average number of parasitic wasp species attacking a given beetle or other insect host species. Synthesis of our model with data from real host systems suggests that the Hymenoptera may have 2.5 - 3.2x more species than the Coleoptera. While there are more described species of beetles than all other animals, the Hymenoptera are almost certainly the larger order.

zoology↗