Biology subjects

Green, E. M.

Publications and source records attributed to Green, E. M..

10 recordsLinked to original sources

bioRxiv2026

TDP-43 dysfunction induces cryptic circular RNAs in ALS/FTD

Nuclear depletion of TDP-43 is a defining pathological feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leading to widespread RNA misprocessing, including the formation of cryptic exons. Here, we identified TDP-43 as a regulator of circular RNA (circRNA) biogenesis in multiple human neuronal cell models, and showed that its dysfunction induces the de novo formation of cryptic circular RNAs (c-circRNAs). Analysis of post-mortem brain transcriptomic data identified a subset of c-circRNAs which are specific for ALS and FTD cases with TDP-43 pathology. Further, we developed highly sensitive rolling-circle amplification-based circRNA detection assays that allow to distinguish TDP-43 pathology in human CNS tissues with a 0.99 AUC. We found that c-circRNAs can co-occur with cryptic linear splicing events, uncovering complex RNA misprocessing hotspots that induce loss of disease-relevant proteins, including RPTOR and EHMT1. Notably, one of these c-circRNAs originates from UNC13A, a gene whose cryptic exon has previously been linked to one of the major GWAS hits in ALS/FTD and that is being pursued as a therapeutic target through splice-switching ASOs. We showed that c-circUNC13A is co-regulated with the linear cryptic transcript and suppression of UNC13A cryptic exon results in c-circUNC13A reduction in cultured neurons and in vivo, highlighting its potential as a target engagement biomarker for emerging UNC13A-directed therapies. Overall, this work identifies a novel molecular mechanism for TDP-43 dysfunction, opening novel avenues for understanding disease pathogenesis and developing much needed pathology biomarkers.

neuroscience↗

bioRxiv2026

Alterations to the yeast chromatin landscape during hypoxia

When cells encounter low oxygen conditions, signaling events target key transcription factors to reprogram gene expression to promote cell survival and adaptation under this challenging environmental stress. The substantial metabolic changes that occur in hypoxia also drive changes to the chromatin environment due to altered cofactor availability and direct oxygen sensitivity of chromatin-modifying enzymes, among other factors. Given the links between hypoxic cellular environments and numerous pathophysiological processes, including tumor progression and metastasis, cardiovascular disorders, and aberrant development, it is critical to understand the chromatin states and chromatin-based regulatory mechanisms associated with cellular responses to hypoxia. Here, we used the model eukaryote Saccharomyces cerevisiae to interrogate the requirement for key transcription factors and chromatin regulators in survival during hypoxia and we assessed the distribution of major histone modifications associated with the response to stress, H3K9 acetylation and H3K4 methylation, throughout the genome during hypoxia. Our results show that only a small number of chromatin modifiers in yeast contribute to survival in hypoxia; however, there are substantial changes to the abundance and distribution of both H3K9ac and H3K4me3 during hypoxic growth. Specifically, H3K9ac levels are reduced throughout the genome though still maintain association with transcriptionally active genes. H3K4me3 has increased abundance genome-wide and shows a wider distribution with greater abundance in coding sequences particularly of genes upregulated in hypoxic conditions. Altogether, our data demonstrate substantial changes to the chromatin landscape in hypoxic conditions in the budding yeast model, providing key insights into physiological chromatin changes caused by limiting oxygen in the environment.

molecular biology↗

bioRxiv2025

Cryptic splicing in synaptic and membrane excitability genes links TDP-43 loss to neuronal dysfunction

TDP-43 pathology is a defining pathological hallmark of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major feature of TDP-43 pathology is its nuclear depletion, leading to the aberrant inclusion of cryptic exons during RNA splicing. STMN2 and UNC13A have emerged as prominent TDP-43 splicing targets, but the broader impact of TDP-43-dependent cryptic splicing on neuronal function remains unclear. Here, we report new TDP-43 splicing targets critical for membrane excitability and synaptic function, including KALRN, RAP1GAP, SYT7 and KCNQ2. Using human stem cell-derived neurons, we show that TDP-43 reduction induces cryptic splicing and downregulation of these genes, resulting in impaired excitability and synaptic transmission. In postmortem brains from patients with FTD, these cryptic splicing events occur selectively in neurons with TDP-43 pathology. Importantly, suppressing individual cryptic splicing events using antisense oligonucleotides partially restores neuronal function, and combined targeting almost fully rescues the synaptic deficit caused by TDP-43 loss. Together, our findings provide evidence that cryptic splicing in these synaptic and membrane excitability genes is not only a downstream marker but instead a direct driver of neuronal dysfunction, establishing a mechanistic link between TDP-43 pathology and neurodegeneration in ALS and FTD.

neuroscience↗

bioRxiv2025

Defined human tri-lineage brain microtissues

Microglia are the immune cells of the central nervous system and are thought to be key players in both physiological and disease conditions. Several microglial features are poorly conserved between mice and human, such as the function of the neurodegeneration-associated immune receptor Trem2. Induced pluripotent stem cell (iPSC)-derived microglia offer a powerful opportunity to generate and study human microglia. However, human iPSC-derived microglia often exhibit activated phenotypes in vitro, and assessing their impact on other brain cell types remains challenging due to limitations in current co-culture systems. Here, we developed fully defined brain microtissues, composed of human iPSC-derived neurons, astrocytes, and microglia, co-cultured in 2D or 3D formats. Our microtissues are stable and self-sufficient over time, requiring no exogenous cytokines or growth factors. All three cell types exhibit morphologies characteristic of their in vivo environment and show functional properties. Co-cultured microglia develop more homeostatic phenotypes compared to microglia exposed to exogenous cytokines. Hence, these tri-cultures provide a unique approach to investigate cell-cell interactions between brain cell types. We found that astrocytes and not neurons are sufficient for microglial survival and maturation, and that astrocyte-derived M-CSF is essential for microglial survival. Single-cell and single-nucleus RNA sequencing analyses nominated a network of reciprocal communication between cell types. Brain microtissues faithfully recapitulated pathogenic -synuclein seeding and aggregation, suggesting their usefulness as human cell models to study not only normal but also pathological cell biological processes.

neuroscience↗

bioRxiv2024

The External Microbiome Communicates with the Developing Zebrafish (Danio rerio) Embryo Through the Protective Chorion and Influences Developmental Trajectory

The microbiome has a significant influence on host physiological processes including energy metabolism and neurobiology. However, current knowledge is largely limited to post-embryonic development, highlighting a notable gap in host-microbe communication during embryonic development, particularly in oviparous organisms. This is because the developing embryo is protected from the external environment by the chorion and typically considered to be sterile. We hypothesized the external microbiome influences embryonic development in oviparous organisms despite lack of physical contact with microbes, shaping host physiology beyond embryogenesis. To test this interaction, we utilized zebrafish (Danio rerio) reared germ-free or conventionalized with microbes at different times during embryonic development (6 and 24 hours post fertilization) to examine changes in transcriptomics, proteomics, and physiology at 32 hours post-fertilization. In contrast to the prevailing notion, we reveal a significant role of the external aquatic microbial community in regulating embryonic transcript and protein abundance associated with critical developmental processes including energy metabolism and neurodevelopment. Furthermore, we demonstrate the external microbial community drives differential expression of genes involved in cytochrome P450 directed xenobiotic metabolism and associated bioenergetic and behavioral responses following exposure to a CYP1A activator during embryogenesis. These findings reveal embryonic development is an integration of host genetic blueprints and external microbial cues, enhancing knowledge of fundamental developmental processes influenced by embryo-microbe interactions that shape developmental susceptibility to environmental stressors. Significance StatementHost-microbiome interactions play a crucial role in shaping vertebrate physiology. However, the impact of these interactions during embryonic development remains poorly understood which has limited our evaluation of environmental drivers of developmental disorders and disease. Here, we provide evidence that the external microbiome indirectly communicates with the developing zebrafish (Danio rerio) embryo through the chorion, influencing physiological processes including bioenergetics, neurodevelopment, and xenobiotic responses. These findings signify a critical role of the external microbiome during the early stages of embryonic development and may inform research addressing the effects of the maternal microbiome on human embryonic and fetal development, particularly in the context of developmental origins of disease and prenatal chemical exposures.

developmental biology↗

bioRxiv2022

Set1 regulates telomere function via H3K4 methylation-dependent and independent pathways and calibrates the abundance of telomere maintenance factors

Set1 is an H3K4 methyltransferase which comprises the catalytic subunit of the COMPASS complex and has been implicated in transcription, DNA repair, cell cycle control, and numerous other genomic functions. Set1 also promotes proper telomere maintenance, as cells lacking Set1 have short telomeres and disrupted subtelomeric gene repression; however, the precise role for Set1 in these processes has not been fully defined. In this study, we have tested mutants of Set1 and the COMPASS complex which differentially alter H3K4 methylation status and attempt to separate catalytic and non-catalytic functions of Set1. Our data reveal that Set1-dependent subtelomeric gene repression relies on its catalytic activity towards H3K4, whereas telomere length is regulated by Set1 catalytic activity but likely independent of the H3K4 substrate. Furthermore, we uncover a role for Set1 in calibrating the abundance of critical telomere maintenance proteins, including components of the telomerase holoenzyme and members of the telomere capping CST (Cdc13-Stn1-Ten1) complex, through both transcriptional and post-transcriptional pathways. Altogether, our data provide new insights into the H3K4 methylation-dependent and independent roles for Set1 in telomere maintenance in yeast and shed light on possible roles for Set1-related methyltransferases in other systems.

molecular biology↗

bioRxiv2021

Set4 coordinates the activity of histone deacetylases and regulates stress-responsive gene expression within subtelomeric regions in yeast

The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts, although they appear to lack methyltransferase activity. The molecular functions of Set4 are relatively unexplored, likely due to its low abundance in standard growth conditions. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status has revealed a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, and the loss of Set4 decreases cell fitness and cell wall integrity in hypoxia. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for yeast Set4 in regulating repressive, heterochromatin-like environments.

genetics↗

bioRxiv2021

TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

A hallmark pathological feature of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function.

neuroscience↗

bioRxiv2018

Mavacamten stabilizes a folded-back sequestered super-relaxed state of β-cardiac myosin

SummaryMutations in {beta}-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity and ATPase activityof myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative modelpositsthat mutations in myosin affect the stability ofa sequestered, super-relaxed state (SRX) of the proteinwith very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here, using a combination of biochemical and structural approaches, we show that purified myosin enters aSRX thatcorresponds to a folded-back conformation, which in muscle fibersresults insequestration of heads around the thick filament backbone. Mutations that cause HCM destabilize this state, while the small molecule mavacamtenpromotes it. These findings provide a biochemical and structural link between the genetics and physiology ofcardiomyopathywith implications for therapeutic strategies.

biochemistry↗

bioRxiv2017

Extending Chemical Perturbations Of The Ubiquitin Fitness Landscape In A Classroom Setting

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.\n\nBuilds onMavor D, Barlow KA, Thompson S, Barad BA, Bonny AR, Cario CL, Gaskins G, Liu Z, Deming L, Axen SD, Caceres E, Chen W, Cuesta A, Gate R, Green EM, Hulce KR, Ji W, Kenner LR, Mensa B, Morinishi LS, Moss SM, Mravic M, Muir RK, Niekamp S, Nnadi CI, Palovcak E, Poss EM, Ross TD, Salcedo E, See S, Subramaniam M, Wong AW, Li J, Thorn KS, Conchuir SO, Roscoe BP, Chow ED, DeRisi JL, Kortemme T, Bolon DN, Fraser JS. Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting. eLife. 2016.\n\nImpact StatementWe organized a project-based course that used deep mutational scanning in multiple chemical conditions to resolve the inconsistencies between tolerance to mutations in laboratory conditions and sequence conservation over evolutionary timescales.

bioinformatics↗