Biology subjects

Schubert, D.

Publications and source records attributed to Schubert, D..

At least 19 recordsLinked to original sources

bioRxiv2026

PWO1 and TRB proteins coordinate chromatin regulation to prevent premature differentiation and ectopic lignin deposition in Arabidopsis

The Arabidopsis PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 (PWO1) and Telomere Repeat-Binding Proteins 1-3 (TRB1-3, TRBs) associate with distinct and shared protein complexes involved in epigenetic regulation, yet their cooperative roles in chromatin control and plant development remain largely unexplored. Here, we show that the interaction between PWO1 and TRBs is evolutionarily conserved. Both PWO1 and TRBs associate with plant telomeres, interact at these regions, and are co-enriched at subsets of interspersed telo-box motifs across regulatory regions genome-wide. TRBs facilitate PWO1 binding at shared genomic regions, including telo-box motifs. PWO1 and TRBs share a substantial number of genomic targets and preferentially bind chromatin regions associated with transcriptionally active states, whereas TRBs alone associate with repressive marks at thousands of loci. Genetic analyses show that the pwo1 trb1 trb3 triple mutant displays severe developmental defects, including main stem arrest and early maturation associated with aberrant lignin deposition in interfascicular tissues. In the triple mutant, key enzymes in the lignin biosynthesis pathway are upregulated, indicating that PWO1, TRB1, and TRB3 cooperatively regulate secondary cell wall formation. Together, our findings provide new insights into how PWO1 and TRBs cooperate to regulate chromatin states and orchestrate plant development, highlighting their central role in controlling gene expression programs. Significance statementThis study shows that PWO1 and TRB proteins co-occupy telomeres, including interspersed telo-box motifs, to regulate chromatin organization and plant development, particularly ectopic lignin deposition. Our findings reveal how these nuclear protein factors coordinate epigenetic states in Arabidopsis thaliana, providing a framework for understanding the control of developmental programs. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/740627v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a3d544org.highwire.dtl.DTLVardef@1067082org.highwire.dtl.DTLVardef@1c49163org.highwire.dtl.DTLVardef@252171_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Evolutionarily conserved PWO-TRB interactions and their shared roles in chromatin regulation and plant development. Created with BioRender.com. C_FIG

plant biology↗

bioRxiv2026

Epigenetic regulation of a heat-trainable sHSP locus controls thermomemory in a unicellular alga

A powerful way to enhance heat tolerance is to prime organisms with a moderate heat treatment to establish a molecular stress memory permitting the survival of the organism when exposed to subsequent heat shocks. While this has been extensively studied in multicellular organisms, we demonstrate that the unicellular red alga Cyanidioschyzon merolae exhibits heat stress memory. We show that, similarly to more complex organisms, thermomemory in this alga is underpinned by transcriptomic reprogramming, with the chloroplast emerging as the main site of gene trainability. Additionally, we find a conserved small heat shock protein (sHSP)-encoding locus in the nuclear genome to be heat-trainable, likely by histone depletion and sustained removal of the repressive mark histone H3 Lysine 27 trimethylation (H3K27me3). Of C. merolaes two sHSPs, only the nuclear-localizing CmsHSP2 is necessary for proper HS memory establishment. Finally, we reveal a role for the H3K27me3-transferase CmE(z) (Enhancer of zeste) in heat stress memory which shapes the transcriptome to recurring heat exposures, beyond regulating the trainable sHSP locus. Overall, our work provides a molecular framework for the regulation of heat stress memory in a unicellular eukaryote.

genetics↗

bioRxiv2026

MEA-LINK identifies the CCL4-CCR5 axis in neuronal hyperactivity control by human microglia

Microglia, the resident immune cells of the brain, act along a spectrum to maintain CNS homeostasis, respond to perturbations, and control neuronal activity. Disentangling the molecular mechanisms of human microglia-neuron crosstalk remains challenging due to the context-dependent, dynamic nature of their interaction. We introduce MEA-LINK, a systems-approach leveraging natural variation to screen for immune modulators of neuronal activity. This multi-modal platform integrates human induced pluripotent stem cell (hiPSC) technology with micro-electrode array (MEA) recordings and proteomic analyses of secreted immune factors, allowing for longitudinal samples and correlations across modalities. We applied MEA-LINK to explore microglia-neuron interactions during development and hyperactivity challenges. We show that human microglia accelerate neuronal network development and rescue hyperactive network phenotypes. Linking the secretome adaptations to neuronal network activity variations, we identified CCL4 as a top candidate in microglia-mediated hyperactivity control. Then, we functionally validated the context-dependent role of microglial CCL4 to neuronal CCR5 signaling in human neuronal networks. Our findings support a neuron-specific function of chemokines and their receptors in the brain and provide a new perspective for immune signaling in neuronal hyperactivity control. The MEA-LINK platform thus offers a foundation for comprehensive, systematic studies of human microglia-neuron interactions. HighlightsO_LIMEA-LINK integrates micro-electrode array recordings with proteomics of longitudinal samples to identify immune modulators of neuronal activity. C_LIO_LIHuman microglia rescue neuronal hyperactivity induced by pharmacological and genetic challenges. C_LIO_LIMicroglial CCL4 dampens neuronal activity via CCR5 signaling in a context-dependent manner. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/703799v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@18e44f7org.highwire.dtl.DTLVardef@151c450org.highwire.dtl.DTLVardef@12f7b89org.highwire.dtl.DTLVardef@57651b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Long-term co-maturation of stem cell-derived microglia and neuronal networks: an optimized platform to assess human microglial contribution to neuronal function

Microglia-neuron interactions play a central role in a variety of central nervous system disorders. Technologies using human induced pluripotent stem cells (hiPSCs) have been developed to model human brain cells with the goal to understand their function. To effectively study neuro-immune crosstalk and investigate microglial contributions to neuronal network development and function, both microglia and neurons should co-mature allowing for long-term interactions throughout their differentiation. Here, we present a co-maturation protocol that robustly generates glutamatergic neuronal networks containing human iPSC-derived microglia. We validated the long-term co-cultures using single-cell transcriptomics, imaging and neuronal activity readouts. We show that astrocytes were required for long-term survival of microglia and for their integration into neuronal networks. Our co-maturation approach induced the typical ramified microglia morphology and characteristic microglia-neuron interactions. Homeostatic markers like P2RY12 and TMEM119 and neuronal remodeling associated genes were upregulated compared to microglia monocultures, highlighting the necessity of the environment to generate and maintain the context-dependent microglia signature in vitro. In this manuscript, we include the full optimization process of our co-maturation approach, a comprehensive description of the protocol, practical guidelines and troubleshooting tips. Our co-maturation model provides a powerful tool to assess the role of human microglia in modulating neuronal function and development in health and disease. HighlightsO_LIptimized human iPSC differentiation protocol that allows for co-maturation of microglia and Neurogenin2-induced neuronal networks. C_LIO_LIare required for long-term survival and integration of microglia into neuronal networks. C_LIO_LICo-maturation approach enables characteristic neuron-microglia interactions and induces signature morphology, transcriptome and proteins of human microglia. C_LI

neuroscience↗

bioRxiv2025

Whole-brain clearing reveals region- and cell type-specific imbalances in inhibitory neurons in a mouse model for Kleefstra Syndrome

GABAergic inhibition is essential for balanced brain function and is frequently disrupted in neurodevelopmental disorders such as autism spectrum disorder (ASD). The inhibition is generated by a diverse population of GABAergic interneurons, whose composition and density differs throughout the brain. Here, we applied an unbiased whole-brain clearing and light-sheet imaging approach to systematically map the distribution of the three major GABAergic neuron classes - Parvalbumin-positive (PV), Somatostatin-positive (SST), and Vasoactive Intestinal Polypeptide-positive (VIP) cells - across the mouse brain in a model of Kleefstra Syndrome (Ehmt1+/-), a monogenic intellectual disability disorder with a strong ASD component. Analyzing 895 brain regions we identified widespread, cell type- and region-specific alterations in GABAergic classes. Notably, we observed increased VIP neuron density in the Ehmt1+/- cortex and decreased SST neuron density in sensory cortical and subcortical regions. In the basolateral amygdala (BLA), PV neurons exhibited precocious maturation already at the juvenile stage, which persisted into adulthood and was associated with enhanced inhibitory input onto BLA principal neurons. We here demonstrate that Ehmt1 haploinsufficiency results in region- and cell-type specific changes throughout the brain. These results underscore the value of whole-brain, high-resolution mapping approaches in uncovering previously unrecognized patterns of neural vulnerability in neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

PWO proteins are associated with PRC2 since their emergence in vascular plants

PWWP-Domain Interactor of Polycombs 1 (PWO1), also known as PWWP1, interacts with the catalytic subunits of the Polycomb Repressive Complex 2 (PRC2) and, together with PWO2/3 proteins, plays a critical role in the development of Arabidopsis thaliana (At). PWOs are unique to plants and impact chromatin structure by enabling crosstalk between active and repressive epigenetic marks through mechanisms that are not yet fully understood. We aimed to understand the evolution of PWO proteins and whether their interaction with PRC2 has been conserved through evolution. Our study reveals that PWO proteins are present in vascular plants, but absent in bryophytes and green algae. The ancestral clade of PWO proteins includes the Selaginella moellendorffii (Sm) PWO orthologs SmPWOa and SmPWOb. Transient expression assays showed that both AtPWO1 and SmPWOa form nuclear speckles where they tether At, Sm, but also Physcomitrium patens (Pp) PRC2 catalytic subunits, despite the absence of PWO proteins in Pp. The PWO-PRC2 interactions were confirmed by protein-protein analyses. A newly identified evolutionarily conserved short C-terminal alpha-helix (c-motif) in PWO proteins contributes to an interaction interface for PWO-PRC2 binding. SmPWOs partially rescue the pwo1;pwo2 mutant phenotype in Arabidopsis, highlighting the functional conservation of PWOs in vascular plants.

plant biology↗

bioRxiv2024

CACNA1A haploinsufficiency leads to reduced synaptic function and increased intrinsic excitability

Haploinsufficiency of the CACNA1A gene, encoding the pore-forming 1 subunit of P/Q-type voltage-gated calcium channels, is associated with a clinically variable phenotype ranging from cerebellar ataxia, to neurodevelopmental syndromes with epilepsy and intellectual disability. To understand the pathological mechanisms of CACNA1A loss-of-function variants, we characterized a human neuronal model for CACNA1A haploinsufficiency, by differentiating isogenic induced pluripotent stem cell lines into glutamatergic neurons, and investigated the effect of CACNA1A haploinsufficiency on mature neuronal networks through a combination of electrophysiology, gene expression analysis, and in silico modeling. We observed an altered network synchronization in CACNA1A+/- networks alongside synaptic deficits, notably marked by an augmented contribution of GluA2-lacking AMPA receptors. Intriguingly, these synaptic perturbations coexisted with increased non-synaptically driven activity, as characterized by inhibition of NMDA and AMPA receptors on micro-electrode arrays. Single-cell electrophysiology and gene expression analysis corroborated this increased intrinsic excitability through reduced potassium channel function and expression. Moreover, we observed partial mitigation of the CACNA1A+/- network phenotype by 4-aminopyridine, a therapeutic intervention for episodic ataxia type 2. In summary, our study pioneers the characterization of a human induced pluripotent stem cell-derived neuronal model for CACNA1A haploinsufficiency, and has unveiled novel mechanistic insights. Beyond showcasing synaptic deficits, this neuronal model exhibited increased intrinsic excitability mediated by diminished potassium channel function, underscoring its potential as a therapeutic discovery platform with predictive validity.

neuroscience↗

bioRxiv2024

Cold stress induces a rapid redistribution of the antagonistic marks H3K4me3 and H3K27me3 in Arabidopsis thaliana

When exposed to low temperatures, plants undergo a drastic reprogramming of their transcriptome in order to adapt to their new environmental conditions, which primes them for potential freezing temperatures. While the involvement of transcription factors in this process, termed cold acclimation, has been deeply investigated, the potential contribution of chromatin regulation remains largely unclear. A large proportion of cold-inducible genes carries the repressive mark histone 3 lysine 27 trimethylation (H3K27me3), which has been hypothesized as maintaining them in a silenced state in the absence of stress, but which would need to be removed or counteracted upon stress perception. However, the fate of H3K27me3 during cold exposure has not been studied genome-wide. In this study, we offer an epigenome profiling of H3K27me3 and its antagonistic active mark H3K4me3 during short-term cold exposure. Both chromatin marks undergo rapid redistribution upon cold exposure, however, the gene sets undergoing H3K4me3 or H3K27me3 differential methylation are distinct, refuting the simplistic idea that gene activation relies on a switch from an H3K27me3 repressed chromatin to an active form enriched in H3K4me3. Coupling the ChIP-seq experiments with transcriptome profiling reveals that differential histone methylation correlates with changes in expression. Interestingly, only a subset of cold-regulated genes lose H3K27me3 during their induction, indicating that H3K27me3 is not an obstacle to transcriptional activation. In the H3K27me3 methyltransferase curly leaf (clf) mutant, many cold regulated genes display reduced H3K27me3 levels but their transcriptional activity is not altered prior or during a cold exposure, suggesting that H3K27me3 may serve a more intricate role in the cold response than simply repressing the cold-inducible genes in naive conditions.

plant biology↗

bioRxiv2023

Human Th17- and IgG3-associated autoimmunity induced by a translocating gut pathobiont

Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFN{gamma}+ Th17 differentiation and IgG3 subclass switch of anti-E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases. One Sentence SummaryTranslocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.

immunology↗

bioRxiv2023

Non-pathogenic leaf-colonising bacteria elicit pathogen-like responses in a colonisation density-dependent manner

Leaves are colonised by a complex mix of microbes, termed the leaf microbiota. Even though the leaf microbiota is increasingly recognised as an integral part of plant life and health, our understanding of its interactions with the plant host is still limited. Here, mature, axenically grown Arabidopsis thaliana plants were spray-inoculated with six diverse leaf-colonising bacteria. The transcriptomic changes in leaves were tracked over time and significant changes in ethylene marker (ARL2) expression were observed only two to four days after spray-inoculation. Whole transcriptome sequencing revealed that four days after inoculation, leaf transcriptional changes to colonisation by non-pathogenic and pathogenic bacteria differed in strength but not in the type of response. Inoculation of plants with different densities of the non-pathogenic bacterium Williamsia sp. Leaf354 showed that high bacterial titers caused disease phenotypes and led to severe transcriptional reprogramming with a strong focus on plant defence. An in silico epigenetic analysis of the data was congruent with the transcriptomic analysis. These findings suggest (1) that plant responses are not rapid after spray-inoculation, (2) that plant responses only differ in strength and (3) that plants respond to high titers of non-pathogenic bacteria with pathogen-like responses. Plain Language SummaryPlants are colonised by diverse bacteria affecting many aspects of plant life. Here we show that plants do not differentiate between different bacteria but measure their quantities to keep bacterial numbers in check.

plant biology↗

bioRxiv2023

A human in vitro neuronal model for studying homeostatic plasticity at the network level

Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliable in vitro platform to measure and characterize homeostatic plasticity at network and single-cell level; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.

neuroscience↗

bioRxiv2023

FriendlyClearMap: An optimized toolkit for mouse brain mapping and analysis

Tissue clearing is currently revolutionizing neuroanatomy by enabling organ-level imaging with cellular resolution. However, currently available tools for data analysis require a significant time investment for training and adaptation to each laboratorys use case, which limits productivity. Here, we present FriendlyClearMap, an integrated toolset that makes ClearMap1 and ClearMap2s CellMap pipeline easier to use, extends its functions, and provides Docker Images from which it can be run with minimal time investment. We also provide detailed tutorials for each step of the pipeline. For more precise alignment, we add a landmark-based atlas registration to ClearMaps functions as well as include young mouse reference atlases for developmental studies. We provide alternative cell segmentation method besides ClearMaps threshold-based approach: Ilastiks Pixel Classification, importing segmentations from commercial image analysis packages and even manual annotations. Finally, we integrate BrainRender, a recently released visualization tool for advanced 3D visualization of the annotated cells. As a proof-of-principle, we use FriendlyClearMap to quantify the distribution of the three main GABAergic interneuron subclasses (Parvalbumin+, Somatostatin+, and VIP+) in the mouse fore- and midbrain. For PV+ neurons, we provide an additional dataset with adolescent vs. adult PV+ neuron density, showcasing the use for developmental studies. When combined with the analysis pipeline outlined above, our toolkit improves on the state-of-the-art packages by extending their function and making them easier to deploy at scale.

neuroscience↗

bioRxiv2023

SCN1A-deficient hiPSC-derived excitatory neuronal networks display mutation-specific phenotypes

Dravet syndrome is a severe epileptic encephalopathy, characterized by (febrile) seizures, behavioral problems and developmental delay. 80% of Dravet syndrome patients have a mutation in SCN1A, encoding NaV1.1. Milder clinical phenotypes, such as GEFS+ (generalized epilepsy with febrile seizures plus), can also arise from SCN1A mutations. Predicting the clinical phenotypic outcome based on the type of mutation remains challenging, even when the same mutation is inherited within one family. Both this clinical and genetic heterogeneity add to the difficulties of predicting disease progression and tailored prescription of anti-seizure medication. A better understanding of the neuropathology of different SCN1A mutations, might give insight in differentiating the expected clinical phenotype and best fit treatment choice. Initially it was recognized that loss of Na+ -current in inhibitory neurons specifically resulted in disinhibition and consequently seizure generation. However, the extent to which excitatory neurons contribute to the pathophysiology is currently debated, and might depend on the patient clinical phenotype or the specific mutation in SCN1A. To examine the genotype-phenotype correlations of SCN1A mutations in relation to excitatory neurons, we investigated a panel of patient-derived excitatory neuronal networks differentiated on multi-electrode arrays. We included patients with different clinical phenotypes, harboring different mutations in SCN1A, plus a family where the same mutation leads to both GEFS+ and Dravet syndrome. We hitherto describe a previously unidentified functional excitatory neuronal network phenotype in the context of epilepsy, which corresponded to seizurogenic network prediction patterns elicited by proconvulsive compounds. We find that excitatory neuronal networks were differently affected, dependent on the type of SCN1A mutation, but not on clinical severity. Specifically, pore domain mutations could be distinguished from voltage sensing domain mutations. Furthermore, all patients showed aggravated neuronal network responses upon febrile temperatures. While the basal neuronal network phenotypes could not be distinguished based on patient clinical severity, retrospective drug screening revealed that anti-seizure medication only affected GEFS+ patient-, but not Dravet patient-derived neuronal networks in a patient specific and clinically relevant manner. In conclusion, our results indicate a mutation-specific excitatory neuronal network phenotype, which recapitulates the foremost clinically relevant features, providing future opportunities for precision therapies. HighlightsO_LIHuman stem cell derived excitatory neurons are affected by mutations in SCN1A and display mutation-specific, but not clinical phenotype specific, neuronal network phenotypes C_LIO_LIThe neuronal network phenotype we describe corresponds to seizurogenic network prediction patterns in vitro C_LIO_LIExcitatory neuronal networks respond to Dravet syndrome clinically relevant triggers, like febrile temperatures and Dravet-contraindicated ASM Carbamazepine C_LIO_LIRetrospective drug screening revealed that GEFS+ neuronal networks, but not Dravet neuronal networks respond to ASM in a patient-specific and clinical relevant manner C_LI

neuroscience↗

bioRxiv2022

Completing the TRB family: newly characterized members show ancient evolutionary origins and distinct localization, yet similar interactions

Telomere repeat binding proteins (TRBs) belong to a family of proteins possessing a Myb-like domain which binds to telomeric repeats. Three members of this family (TRB1, TRB2, TRB3) from Arabidopsis thaliana have already been described as associated with terminal telomeric repeats (telomeres) or short interstitial telomeric repeats in gene promoters (telo-boxes). They are also known to interact with several protein complexes: telomerase, Polycomb repressive complex 2 (PRC2) E(z) subunits and the PEAT complex (PWOs-EPCRs-ARIDs-TRBs). Here we characterize two novel members of the TRB family (TRB4 and TRB5). Our wide phylogenetic analyses have shown that TRB proteins evolved in the plant kingdom after the transition to a terrestrial habitat in Streptophyta, and consequently TRBs diversified in seed plants. TRB4-5 share common TRB motifs while differing in several others and seem to have an earlier phylogenetic origin than TRB1-3. Their common Myb-like domains bind long arrays of telomeric repeats in vitro, and we have determined the minimal recognition motif of all TRBs as one telo-box. Our data indicate that despite the distinct localization patterns of TRB1-3 and TRB4-5 in situ, all members of TRB family mutually interact and also bind to telomerase/PRC2/PEAT complexes. Additionally, we have detected novel interactions between TRB4-5 and EMF2 and VRN2, which are Su(z)12 subunits of PRC2.

plant biology↗

bioRxiv2022

The UBP5 histone H2A deubiquitinase counteracts PRC2-mediated repression to regulate Arabidopsis development and stress responses

Polycomb Repressive Complexes (PRCs) control gene expression through the incorporation of H2Aub and H3K27me3. However, there is limited knowledge about PRCs interacting proteins and their interplay with PRCs in epigenome reshaping, which is fundamental to understand gene regulatory mechanisms. Here, we identified UBIQUITIN SPECIFIC PROTEASE 5 (UBP5) as a novel interactor of the PRC2 subunit SWINGER and its associated factor PWO1 in Arabidopsis thaliana. As inferred from the functional analyses of ubp5 CRISPR-Cas9 mutant plants, UBP5 regulates plant development and stress responses, notably by promoting H2A monoubiquitination erasure, leading to transcriptional de-repression. Preferential association of UBP5 at PRC2 recruiting motifs and local H3K27me3 gaining in ubp5 mutant plants further suggest the existence of functional interplays between UBP5 and PRC2 in regulating epigenome dynamics. In summary, UBP5 provides novel insights to disentangle the complex PRC2 interaction network and is a crucial regulator of the pivotal epigenetic repressive marks H2Aub and H3K27me3.

developmental biology↗

bioRxiv2022

Transposons repressed by H3K27me3 were co-opted as cis-regulatory elements of H3K27me3 controlled protein coding genes during evolution of plants

The mobility of transposable elements (TEs) contributes to evolution of genomes 1,2. Meanwhile, their uncontrolled activity causes genomic instability and therefore expression of TEs is silenced by host genomes 3,4. TEs are marked with DNA and H3K9 methylation that are associated with silencing in flowering plants 5, animals, and fungi 6. Yet, in distantly related eukaryotes TEs are instead marked by H3K27me3 deposited by the Polycomb Repressive Complex 2 (PRC2) 7-11, an epigenetic mark associated with gene silencing in multicellular eukaryotes 12-15. It was therefore proposed that the ancestral activity of PRC2 was the deposition of H3K27me3 to silence TEs 16. To test this hypothesis we obtained mutants deprived of PRC2 activity and used genomics to analyze the role of PRC2 in extant species along the lineage of Archaeplastida. While in the red alga Cyanidioschyzon merolae more TEs than genes were repressed by PRC2, an opposite trend was observed in bryophytes Marchantia polymorpha and Anthoceros agrestis. In the red alga, TEs silenced by H3K27me3 are in subtelomeres but in bryophytes, TEs and genes marked by H3K27me3 form coregulated transcriptional units. The latter trend was also observed in the flowering plant Arabidopsis thaliana, and we identified cis-elements recognised by transcription factors in TEs flanking genes repressed by PRC2. Together with the silencing of TEs by PRC2 in ciliates that diverged early from an ancestor common with Archaeplastida, our findings support the hypothesis that PRC2 deposited H3K27me3 to silence TEs in early lineages of eukaryotes. During evolution, TE fragments marked with H3K27me3 were selected to shape transcriptional regulation that control networks of genes regulated by PRC2. HighlightsH3K27me3 marks a decreasing proportion of TEs during evolution of plants The polycomb repressive complex 2 represses TEs in red algae and bryophytes H3K27me3-marked TEs in flowering plants contain transcription factor binding sites Transcription factors bind TEs and regulate networks of genes controlled by PRC2

evolutionary biology↗

bioRxiv2021

Loss-of-function variants in the schizophrenia risk gene SETD1A alter neuronal network activity in human neurons through cAMP/PKA pathway

Heterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, were shown to cause a novel neurodevelopmental syndrome and increase the risk for schizophrenia. We generated excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/-) using CRISPR/Cas9. Our data show that SETD1A haploinsufficiency resulted in morphologically increased dendritic complexity and functionally increased bursting activity. This network phenotype was primarily driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling revealed perturbations in gene sets associated with glutamatergic synaptic function. At the molecular level, we identified specific changes in the cAMP/PKA pathway pointing toward a hyperactive cAMP pathway in SETD1A+/- neurons. Finally, by pharmacologically targeting the cAMP pathway we were able to rescue the network deficits in SETD1A+/- cultures. Our results demonstrate a link between SETD1A and the cAMP-dependent pathway in human neurons.

neuroscience↗

bioRxiv2021

Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations in vitro

Micro-electrode arrays (MEAs) are increasingly used to characterize neuronal network activity of human induced pluripotent stem-cell (hiPSC)-derived neurons. Despite their gain in popularity, MEA recordings from hiPSC-derived neuronal networks are not always used to their full potential in respect to experimental design, execution and data analysis. Therefore, we benchmarked the robustness and sensitivity of MEA-derived neuronal activity patterns derived from ten healthy individual control lines. We provide recommendations on experimental design and analysis to achieve standardization. With such standardization, MEAs can be used as a reliable platform to distinguish (disease-specific) network phenotypes. In conclusion, we show that MEAs are a powerful and robust tool to uncover functional neuronal network phenotypes from hiPSC-derived neuronal networks, and provide an important resource to advance the hiPSC field towards the use of MEAs for disease-phenotyping and drug discovery.

neuroscience↗