During cortical development, neural stem cells (NSCs) combine self-renewal with the sequential production of different subtypes of projection neurons as well as glia cells. How the NSC epigenome accommodates this over time remains unresolved. Here, we address this gap by multimodal epigenomic profiling of mouse cortical development across six time points and five embryonic days. Single-cell gene expression and temporal modeling reveal that NSC self-renewal is not homeostatic, showing progressively stronger astrocytic preference over time. Chromosome accessibility, DNA methylation, and Hi-C show that this process involves major reorganization of the NSC epigenome. A model combining transcription factor motif affinities with epigenetic features, as well as integration of the results with a reporter assay in vivo, show that activation of the NSC neuronal fate regulatory program may be affected by a changing epigenome. Collectively, our findings uncover temporal epigenomic reprogramming that underlies the evolving differentiation potential of NSCs, providing insights into the intrinsic and extrinsic mechanisms that pattern cortical lineages.

In contrast to the well-understood role of core histones in DNA packaging, the function of the linker histone (H1) remains enigmatic. Challenging the prevailing view that linker histones are a general feature of heterochromatin, here we show a critical requirement for H1 in Polycomb repressive complex 2 (PRC2) function. A CRISPR-Cas9 genetic screen using a fluorescent PRC2 reporter identified an essential role for the poorly characterized gene CRAMP1 in PRC2-mediated repression. CRAMP1 localizes to the promoters of expressed H1 genes and positively regulates their transcription. CRAMP1 ablation simultaneously depletes all linker histones, which results in selective decompaction of H3K27me3-marked loci and derepression of PRC2 target genes without concomitant loss of PRC2 occupancy or enzymatic activity. Strikingly, we find that linker histones preferentially localize to genomic loci marked by H3K27me3 across diverse cell types and organisms. Altogether, these data demonstrate a prominent role for linker histones in epigenetic repression by PRC2.

As the seat of our cognition, the human neocortex is an object of immense fascination. Human neocortex expansion during evolution has been attributed to an increase in the proliferative capacity of neural progenitor cells during development, particularly basal radial glia (bRG). Despite their evolutionary relevance, the genomic changes driving human-specific bRG biology remain uncharacterized. We used comparative chromatin and transcriptional profiling of neural progenitor cells isolated from gorilla, chimpanzee and human cerebral organoids to identify cis-regulatory elements that have gained activity in humans. Focusing specifically on bRG, we discovered that morphoregulatory enhancer activity and gene expression signatures distinguish human bRG from other great apes. Functional analysis of the morphoregulatory genes FAM107A and CNGA3 in human organoids revealed that these genes are required for the morphological complexity of human bRG. Taken together, our interspecies comparison of basal radial glia suggests that human-specific morphoregulatory signatures characterize neocortex evolution. ### Competing Interest Statement The authors have declared no competing interest.

The cerebral cortex orchestrates complex cognitive functions, yet how its distinct temporal lineages are molecularly patterned during development remains unresolved. Here, we integrate single-cell transcriptomics and chromatin accessibility, together with genome-wide profiling of DNA methylation and 3D chromosomal contact across mouse corticogenesis (E13–E18) to elucidate cell fate transitions. Using metacell flow analysis, we reveal that neural stem cells (NSCs) progressively shift from a progenitor–biased state toward an astrocytic lineage and that this process is accompanied with changes in DNA methylation and 3D genome organization. A model integrating transcription factor motif affinities with epigenetic features identifies key regulators of cis-regulatory element (CRE) activation. In vivo reporter assays further decouple the intrinsic regulatory potential of CREs from context-dependent synergistic activation. Collectively, our findings uncover temporal epigenomic reprogramming that underlies the evolving differentiation potential of NSCs, providing insights into the intrinsic and extrinsic mechanisms that pattern cortical lineages. ### Competing Interest Statement The authors have declared no competing interest.

Performance plastics, such as poly(methyl methacrylate), underpin the modern economy. Global manufacturing of plastic precursors relies on fossil carbon sources, and the urgently needed shift toward renewable carbon use through biofermentation is hindered by the low tolerance of producer strains to methacrylate esters. The principal mode of butyl methacrylate cellular toxicity is membrane disruption. To understand this process, the conditions for membrane stability, and recovery after solvent shock, we investigate the phase stability of hydrated lipid membranes at high levels of a key intermediate, butyl methacrylate. We assess the role of cis- vs trans-unsaturation in 18-carbon chain phospholipids on butyl methacrylate-induced phase conversion and polymorphism. Using ssNMR, SAXS and cryoEM, we demonstrate the formation of stable lipidic cubic phases in hydrated lipid/solvent (cis-chain phospholipid lipid/butyl methacrylate) systems at a 1:6 molar ratio entirely lacking monoolein. Transient lipidic cubic phases form in trans-chain phospholipid/butyl methacrylate systems, which slowly convert to bilayers through a spontaneous "membrane healing" process during recovery after solvent shock. The observed bicontinuous nanostructures with a cubic phase architecture coexist with a stable, monocontinuous hydrated phase of the same morphology but with simpler topological connectivity, which demonstrates that phase stability in cubic phases does not require topological complementarity. We propose trans-lipid substitutions in membranes of fermentative strains as a key step toward sustainable production of methacrylate esters.

The expansion of the neocortex is a hallmark of human evolution and is closely linked to neural stem cell biology. Yet, the epigenetic mechanisms driving divergent gene regulation during primate neurogenesis remain elusive. Here, we comprehensively mapped 3D genome organization, chromatin accessibility and gene expression in induced pluripotent stem cells and derived neural stem cells from human, chimpanzee, gorilla and macaque. We identified human-specific epigenetic signatures including cis-regulatory regions and enhancer-promoter interactions and linked them to gene regulatory dynamics. Deep learning models revealed that complex regulatory grammar at cis-regulatory regions, including transcription factor binding sites, local context and higher-order chromatin organization, underlies species and cell type-specific differences. High-resolution Hi-C uncovered unexpected global shifts in 3D genome architecture in chimpanzee and gorilla neural stem cells while topologically associating domains remain remarkably conserved. Notably, species-specific genes interacted with multiple differentially accessible regions, suggesting that synergistic enhancer activation is a key mechanism driving epigenome evolution. These findings provide new insights into the epigenetic basis of primate brain evolution. ### Competing Interest Statement The authors have declared no competing interest.

Membrane-inserting materials bearing a lipid residue at one end of their macromolecular chain, α-lipid polymers, are increasingly utilized in biological and pharmaceutical fields. Insertion of these materials into lipid membranes underlines several clinically available liposomal formulations and led to the identification of cellular targets in drug discovery. Herein, we approach this concept from the perspective of a lipid membrane to investigate the relationship between the molecular structure of the inserting α-lipid polymer and the effects that the insertion has on the membrane properties. We synthesized libraries of hydrophilic (co)-polymers comprising neutral or acidic monomers, including N-hydroxyethyl acrylamide (HEA), acrylic acid (AA), and 3-acrylamide propanoic acid (3-AAPA), and either of two terminal membrane-inserting moieties with different molecular structures, cholesteryl (Chol) or 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) phospholipid. We investigated the structure-function relationships combining experimental methods (laurdan generalized polarization, flow cytometry, 13C and wide-line 31P solid-state NMR, and surface plasmon resonance) in conjunction with in silico modeling. Our data indicate that insertion of α-lipid polymers increases the fluidity of a range of artificial lipid membranes as well as cell plasma membranes in Caco-2 cell culture. The extent of α-lipid polymer-membrane association, described by kinetic and thermodynamic parameters K a and K d, and K D, respectively, depends on both (i) the nature of the membrane-inserting anchor and (ii) the length of the hydrophilic chain. Hexadecahydro-3H-cyclopenta-[a]-phenanthrene-structure-based cholesterol anchor shows faster and stronger membrane association than phospholipid (DOPE) ones. In addition, the shorter polymers (targeted DP = 50 as opposed to DP = 100) display a higher level of membrane association which leads to a consequent larger, from 1.3- to 2.2-fold depending on the polymer, increase of bilayer fluidity. In silico molecular modeling of Chol-(HEA)n polymers suggests that an increase in overall membrane fluidity results from a significant disruption of the lipid organization occurring near the point of insertion of the cholesterol anchor into the membrane. This effect decreases rapidly further away from the insertion point. Our work hence shows that the insertion of α-lipid polymers has a significant effect on lipid bilayer membranes, whereby the observed increase in membrane fluidity needs to be considered when, for example, designing drug formulations or modifying biological systems where it may impact on liposomal membrane stability in biological environment or ability to retain therapeutic cargo.

Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell response to diabetic conditions. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.

Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming. The molecular mechanisms underlying direct neuronal reprogramming are unclear. Here the authors show Ngn2-mediated chromatin remodeling and its binding sites underlying mouse astrocyte-to-neuron reprogramming and identify Yy1, a transcription co-factor, as an important regulator.

The medical burden of stroke extends beyond the brain injury itself and is largely determined by chronic comorbidities that develop secondarily. We hypothesized that these comorbidities might share a common immunological cause, yet chronic effects post-stroke on systemic immunity are underexplored. Here, we identify myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Single-cell sequencing revealed persistent pro-inflammatory changes in monocytes/macrophages in multiple organs up to 3 months after brain injury, notably in the heart, leading to cardiac fibrosis and dysfunction in both mice and stroke patients. IL-1β was identified as a key driver of epigenetic changes in innate immune memory. These changes could be transplanted to naive mice, inducing cardiac dysfunction. By neutralizing post-stroke IL-1β or blocking pro-inflammatory monocyte trafficking with a CCR2/5 inhibitor, we prevented post-stroke cardiac dysfunction. Such immune-targeted therapies could potentially prevent various IL-1β-mediated comorbidities, offering a framework for secondary prevention immunotherapy.

Transcription factors (TFs) are pivotal in orchestrating lineage decisions and their binding is often accompanied by chromatin remodeling. However, a comprehensive understanding of how cellular and epigenetic contexts influence TF binding and the subsequent activation of lineage-specific programs remains lacking. Here, we dissected the molecular mechanisms underlying the binding of one such TF - Neurog2 and its functional consequences in two cell-types: mouse embryonic stem cells and neural progenitor cells. Our findings reveal that cell-type-specific chromatin accessibility and motif syntax are key determinants for TF binding. While Neurog2 binding primarily leads to chromatin opening, DNA demethylation and increased chromatin interactions, we also uncovered strong indirect, cell-type-specific effects, which ultimately result in vastly different epigenetic landscape. Furthermore, we identify shared and cell-type-specific Neurog2 interactors, including the SWI/SNF and NuRD complexes. Our study shed light on how cellular environment can modulate TF function to establish lineage identity during development. ### Competing Interest Statement The authors have declared no competing interest.

Over the past decade, single-cell genomics technologies have allowed scalable profiling of cell-type-specific features, which has substantially increased our ability to study cellular diversity and transcriptional programs in heterogeneous tissues. Yet our understanding of mechanisms of gene regulation or the rules that govern interactions between cell types is still limited. The advent of new computational pipelines and technologies, such as single-cell epigenomics and spatially resolved transcriptomics, has created opportunities to explore two new axes of biological variation: cell-intrinsic regulation of cell states and expression programs and interactions between cells. Here, we summarize the most promising and robust technologies in these areas, discuss their strengths and limitations and discuss key computational approaches for analysis of these complex datasets. We highlight how data sharing and integration, documentation, visualization and benchmarking of results contribute to transparency, reproducibility, collaboration and democratization in neuroscience, and discuss needs and opportunities for future technology development and analysis.

In Gram-positive bacteria such as Staphylococcus aureus and the coagulase-negative staphylococci (CoNS), the accessory gene regulator ( agr ) is a highly conserved but polymorphic quorum-sensing system involved in colonization, virulence and biofilm development. Signalling via agr depends on the interaction of an autoinducing peptide (AIP) with AgrC, a transmembrane sensor kinase that, once phosphorylated activates the response regulator AgrA. This in turn autoinduces AIP biosynthesis and drives target gene expression directly via AgrA or via the post-transcriptional regulator, RNAIII. In this review we describe the molecular mechanisms underlying the agr -mediated generation of, and response to, AIPs and the molecular basis of AIP-dependent activation and inhibition of AgrC. How the environment impacts on agr functionality is considered and the consequences of agr dysfunction for infection explored. We also discuss the concept of AIP-driven competitive interference between S. aureus and the CoNS and its anti-infective potential.

Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the center of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signaling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC-sequencing identified the GR as a master regulator of Müller cell gliosis in DR. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand. Graphical abstract

Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.

Gene expression is regulated by multiple epigenetic mechanisms, which are coordinated in development and disease. However, current multiomics methods are frequently limited to one or two modalities at a time, making it challenging to obtain a comprehensive gene regulatory signature. Here, we describe a method—3D genome, RNA, accessibility and methylation sequencing (3DRAM-seq)—that simultaneously interrogates spatial genome organization, chromatin accessibility and DNA methylation genome-wide and at high resolution. We combine 3DRAM-seq with immunoFACS and RNA sequencing in cortical organoids to map the cell-type-specific regulatory landscape of human neural development across multiple epigenetic layers. Finally, we apply a massively parallel reporter assay to profile cell-type-specific enhancer activity in organoids and to functionally assess the role of key transcription factors for human enhancer activation and function. More broadly, 3DRAM-seq can be used to profile the multimodal epigenetic landscape in rare cell types and different tissues.

Despite huge advances in stem-cell, single-cell and epigenetic technologies, the precise molecular mechanisms that determine lineage specification remain largely unknown. Applying an integrative multiomics approach, e.g. combining single-cell RNA-seq, single-cell ATAC-seq together with cell-type-specific DNA methylation and 3D genome measurements, we systematically map the regulatory landscape in the mouse neocortex in vivo . Our analysis identifies thousands of novel enhancer-gene pairs associated with dynamic changes in chromatin accessibility and gene expression along the differentiation trajectory. Crucially, we provide evidence that epigenetic remodeling generally precedes transcriptional activation, yet true priming appears limited to a subset of lineage-determining enhancers. Notably, we reveal considerable heterogeneity in both contact strength and dynamics of the generally cell-type-specific enhancer-promoter contacts. Finally, our work suggests a so far unrecognized function of several key transcription factors which act as putative “molecular bridges” and facilitate the dynamic reorganization of the chromatin landscape accompanying lineage specification in the brain.### Competing Interest StatementThe authors have declared no competing interest.

Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations. As a case study, MOBpsi was applied to improve the performance of an Escherichia coli cell factory producing the commodity chemical styrene. Styrene can be bio-manufactured from phenylalanine via an engineered pathway comprised of the enzymes phenylalanine ammonia lyase and ferulic acid decarboxylase. The toxicity, hydrophobicity, and volatility of styrene combine to make bio-production challenging. Previous attempts to create styrene tolerant E. coli strains by targeted genetic interventions have met with modest success. Application of MOBpsi identified new potential targets for improving performance, resulting in two host strains (E. coli NST74ΔaaeA and NST74ΔaaeA cpxPo) with increased styrene production. The best performing re-engineered chassis, NST74ΔaaeA cpxPo, produced ∼3 × more styrene and exhibited increased viability in fed-batch fermentations. Thus, this case study demonstrates the utility of MOBpsi as a systematic tool for improving the bio-manufacturing of toxic chemicals.

In the cell nucleus, radial genome organization relative to the nuclear lamina exhibits both cell type-specific variability and evolutionarily conserved distribution. A recent study by Ahanger, Delgado et al. explores the in vivo spatial distribution of the genome in the brain and identifies conserved genomic localization across mammalian species that are correlated with gene expression states.

Biofermentative production of styrene from renewable carbon sources is crucially dependent on strain tolerance and viability at elevated styrene concentrations. Solvent-driven collapse of bacterial plasma membranes limits yields and is technologically restrictive. Styrene is a hydrophobic solvent that readily partitions into the membrane interior and alters membrane-chain order and packing. We investigate styrene incorporation into model membranes and the role lipid chains play as determinants of membrane stability in the presence of styrene. MD simulations reveal styrene phase separation followed by irreversible segregation into the membrane interior. Solid state NMR shows committed partitioning of styrene into the membrane interior with persistence of the bilayer phase up to 67 mol % styrene. Saturated-chain lipid membranes were able to retain integrity even at 80 mol % styrene, whereas in unsaturated lipid membranes, we observe the onset of a non-bilayer phase of small lipid aggregates in coexistence with styrene-saturated membranes. Shorter-chain saturated lipid membranes were seen to tolerate styrene better, which is consistent with observed chain length reduction in bacteria grown in the presence of small molecule solvents. Unsaturation at mid-chain position appears to reduce the membrane tolerance to styrene and conversion from cis- to trans-chain unsaturation does not alter membrane phase stability but the lipid order in trans-chains is less affected than cis.