RESUMO
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-1ß 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-1ß 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-1ß-mediated comorbidities, offering a framework for secondary prevention immunotherapy.
Assuntos
Lesões Encefálicas , Imunidade Inata , Memória Imunológica , Inflamação , Interleucina-1beta , Camundongos Endogâmicos C57BL , Monócitos , Animais , Camundongos , Interleucina-1beta/metabolismo , Lesões Encefálicas/imunologia , Humanos , Masculino , Monócitos/metabolismo , Monócitos/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/imunologia , Cardiopatias/imunologia , Feminino , Receptores CCR2/metabolismo , Fibrose , Epigênese Genética , Imunidade TreinadaRESUMO
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.
Assuntos
Astrócitos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Reprogramação Celular , Proteínas do Tecido Nervoso , Neurônios , Fator de Transcrição YY1 , Animais , Fator de Transcrição YY1/metabolismo , Fator de Transcrição YY1/genética , Astrócitos/metabolismo , Camundongos , Reprogramação Celular/fisiologia , Neurônios/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Epigenoma , Montagem e Desmontagem da Cromatina , Epigênese Genética , Células CultivadasRESUMO
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.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Animais , Camundongos , Diabetes Mellitus/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Células Ependimogliais/metabolismo , Neuroglia/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Retina/metabolismoRESUMO
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.
Assuntos
Cromatina , Epigenoma , Humanos , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Metilação de DNA/genética , Organoides/metabolismoRESUMO
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.
Assuntos
Staphylococcus aureus , Staphylococcus , Staphylococcus/genética , Staphylococcus aureus/metabolismo , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Proteínas Quinases/genética , Peptídeos/metabolismo , Percepção de Quorum , Proteínas de Bactérias/metabolismoRESUMO
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.
RESUMO
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 â¼3 × 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.
Assuntos
Escherichia coli , Engenharia Metabólica , Escherichia coli/metabolismo , Fermentação , Engenharia Metabólica/métodos , Fenilalanina/genética , Fenilalanina/metabolismo , Estireno/metabolismoRESUMO
How multiple epigenetic layers and transcription factors (TFs) interact to facilitate brain development is largely unknown. Here, to systematically map the regulatory landscape of neural differentiation in the mouse neocortex, we profiled gene expression and chromatin accessibility in single cells and integrated these data with measurements of enhancer activity, DNA methylation and three-dimensional genome architecture in purified cell populations. This allowed us to identify thousands of new enhancers, their predicted target genes and the temporal relationships between enhancer activation, epigenome remodeling and gene expression. We characterize specific neuronal transcription factors associated with extensive and frequently coordinated changes across multiple epigenetic modalities. In addition, we functionally demonstrate a new role for Neurog2 in directly mediating enhancer activity, DNA demethylation, increasing chromatin accessibility and facilitating chromatin looping in vivo. Our work provides a global view of the gene regulatory logic of lineage specification in the cerebral cortex.
Assuntos
Cromatina , Epigenoma , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Bicamadas Lipídicas , Fosfatidilcolinas , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Membranas/metabolismo , Fosfatidilcolinas/química , EstirenoRESUMO
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.
Assuntos
Núcleo Celular , Genoma , Animais , Evolução Biológica , Encéfalo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Mamíferos/genética , Lâmina Nuclear/genética , Lâmina Nuclear/metabolismoRESUMO
Bacterial resistance to antibiotics constantly remodels the battlefront between infections and antibiotic therapy. Polymyxin B, a cationic peptide with an anti-Gram-negative spectrum of activity is re-entering use as a last resort measure and as an adjuvant. We use fluorescence dequenching to investigate the role of the rough chemotype bacterial lipopolysaccharide from E. coli BL21 as a molecular facilitator of membrane disruption by LPS. The minimal polymyxin B/lipid ratio required for leakage onset increased from 5.9 × 10-4 to 1.9 × 10-7 in the presence of rLPS. We confirm polymyxin B activity against E. coli BL21 by the agar diffusion method and determined a MIC of 291 µg ml-1. Changes in lipid membrane stability and dynamics in response to polymyxin and the role of LPS are investigated by 31P NMR and high resolution 31P MAS NMR relaxation is used to monitor selective molecular interactions between polymyxin B and rLPS within bilayer lipid membranes. We observe a strong facilitating effect from rLPS on the membrane lytic properties of polymyxin B and a specific, pyrophosphate-mediated process of molecular recognition of LPS by polymyxin B.
Assuntos
Lipopolissacarídeos , Polimixina B , Antibacterianos/farmacologia , Escherichia coli , Polimixina B/farmacologia , PolimixinasRESUMO
Membranes of cells are active barriers, in which membrane proteins perform essential remodelling, transport and recognition functions that are vital to cells. Membrane proteins are key regulatory components of cells and represent essential targets for the modulation of cell function and pharmacological intervention. However, novel folds, low molarity and the need for lipid membrane support present serious challenges to the characterisation of their structure and interactions. We describe the use of solid state NMR as a versatile and informative approach for membrane and membrane protein studies, which uniquely provides information on structure, interactions and dynamics of membrane proteins. High resolution approaches are discussed in conjunction with applications of NMR methods to studies of membrane lipid and protein structure and interactions. Signal enhancement in high resolution NMR spectra through DNP is discussed as a tool for whole cell and interaction studies.
Assuntos
Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Bicamadas Lipídicas/metabolismo , Conformação ProteicaRESUMO
Understanding how complex cell-fate decisions emerge at the molecular level is a key challenge in developmental biology. Despite remarkable progress in decoding the contribution of the linear epigenome, how spatial genome architecture functionally informs changes in gene expression remains unclear. In this review, we discuss recent insights in elucidating the molecular landscape of genome folding, emphasizing the multilayered nature of the 3D genome, its importance for gene regulation, and its spatiotemporal dynamics. Finally, we discuss how these new concepts and emergent technologies will enable us to address some of the outstanding questions in development and disease.
Assuntos
Linhagem da Célula/genética , Cromatina/ultraestrutura , Epigenoma/genética , Genoma Humano/genética , Diferenciação Celular/genética , Cromatina/genética , Regulação da Expressão Gênica/genética , HumanosRESUMO
Biological membranes define the interface of life and its basic unit, the cell. Membrane proteins play key roles in membrane functions, yet their structure and mechanisms remain poorly understood. Breakthroughs in crystallography and electron microscopy have invigorated structural analysis while failing to characterise key functional interactions with lipids, small molecules and membrane modulators, as well as their conformational polymorphism and dynamics. NMR is uniquely suited to resolving atomic environments within complex molecular assemblies and reporting on membrane organisation, protein structure, lipid and polysaccharide composition, conformational variations and molecular interactions. The main challenge in membrane protein studies at the atomic level remains the need for a membrane environment to support their fold. NMR studies in membrane mimetics and membranes of increasing complexity offer close to native environments for structural and molecular studies of membrane proteins. Solution NMR inherits high resolution from small molecule analysis, providing insights from detergent solubilised proteins and small molecular assemblies. Solid-state NMR achieves high resolution in membrane samples through fast sample spinning or sample alignment. Recent developments in dynamic nuclear polarisation NMR allow signal enhancement by orders of magnitude opening new opportunities for expanding the applications of NMR to studies of native membranes and whole cells.
RESUMO
The genome folds into a hierarchy of three-dimensional structures within the nucleus. At the sub-megabase scale, chromosomes form topologically associating domains (TADs)1-4. However, how TADs fold in single cells is elusive. Here, we reveal TAD features inaccessible to cell population analysis by using super-resolution microscopy. TAD structures and physical insulation associated with their borders are variable between individual cells, yet chromatin intermingling is enriched within TADs compared to adjacent TADs in most cells. The spatial segregation of TADs is further exacerbated during cell differentiation. Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms: cohesin generates chromatin contacts and intermingling while CTCF prevents inter-TAD contacts. Furthermore, TADs are subdivided into discrete nanodomains, which persist in cells depleted of CTCF or cohesin, whereas disruption of nucleosome contacts alters their structural organization. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale.
Assuntos
Cromatina/química , Células-Tronco Embrionárias/ultraestrutura , Domínios Proteicos , Animais , Diferenciação Celular/genética , Linhagem Celular , Coloração Cromossômica , Drosophila/genética , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Conformação Molecular , Nanoestruturas , Microscopia NuclearRESUMO
The extracellular signal-regulated kinases (ERKs) serve as important determinants of cellular signal transduction pathways, and hence may play important roles during infections. Previous work suggested that putative ERK7 of Toxoplasma gondii is required for efficient intracellular replication of the parasite. However, the antigenic and immunostimulatory properties of TgERK7 protein remain unknown. The objective of this study was to produce a recombinant TgERK7 protein in vitro and to evaluate its effect on the induction of humoral and T cell-mediated immune responses against T. gondii infection in BALB/c mice. Immunization using TgERK7 mixed with Freund's adjuvants significantly increased the ratio of CD3e+CD4+ T/CD3e+CD8a+ T lymphocytes in spleen and elevated serum cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-12p70, IL-23, MCP-1, and TNF-α) in immunized mice compared to control mice. On the contrary, immunization did not induce high levels of serum IgG antibodies. Five predicted peptides of TgERK7 were synthesized and conjugated with KLH and used to analyze the antibody specificity in the sera of immunized mice. We detected a progressive increase in the antibody level only against TgERK7 peptide A (DEVDKHVLRKYD). Antibody raised against this peptide significantly decreased intracellular proliferation of T. gondii in vitro, suggesting that peptide A can potentially induce a protective antibody response. We also showed that immunization improved the survival rate of mice challenged with a virulent strain and significantly reduced the parasite cyst burden within the brains of chronically infected mice. Our data show that TgERK7-based immunization induced TgERK7 peptide A-specific immune responses that can impart protective immunity against T. gondii infection. The therapeutic potential of targeting ERK7 signaling pathway for future toxoplasmosis treatment is warranted.
Assuntos
Antígenos de Protozoários/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Citocinas/sangue , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Imunidade Celular , Imunidade Humoral , Imunização , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/química , Peptídeos/genética , Conformação Proteica , Vacinas Protozoárias/imunologia , Coelhos , Proteínas Recombinantes/imunologia , Toxoplasma/genéticaRESUMO
To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.