RESUMEN
Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs. How this occurs remains unclear. Here we demonstrate that in small pre-B cells, the lineage and stage-specific epigenetic reader bromodomain and WD repeat-containing protein 1 (BRWD1) reorders three-dimensional chromatin topology to affect the transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters, and coordinated this switch with Igk locus contraction. BRWD1 did so by converting chromatin-bound static to dynamic cohesin competent to mediate long-range looping. ATP-depletion revealed cohesin conversion to be the main energetic mechanism dictating dynamic chromatin looping. Our findings provide a new mechanism of cohesin regulation and reveal how cohesin function can be dictated by lineage contextual mechanisms to facilitate specific cell fate transitions.
Asunto(s)
Cromatina , Cohesinas , Cromatina/genética , Células Precursoras de Linfocitos B , Regulación de la Expresión Génica , Diferenciación Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
The T cell repertoire of healthy mice and humans harbors self-reactive CD4+ conventional T (Tconv) cells capable of inducing autoimmunity. Using T cell receptor profiling paired with in vivo clonal analysis of T cell differentiation, we identified Tconv cell clones that are recurrently enriched in non-lymphoid organs following ablation of Foxp3+ regulatory T (Treg) cells. A subset of these clones was highly proliferative in the lymphoid organs at steady state and exhibited overt reactivity to self-ligands displayed by dendritic cells, yet were not purged by clonal deletion. These clones spontaneously adopted numerous hallmarks of follicular helper T (TFH) cells, including expression of Bcl6 and PD-1, exhibited an elevated propensity to localize within B cell follicles at steady state, and produced interferon-γ in non-lymphoid organs following sustained Treg cell depletion. Our work identifies a naturally occurring population of self-reactive TFH-like cells and delineates a previously unappreciated fate for self-specific Tconv cells.
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Linfocitos T CD4-Positivos , Células T Auxiliares Foliculares , Linfocitos T Reguladores , Animales , Humanos , Ratones , Autoinmunidad , Diferenciación Celular , Células Clonales , Fenotipo , Linfocitos T Colaboradores-Inductores , Linfocitos T CD4-Positivos/inmunologíaRESUMEN
The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.
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Movimiento Celular , Pulmón , Mecanotransducción Celular , Neutrófilos , Animales , Ratones , Membrana Celular , Canales Iónicos/genética , Neutrófilos/metabolismo , Neutrófilos/microbiología , Actividad Bactericida de la Sangre/genética , Mecanotransducción Celular/genéticaRESUMEN
Within germinal centers (GCs), complex and highly orchestrated molecular programs must balance proliferation, somatic hypermutation and selection to both provide effective humoral immunity and to protect against genomic instability and neoplastic transformation. In contrast to this complexity, GC B cells are canonically divided into two principal populations, dark zone (DZ) and light zone (LZ) cells. We now demonstrate that, following selection in the LZ, B cells migrated to specialized sites within the canonical DZ that contained tingible body macrophages and were sites of ongoing cell division. Proliferating DZ (DZp) cells then transited into the larger DZ to become differentiating DZ (DZd) cells before re-entering the LZ. Multidimensional analysis revealed distinct molecular programs in each population commensurate with observed compartmentalization of noncompatible functions. These data provide a new three-cell population model that both orders critical GC functions and reveals essential molecular programs of humoral adaptive immunity.
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Microambiente Celular/genética , Microambiente Celular/inmunología , Centro Germinal/citología , Centro Germinal/fisiología , Animales , Biomarcadores , Biología Computacional/métodos , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Genómica/métodos , Ratones , Fosforilación , Proteoma , Proteómica/métodos , TranscriptomaRESUMEN
In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet how the pre-BCR mediates these functions remains unclear. Here, we demonstrate that the pre-BCR initiates a feed-forward amplification loop mediated by the transcription factor interferon regulatory factor 4 and the chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 ligation by C-X-C motif chemokine ligand 12 activates the mitogen-activated protein kinase extracellular-signal-regulated kinase, which then directs the development of small pre- and immature B cells, including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin-7 have only modest effects on B cell developmental transcriptional and epigenetic programs. These data show a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.
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Linfocitos B/inmunología , Cadenas kappa de Inmunoglobulina/genética , Células Precursoras de Linfocitos B/fisiología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores CXCR4/metabolismo , Animales , Puntos de Control del Ciclo Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Femenino , Factores Reguladores del Interferón/genética , Linfopoyesis , Masculino , Ratones , Receptores de Antígenos de Linfocitos B/genética , Receptores CXCR4/genética , Recombinación GenéticaRESUMEN
Thymocyte development requires a complex orchestration of multiple transcription factors. Ablating either TCF-1 or HEB in CD4+CD8+ thymocytes elicits similar developmental outcomes including increased proliferation, decreased survival, and fewer late Tcra rearrangements. Here, we provide a mechanistic explanation for these similarities by showing that TCF-1 and HEB share ~7,000 DNA-binding sites genome wide and promote chromatin accessibility. The binding of both TCF-1 and HEB was required at these shared sites for epigenetic and transcriptional gene regulation. Binding of TCF-1 and HEB to their conserved motifs in the enhancer regions of genes associated with T cell differentiation promoted their expression. Binding to sites lacking conserved motifs in the promoter regions of cell-cycle-associated genes limited proliferation. TCF-1 displaced nucleosomes, allowing for chromatin accessibility. Importantly, TCF-1 inhibited Notch signaling and consequently protected HEB from Notch-mediated proteasomal degradation. Thus, TCF-1 shifts nucleosomes and safeguards HEB, thereby enabling their cooperation in establishing the epigenetic and transcription profiles of CD4+CD8+ thymocytes.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Regulación de la Expresión Génica/inmunología , Factor Nuclear 1-alfa del Hepatocito/inmunología , Linfopoyesis/inmunología , Timocitos/inmunología , Animales , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-B cell receptor (pre-BCR)-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5' to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which, at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination.
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Histona Acetiltransferasas/metabolismo , Inmunoglobulinas/genética , Recombinación Genética/inmunología , Animales , Apoptosis , Regulación hacia Abajo/inmunología , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/inmunología , Interleucina-7/genética , Interleucina-7/inmunología , Ratones , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Transcriptional regulation during CD4+ T cell fate decisions enables their differentiation into distinct states, guiding immune responses toward antibody production via Tfh cells or inflammation by Teff cells. Tfh-Teff cell fate commitment is regulated by mutual antagonism between the transcription factors Bcl6 and Blimp-1. Here we examined how T cell receptor (TCR) signals establish and arbitrate Bcl6-Blimp-1 counter-antagonism. We found that the TCR-signal-induced transcription factor Irf4 is essential for the differentiation of Bcl6-expressing Tfh and Blimp-1-expressing Teff cells. Increased TCR signaling raised Irf4 amounts and promoted Teff cell fates at the expense of Tfh ones. Importantly, orthogonal induction of Irf4 expression redirected Tfh cell fate trajectories toward those of Teff. Mechanistically, we linked greater Irf4 abundance with its recruitment toward low-affinity binding sites within Teff cell cis-regulatory elements, including those of Prdm1. We propose that the Irf4 locus functions as the "reader" of TCR signal strength, and in turn, concentration-dependent activity of Irf4 "writes" T helper fate choice.
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Regulación de la Expresión Génica , Redes Reguladoras de Genes , Factores Reguladores del Interferón/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Antígenos/inmunología , Sitios de Unión , Diferenciación Celular/inmunología , Línea Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunización , Factores Reguladores del Interferón/genética , Interleucina-2/metabolismo , Masculino , Ratones , Ratones Noqueados , Motivos de Nucleótidos , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
Liver-specific ten-eleven translocation (Tet) methylcytosine dioxygenases 2 and 3 (Tet2 plus Tet3)-deficient hepatitis B virus (HBV) transgenic mice fail to support viral biosynthesis. The levels of viral transcription and replication intermediates are dramatically reduced. Hepatitis B core antigen is only observed in a very limited number of pericentral hepatocytes in a pattern that is similar to glutamate-ammonia ligase (Glul), a ß-catenin target gene. HBV transcript abundance in adult Tet-deficient mice resembles that observed in wild-type neonatal mice. Furthermore, the RNA levels of several ß-catenin target genes including Glul, Lhpp, Notun, Oat, Slc1a2, and Tbx3 in Tet-deficient mice were also similar to that observed in wild-type neonatal mice. As HBV transcription is regulated by ß-catenin, these findings support the suggestion that neonatal Tet deficiency might limit ß-catenin target gene expression, limiting viral biosynthesis. Additionally, HBV transgene DNA displays increased 5-methylcytosine (5mC) frequency at CpG sequences consistent with neonatal Tet deficiency being responsible for decreased developmental viral DNA demethylation mediated by 5mC oxidation to 5-hydroxymethylcytosine, a process that might be responsible for the reduction in cellular ß-catenin target gene expression and viral transcription and replication.IMPORTANCEChronic hepatitis B virus (HBV) infection causes significant worldwide morbidity and mortality. There are no curative therapies available to resolve chronic HBV infections, and the small viral genome limits molecular targets for drug development. An alternative approach to drug development is to target cellular genes essential for HBV biosynthesis. In the liver, ten-eleven translocation (Tet) genes encode cellular enzymes that are not essential for postnatal mouse development but represent essential activities for viral DNA demethylation and transcription. Consequently, Tet inhibitors may potentially be developed into therapeutic agents capable of inducing and/or maintaining HBV covalently closed circular DNA methylation, resulting in transcriptional silencing and the resolution of chronic viral infection.
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Proteínas de Unión al ADN , Dioxigenasas , Virus de la Hepatitis B , Animales , Ratones , beta Catenina/genética , Dioxigenasas/genética , Dioxigenasas/metabolismo , Desmetilación del ADN , Metilación de ADN , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Virus de la Hepatitis B/metabolismo , Ratones TransgénicosRESUMEN
Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare and relatively indolent B-cell lymphoma. Characteristically, the [lymphocyte-predominant (LP)] tumor cells are embedded in a microenvironment enriched in lymphocytes. More aggressive variants of mature B-cell and peripheral T-cell lymphomas exhibit nuclear expression of the polo-like kinase 1 (PLK1) protein, stabilizing MYC (alias c-myc) and associated with worse clinical outcomes. This study demonstrated expression of PLK1 in the LP cells in 100% of NLPHL cases (n = 76). In contrast, <5% of classic Hodgkin lymphoma cases (n = 70) showed PLK1 expression within the tumor cells. Loss-of-function approaches demonstrated that the expression of PLK1 promoted cell proliferation and increased MYC stability in NLPHL cell lines. Correlation with clinical parameters revealed that the increased expression of PLK1 was associated with advanced-stage disease in patients with NLPHL. A multiplex immunofluorescence panel coupled with artificial intelligence algorithms was used to correlate the composition of the tumor microenvironment with the proliferative stage of LP cells. The results showed that LP cells with PLK1 (high) expression were associated with increased numbers of cytotoxic and T-regulatory T cells. Overall, the findings demonstrate that PLK1 signaling increases NLPHL proliferation and constitutes a potential vulnerability that can be targeted with PLK1 inhibitors. An active immune surveillance program in NLPHL may be a critical mechanism limiting PLK1-dependent tumor growth.
Asunto(s)
Enfermedad de Hodgkin , Linfoma de Células B , Humanos , Inteligencia Artificial , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Linfocitos/patología , Linfoma de Células B/patología , Quinasa Tipo Polo 1 , Microambiente TumoralRESUMEN
Despite evidence of genetic signatures in normal tissue correlating with disease risk, prospectively identifying genetic drivers and cell types that underlie subsequent pathologies has historically been challenging. The human prostate is an ideal model to investigate this phenomenon because it is anatomically segregated into three glandular zones (central, peripheral, and transition) that develop differential pathologies: prostate cancer in the peripheral zone (PZ) and benign prostatic hyperplasia (BPH) in the transition zone (TZ), with the central zone (CZ) rarely developing disease. More specifically, prostatic basal cells have been implicated in differentiation and proliferation during prostate development and regeneration; however, the contribution of zonal variation and the critical role of basal cells in prostatic disease etiology are not well understood. Using single-cell RNA sequencing of primary prostate epithelial cultures, we elucidated organ-specific, zone-specific, and cluster-specific gene expression differences in basal cells isolated from human prostate and seminal vesicle (SV). Aggregated analysis identified ten distinct basal clusters by Uniform Manifold Approximation and Projection. Organ specificity compared gene expression in SV with the prostate. As expected, SV cells were distinct from prostate cells by clustering, gene expression, and pathway analysis. For prostate zone specificity, we identified two CZ-specific clusters, while the TZ and PZ populations clustered together. Despite these similarities, differential gene expression was identified between PZ and TZ samples that correlated with gene expression profiles in prostate cancer and BPH, respectively. Zone-specific profiles and cell type-specific markers were validated using immunostaining and bioinformatic analyses of publicly available RNA-seq datasets. Understanding the baseline differences at the organ, zonal, and cellular level provides important insight into the potential drivers of prostatic disease and guides the investigation of novel preventive or curative treatments. Importantly, this study identifies multiple prostate basal cell populations and cell type-specific gene signatures within prostate basal epithelial cells that have potential critical roles in driving prostatic diseases. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Hiperplasia Prostática , Neoplasias de la Próstata , Masculino , Humanos , Próstata/patología , Transcriptoma , Hiperplasia Prostática/patología , Neoplasias de la Próstata/patología , Células Epiteliales/patología , Análisis de Secuencia de ARNRESUMEN
The molecular crosstalk between the interleukin 7 receptor (IL-7R) and the precursor to the B cell antigen receptor (pre-BCR) in B lymphopoiesis has not been elucidated. Here we demonstrate that in pre-B cells, the IL-7R but not the pre-BCR was coupled to phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt; signaling by this pathway inhibited expression of recombination-activating gene 1 (Rag1) and Rag2. Attenuation of IL-7 signaling resulted in upregulation of the transcription factors Foxo1 and Pax5, which coactivated many pre-B cell genes, including Rag1, Rag2 and Blnk. Induction of Blnk (which encodes the signaling adaptor BLNK) enabled pre-BCR signaling via the signaling molecule Syk and promoted immunoglobulin light-chain rearrangement. BLNK expression also antagonized Akt activation, thereby augmenting the accumulation of Foxo1 and Pax5. This self-reinforcing molecular circuit seemed to sense limiting concentrations of IL-7 and functioned to constrain the proliferation of pre-B cells and trigger their differentiation.
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Linfocitos B/inmunología , Diferenciación Celular , Interleucina-7/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal , Animales , Linfocitos B/citología , Células Cultivadas , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/inmunología , Ratones , Factor de Transcripción PAX5/inmunología , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
We previously discovered using transcriptomics that rats undergoing withdrawal after chronic ethanol exposure had increased expression of several genes encoding RNA splicing factors in the hippocampus. Here, we examined RNA splicing in the rat hippocampus during withdrawal from chronic ethanol exposure and in postmortem hippocampus of human subjects diagnosed with alcohol use disorder (AUD). We found that expression of the gene encoding the splicing factor, poly r(C) binding protein 1 (PCBP1), was elevated in the hippocampus of rats during withdrawal after chronic ethanol exposure and AUD subjects. We next analyzed the rat RNA-Seq data for differentially expressed (DE) exon junctions. One gene, Hapln2, had increased usage of a novel 3' splice site in exon 4 during withdrawal. This splice site was conserved in human HAPLN2 and was used more frequently in the hippocampus of AUD compared to control subjects. To establish a functional role for PCBP1 in HAPLN2 splicing, we performed RNA immunoprecipitation (RIP) with a PCBP1 antibody in rat and human hippocampus, which showed enriched PCBP1 association near the HAPLN2 exon 4 3' splice site in the hippocampus of rats during ethanol withdrawal and AUD subjects. Our results indicate a conserved role for the splicing factor PCBP1 in aberrant splicing of HAPLN2 after chronic ethanol exposure. As the HAPLN2 gene encodes an extracellular matrix protein involved in nerve conduction velocity, use of this alternative splice site is predicted to result in loss of protein function that could negatively impact hippocampal function in AUD.
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Alcoholismo , Sitios de Empalme de ARN , Humanos , Ratas , Animales , Empalme del ARN/genética , Etanol/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Alcoholismo/genética , Alcoholismo/metabolismo , Hipocampo/metabolismo , Empalme Alternativo/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Monocytes (Mos)/macrophages (MÏs) orchestrate biological processes critical for efficient skin wound healing. However, current understanding of skin wound Mo/MÏ heterogeneity is limited by traditional experimental approaches such as flow cytometry and immunohistochemistry. Therefore, we sought to more fully explore Mo/MÏ heterogeneity and associated state transitions during the course of excisional skin wound healing in mice using single-cell RNA sequencing. The live CD45+CD11b+Ly6G- cells were isolated from skin wounds of C57BL/6 mice on days 3, 6, and 10 postinjury and captured using the 10x Genomics Chromium platform. A total of 2813 high-quality cells were embedded into a uniform manifold approximation and projection space, and eight clusters of distinctive cell populations were identified. Cluster dissimilarity and differentially expressed gene analysis categorized those clusters into three groups: early-stage/proinflammatory, late-stage/prohealing, and Ag-presenting phenotypes. Signature gene and Gene Ontology analysis of each cluster provided clues about the different functions of the Mo/MÏ subsets, including inflammation, chemotaxis, biosynthesis, angiogenesis, proliferation, and cell death. Quantitative PCR assays validated characteristics of early- versus late-stage Mos/MÏs inferred from our single-cell RNA sequencing dataset. Additionally, cell trajectory analysis by pseudotime and RNA velocity and adoptive transfer experiments indicated state transitions between early- and late-state Mos/MÏs as healing progressed. Finally, we show that the chemokine Ccl7, which was a signature gene for early-stage Mos/MÏs, preferentially induced the accumulation of proinflammatory Ly6C+F4/80lo/- Mos/MÏs in mouse skin wounds. In summary, our data demonstrate the complexity of Mo/MÏ phenotypes, their dynamic behavior, and diverse functions during normal skin wound healing.
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Leucocitos , Monocitos , Ratones , Animales , Ratones Endogámicos C57BL , Cicatrización de Heridas/genética , MacrófagosRESUMEN
During B lymphopoiesis, recombination of the locus encoding the immunoglobulin κ-chain complex (Igk) requires expression of the precursor to the B cell antigen receptor (pre-BCR) and escape from signaling via the interleukin 7 receptor (IL-7R). By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that a STAT5 tetramer bound the Igk intronic enhancer (E(κi)), which led to recruitment of the histone methyltransferase Ezh2. Ezh2 marked trimethylation of histone H3 at Lys27 (H3K27me3) throughout the κ-chain joining region (J(κ)) to the κ-chain constant region (C(κ)). In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R-STAT5 signaling, and the transcription factor E2A bound E(κi), which resulted in acquisition of H3K4me1 and acetylated histone H4 (H4Ac). Genome-wide analyses showed a STAT5 tetrameric binding motif associated with transcriptional repression. Our data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression.
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Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Inmunoglobulinas/genética , Factor de Transcripción STAT5/metabolismo , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión/genética , Unión Competitiva , Células COS , Chlorocebus aethiops , Análisis por Conglomerados , Proteína Potenciadora del Homólogo Zeste 2 , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Histonas/metabolismo , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Complejo Represivo Polycomb 2 , Unión Proteica , Receptores de Interleucina-7/metabolismo , Transducción de SeñalRESUMEN
Positive effects of alcohol drinking such as anxiolysis and euphoria appear to be a crucial factor in the initiation and maintenance of alcohol use disorder (AUD). However, the mechanisms that lead from chromatin reorganization to transcriptomic changes after acute ethanol exposure remain unknown. Here, we used Assay for Transposase-Accessible Chromatin followed by high throughput sequencing (ATAC-seq) and RNA-seq to investigate epigenomic and transcriptomic changes that underlie anxiolytic effects of acute ethanol using an animal model. Analysis of ATAC-seq data revealed an overall open or permissive chromatin state that was associated with transcriptomic changes in the amygdala after acute ethanol exposure. We identified a candidate gene, Hif3a (Hypoxia-inducible factor 3, alpha subunit), that had 'open' chromatin regions (ATAC-seq peaks), associated with significantly increased active epigenetic histone acetylation marks and decreased DNA methylation at these regions. The mRNA levels of Hif3a were increased by acute ethanol exposure, but decreased in the amygdala during withdrawal after chronic ethanol exposure. Knockdown of Hif3a expression in the central nucleus of amygdala attenuated acute ethanol-induced increases in Hif3a mRNA levels and blocked anxiolysis in rats. These data indicate that chromatin accessibility and transcriptomic signatures in the amygdala after acute ethanol exposure underlie anxiolysis and possibly prime the chromatin for the development of AUD.
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Alcoholismo , Epigénesis Genética , Animales , Ratas , Epigénesis Genética/genética , Etanol/farmacología , Cromatina , Perfilación de la Expresión Génica , Alcoholismo/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genéticaRESUMEN
[Figure: see text].
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Proliferación Celular , Células Endoteliales/metabolismo , Endotoxemia/metabolismo , Pulmón/irrigación sanguínea , Lisofosfolípidos/metabolismo , Regeneración , Receptores de Esfingosina-1-Fosfato/metabolismo , Esfingosina/análogos & derivados , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Permeabilidad Capilar , Linaje de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Endotoxemia/genética , Endotoxemia/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones Noqueados , Fenotipo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/genéticaRESUMEN
Diabetic wounds are characterized by persistent accumulation of proinflammatory monocytes (Mo)/macrophages (MΦ) and impaired healing. However, the mechanisms underlying the persistent accumulation of Mo/MΦ remain poorly understood. In this study, we report that Ly6C+F4/80lo/- Mo/MΦ proliferate at higher rates in wounds of diabetic mice compared with nondiabetic mice, leading to greater accumulation of these cells. Unbiased single cell RNA sequencing analysis of combined nondiabetic and diabetic wound Mo/MΦ revealed a cluster, populated primarily by cells from diabetic wounds, for which genes associated with the cell cycle were enriched. In a screen of potential regulators, CCL2 levels were increased in wounds of diabetic mice, and subsequent experiments showed that local CCL2 treatment increased Ly6C+F4/80lo/- Mo/MΦ proliferation. Importantly, adoptive transfer of mixtures of CCR2-/- and CCR2+/+ Ly6Chi Mo indicated that CCL2/CCR2 signaling is required for their proliferation in the wound environment. Together, these data demonstrate a novel role for the CCL2/CCR2 signaling pathway in promoting skin Mo/MΦ proliferation, contributing to persistent accumulation of Mo/MΦ and impaired healing in diabetic mice.
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Complicaciones de la Diabetes/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Piel/patología , Animales , Antígenos Ly/metabolismo , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Cicatrización de HeridasRESUMEN
BACKGROUND: Acute ischemic stroke is one of the leading causes of death and disability globally. Recent advances in omics methodology enable lipidomic profiling, which may provide knowledge of the underlying pathology of acute ischemic stroke and its associated outcomes. OBJECTIVE: This study aims to examine the longer-term relationships between symptoms and outcomes following acute ischemic stroke and the underlying lipidomic signatures over 6 months during recovery between acute ischemic stroke patients who received reperfusion therapies and those who did not. METHODS: This prospective cohort study will enroll 104 participants post-acute ischemic stroke in two groups based on their receipt of reperfusion therapy (Group 1) or not (Group 2; n = 52/group). Peripheral plasma samples will be collected from both groups for lipidomic analysis over 6 months. Arterial blood samples will be collected during the procedure for those receiving reperfusion. Self-reported symptoms and outcome data will be collected from both groups. DISCUSSION: We will compare and examine the associations among plasma lipidomic biomarkers and symptoms and cognitive, functional, and health-related quality of life outcomes over 6 months between acute ischemic stroke patients who did and did not receive reperfusion intervention.
Asunto(s)
Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Accidente Cerebrovascular/terapia , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular Isquémico/complicaciones , Isquemia Encefálica/terapia , Isquemia Encefálica/complicaciones , Isquemia Encefálica/diagnóstico , Estudios Prospectivos , Calidad de Vida , Lipidómica , Resultado del Tratamiento , Estudios Observacionales como AsuntoRESUMEN
Combination antiretroviral therapy (cART) dramatically changed the face of the HIV/AIDS pandemic, making it one of the most prominent medical breakthroughs of the past 3 decades. However, as the life span of persons living with HIV (PLWH) continues to approach that of the general population, the same cannot be said regarding their quality of life. PLWH are affected by comorbid conditions such as high blood pressure, diabetes, and neurocognitive impairment at a higher rate and increased severity than their age-matched counterparts. PLWH also have higher levels of inflammation, the drivers of which are not entirely clear. As cART treatment is lifelong, we assessed here the effects of cART, independent of HIV, on primary human monocyte-derived macrophages (MDMs). MDMs were unskewed or skewed to an alternative phenotype and treated with Atripla or Triumeq, two first-line cART treatments. We report that Triumeq skewed alternative MDMs toward an inflammatory nonsenescent phenotype. Both Atripla and Triumeq caused mitochondrial dysfunction, specifically efavirenz and abacavir. Additionally, transcriptome sequencing (RNA-seq) demonstrated that both Atripla and Triumeq caused differential regulation of genes involved in immune regulation and cell cycle and DNA repair. Collectively, our data demonstrate that cART, independent of HIV, alters the MDM phenotype. This suggests that cART may contribute to cell dysregulation in PLWH that subsequently results in increased susceptibility to comorbidities.