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BACKGROUND: An HIV-1 DNA vaccine composed of seven highly conserved, structurally important elements (Conserved Elements, CE) of HIV p24Gag was tested in a phase I randomized, double-blind clinical trial (HVTN 119, NCT03181789) in people without HIV. A CE prime- CE+full-length p55Gag boost DNA vaccine was compared to p55Gag DNA vaccination only. METHODS: Two groups (n=25 each) received 4 DNA vaccinations [2xCE prime- 2xCE+p55Gag boost or 4x p55Gag] by intramuscular injection/electroporation, including IL-12 DNA adjuvant. The placebo group (n=6) received saline. Participants were followed for safety and tolerability. Immunogenicity was assessed for T cell and antibody responses. RESULTS: Both regimens were safe and generally well-tolerated. The p24CE vaccine was immunogenic (29% CD4+ and 4% CD8+ responders) and was significantly boosted by CE+p55Gag (64% CD4+, p=0.037; 42% CD8+, p=0.004). CE+p55Gag induced CD4+ responses to 5 of 7 CE, compared to only 2 CE by p55Gag DNA alone, with a higher reponse to CE5 in 30% of individuals (p=0.006). CE+p55Gag induced significantly higher mean CD4+ CE Tcell breadth (0.68 vs 0.22 CE; p=0.029) and a strong trend for increased CD4+ and CD8+ T-cell breadth (1.14 vs. 0.52 CE; p=0.051) compared to p55Gag alone. Both groups developed high p55Gag T-cell (91% each) and p24Gag antibody (91% vs. 80%) responses. p24CE vaccine-induced CD4+ CE T-cell responses correlated (p=0.007) with p24Gag antibody responses. CONCLUSION: The combination CE/CE+p55Gag DNA vaccine induced T-cell immune responses to conserved regions in p24Gag resulting in significant increases in breadth and epitope recognition throughout p55Gag. Vaccines able to focus immune responses by priming responses to highly conserved regions could be part of a comprehensive HIV vaccine strategy. CLINICAL TRIALS: gov NCT03181789 Study URL: https://www. CLINICALTRIALS: gov/search?term=NCT03181789 FUNDING. HIV vaccine trial network (HVTN), NIAID/NIH.
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Zn2+ is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment. We found 685 transcription factor motifs were differentially enriched, corresponding to 507 unique transcription factors. The pattern of perturbation and the types of transcription factors were notably different at promoters versus intergenic regions, with zinc-finger transcription factors strongly enriched in intergenic regions in elevated Zn2+ To test whether ATAC-seq and transcription factor enrichment analysis predictions correlate with changes in transcription factor binding, we used ChIP-qPCR to profile six p53 binding sites. We found that for five of the six targets, p53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc alter chromatin accessibility and transcription factor binding to DNA.
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Cromatina , DNA , Ligação Proteica , Fatores de Transcrição , Proteína Supressora de Tumor p53 , Zinco , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Cromatina/metabolismo , Cromatina/genética , Zinco/metabolismo , DNA/metabolismo , DNA/genética , Sítios de Ligação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regiões Promotoras Genéticas/genética , Sequenciamento de Cromatina por Imunoprecipitação/métodosRESUMO
BACKGROUND: An effective HIV vaccine will most likely need to have potent immunogenicity and broad cross-subtype coverage. The aim of the HIV Vaccine Trials Network (HVTN) 124 was to evaluate safety and immunogenicity of a unique polyvalent DNA-protein HIV vaccine with matching envelope (Env) immunogens. METHODS: HVTN 124 was a randomised, phase 1, placebo-controlled, double-blind study, including participants who were HIV seronegative and aged 18-50 years at low risk for infection. The DNA vaccine comprised five plasmids: four copies expressing Env gp120 (clades A, B, C, and AE) and one gag p55 (clade C). The protein vaccine included four DNA vaccine-matched GLA-SE-adjuvanted recombinant gp120 proteins. Participants were enrolled across six clinical sites in the USA and were randomly assigned to placebo or one of two vaccine groups (ie, prime-boost or coadministration) in a 5:1 ratio in part A and a 7:1 ratio in part B. Vaccines were delivered via intramuscular needle injection. The primary outcomes were safety and tolerability, assessed via frequency, severity, and attributability of local and systemic reactogenicity and adverse events, laboratory safety measures, and early discontinuations. Part A evaluated safety. Part B evaluated safety and immunogenicity of two regimens: DNA prime (administered at months 0, 1, and 3) with protein boost (months 6 and 8), and DNA-protein coadministration (months 0, 1, 3, 6, and 8). All randomly assigned participants who received at least one dose were included in the safety analysis. The study is registered with ClinicalTrials.gov (NCT03409276) and is closed to new participants. FINDINGS: Between April 19, 2018 and Feb 13, 2019, 60 participants (12 in part A [five men and seven women] and 48 in part B [21 men and 27 women]) were enrolled. All 60 participants received at least one dose, and 14 did not complete follow-up (six of 21 in the prime-boost group and eight of 21 in the coadminstration group). 11 clinical adverse events deemed by investigators as study-related occurred in seven of 48 participants in part B (eight of 21 in the prime-boost group and three of 21 in the coadministration group). Local reactogenicity in the vaccine groups was common, but the frequency and severity of reactogenicity signs or symptoms did not differ between the prime-boost and coadministration groups (eg, 20 [95%] of 21 in the prime-boost group vs 21 [100%] of 21 in the coadministration group had either local pain or tenderness of any severity [p=1·00], and seven [33%] vs nine [43%] had either erythema or induration [p=0·97]), nor did laboratory safety measures. There were no delayed-type hypersensitivity reactions or vasculitis or any severe clinical adverse events related to vaccination. The most frequently reported systemic reactogenicity symptoms in the active vaccine groups were malaise or fatigue (five [50%] of ten in part A and 17 [81%] of 21 in the prime-boost group vs 15 [71%] of 21 in the coadministration group in part B), headache (five [50%] and 18 [86%] vs 12 [57%]), and myalgia (four [40%] and 13 [62%] vs ten [48%]), mostly of mild or moderate severity. INTERPRETATION: Both vaccine regimens were safe, warranting evaluation in larger trials. FUNDING: US National Institutes of Health and US National Institute of Allergy and Infectious Diseases.
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Vacinas contra a AIDS , Anticorpos Anti-HIV , Infecções por HIV , HIV-1 , Vacinas de DNA , Humanos , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/efeitos adversos , Adulto , Masculino , Feminino , Método Duplo-Cego , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas de DNA/efeitos adversos , Infecções por HIV/prevenção & controle , Infecções por HIV/imunologia , Pessoa de Meia-Idade , Adulto Jovem , Anticorpos Anti-HIV/sangue , Adolescente , HIV-1/imunologia , Estados Unidos , Imunização Secundária , Imunogenicidade da Vacina , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/genética , Anticorpos Neutralizantes/sangueRESUMO
TF profiler is a method of inferring transcription factor regulatory activity, i.e. when a TF is present and actively regulating transcription, directly directly from nascent sequencing assays such as PRO-seq and GRO-seq. Transcription factors orchestrate transcription and play a critical role in cellular maintenance, identity and response to external stimuli. While ChIP assays have measured DNA localization, they fall short of identifying when and where transcription factors are actively regulating transcription. Our method, on the other hand, uses RNA polymerase activity to infer TF activity across hundreds of data sets and transcription factors. Based on these classifications we identify three distinct classes of transcription factors: ubiquitous factors that play roles in cellular homeostasis, driving basal gene programs across tissues and cell types, tissue specific factors that act almost exclusively at enhancers and are themselves regulated at transcription, and stimulus responsive TFs which are regulated post-transcriptionally but act predominantly at enhancers. TF profiler is broadly applicable, providing regulatory insights on any PRO-seq sample for any transcription factor with a known binding motif.
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Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.
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Vírus da Dengue , HIV-1 , Células-Tronco Pluripotentes Induzidas , Macrófagos , Modelos Biológicos , Orthomyxoviridae , Virologia , Animais , Humanos , Diferenciação Celular/genética , HIV-1/crescimento & desenvolvimento , HIV-1/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/virologia , Orthomyxoviridae/crescimento & desenvolvimento , Orthomyxoviridae/fisiologia , Pan troglodytes , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/fisiologia , Fibroblastos/citologia , Monócitos/citologia , Replicação Viral , Citometria de Fluxo , Perfilação da Expressão Gênica , Montagem e Desmontagem da Cromatina , Tropismo Viral , Virologia/métodos , Biomarcadores/análise , Biomarcadores/metabolismoRESUMO
Zinc (Zn2+) is an essential metal required by approximately 2500 proteins. Nearly half of these proteins act on DNA, including > 850 human transcription factors, polymerases, DNA damage response factors, and proteins involved in chromatin architecture. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Here, we examine how changes in the labile Zn2+ pool affect chromatin accessibility and transcription factor binding to DNA. We observed both increases and decreases in accessibility in different chromatin regions via ATAC-seq upon treating MCF10A cells with elevated Zn2+ or the Zn2+-specific chelator tris(2-pyridylmethyl)amine (TPA). Transcription factor enrichment analysis was used to correlate changes in chromatin accessibility with transcription factor motifs, revealing 477 transcription factor motifs that were differentially enriched upon Zn2+ perturbation. 186 of these transcription factor motifs were enriched in Zn2+ and depleted in TPA, and the majority correspond to Zn2+ finger transcription factors. We selected TP53 as a candidate to examine how changes in motif enrichment correlate with changes in transcription factor occupancy by ChIP-qPCR. Using publicly available ChIP-seq and nascent transcription datasets, we narrowed the 50,000+ ATAC-seq peaks to 2164 TP53 targets and subsequently selected 6 high-probability TP53 binding sites for testing. ChIP-qPCR revealed that for 5 of the 6 targets, TP53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc directly alter chromatin accessibility and transcription factor binding to DNA.
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Gene transcription is controlled and modulated by regulatory regions, including enhancers and promoters. These regions are abundant in unstable, non-coding bidirectional transcription. Using nascent RNA transcription data across hundreds of human samples, we identified over 800,000 regions containing bidirectional transcription. We then identify highly correlated transcription between bidirectional and gene regions. The identified correlated pairs, a bidirectional region and a gene, are enriched for disease associated SNPs and often supported by independent 3D data. We present these resources as an SQL database which serves as a resource for future studies into gene regulation, enhancer associated RNAs, and transcription factors.
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BACKGROUND: The increase in DNA copy number in Down syndrome (DS; caused by trisomy 21) has led to the DNA dosage hypothesis, which posits that the level of gene expression is proportional to the gene's DNA copy number. Yet many reports have suggested that a proportion of chromosome 21 genes are dosage compensated back towards typical expression levels (1.0×). In contrast, other reports suggest that dosage compensation is not a common mechanism of gene regulation in trisomy 21, providing support to the DNA dosage hypothesis. RESULTS: In our work, we use both simulated and real data to dissect the elements of differential expression analysis that can lead to the appearance of dosage compensation, even when compensation is demonstrably absent. Using lymphoblastoid cell lines derived from a family with an individual with Down syndrome, we demonstrate that dosage compensation is nearly absent at both nascent transcription (GRO-seq) and steady-state RNA (RNA-seq) levels. Furthermore, we link the limited apparent dosage compensation to expected allelic variation in transcription levels. CONCLUSIONS: Transcription dosage compensation does not occur in Down syndrome. Simulated data containing no dosage compensation can appear to have dosage compensation when analyzed via standard methods. Moreover, some chromosome 21 genes that appear to be dosage compensated are consistent with allele specific expression.
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Síndrome de Down , Humanos , Síndrome de Down/genética , Cromossomo X , Mecanismo Genético de Compensação de Dose , Regulação da Expressão Gênica , DNARESUMO
OBJECTIVES: The glucocorticoid receptor (GR) is a well-studied, ligand-activated transcription factor and a common target of anti-inflammatory treatments. Recently, several studies have drawn attention the effects of binding of GR to RNA rather than DNA and the potential implications of this activity for GR function. The objective of our study was to further characterize the relationship between GR function and RNA binding by measuring changes in the glucocorticoid-driven transcriptome in the presence of a GR mutant that exhibited reduced RNA affinity. DATA DESCRIPTION: GR was activated in three cell lines containing GR constructs (GR-HaloTag). One of the cell lines contained a wild-type GR-HaloTag. Another contained GR-HaloTag with a mutation that reduced RNA affinity and slightly reduced DNA affinity. The third cell line contained GR-HaloTag with a mutation that only slightly reduced DNA affinity. All three cell lines were treated with dexamethasone, a GR agonist. RNA-seq samples were collected every hour for 3 h. Moreover, transcriptome quantification was accomplished via labeling of RNAs transcribed in the final hour of dexamethasone treatment using 4-thiouridine. These labeled RNAs were then purified and sequenced. This data set is the first of its kind for GR and contains valuable insights into the function of RNA binding by GR.
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Receptores de Glucocorticoides , Transcriptoma , Receptores de Glucocorticoides/genética , Glucocorticoides/farmacologia , RNA , Dexametasona/farmacologiaRESUMO
Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A, and this occurred through suppression of IFN-responsive transcription factor activity. Moreover, we discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. Kinase inhibition altered splicing in pathway-specific ways and selectively affected IFN-responsive gene splicing in T21 cells. To further probe Mediator kinase function, we completed cytokine screens and untargeted metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways, including broad up-regulation of anti-inflammatory lipid mediators. Elevated levels of lipid mediators persisted at least 24hr after Mediator kinase inhibition, and many identified lipids serve as ligands for nuclear receptors (e.g. PPAR, LXR) or G-protein coupled receptors (GPCRs; e.g. FFAR4). Notably, ligand-dependent activation of these GPCRs or nuclear receptors will propagate anti-inflammatory signaling pathways and gene expression programs, and this mechanistic link suggests that metabolic changes caused by CDK8/CDK19 inhibition can durably and independently suppress pro-inflammatory IFN responses. Collectively, our results establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.
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Background: Trisomy 21, also known as Down syndrome, describes the genetic condition of having an extra copy of chromosome 21. The increase in DNA copy number has led to the "DNA dosage hypothesis", which claims that the level of gene transcription is proportional to the gene's DNA copy number. Yet many reports have suggested that a proportion of chromosome 21 genes are dosage compensated back towards typical expression levels (1.0x). In contrast, other reports suggest that dosage compensation is not a common mechanism of gene regulation in Trisomy 21, providing support to the DNA dosage hypothesis. Results: In our work, we use both simulated and real data to dissect the elements of differential expression analysis that can lead to the appearance of dosage compensation even when compensation is demonstrably absent. Using lymphoblastoid cell lines derived from a family of an individual with Down syndrome, we demonstrate that dosage compensation is nearly absent at both nascent transcription (GRO-seq) and steady-state RNA (RNA-seq) levels. Conclusions: Transcriptional dosage compensation does not occur in Down syndrome. Simulated data containing no dosage compensation can appear to have dosage compensation when analyzed via standard methods. Moreover, some chromosome 21 genes that appear to be dosage compensated are consistent with allele specific expression.
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The glucocorticoid receptor (GR) is a ligand-activated transcription factor that regulates a suite of genes through direct binding of GR to specific DNA promoter elements. GR also interacts with RNA, but the function of this RNA-binding activity remains elusive. Current models speculate that RNA could repress the transcriptional activity of GR. To investigate the function of the GR-RNA interaction on GR's transcriptional activity, we generated cells that stably express a mutant of GR with reduced RNA binding affinity and treated the cells with the GR agonist dexamethasone. Changes in the dexamethasone-driven transcriptome were quantified using 4-thiouridine labeling of RNAs followed by high-throughput sequencing. We find that while many genes are unaffected, GR-RNA binding is repressive for specific subsets of genes in both dexamethasone-dependent and independent contexts. Genes that are dexamethasone-dependent are activated directly by chromatin-bound GR, suggesting a competition-based repression mechanism in which increasing local concentrations of RNA may compete with DNA for binding to GR at sites of transcription. Unexpectedly, genes that are dexamethasone-independent instead display a localization to specific chromosomal regions, which points to changes in chromatin accessibility or architecture. These results show that RNA binding plays a fundamental role in regulating GR function and highlights potential functions for transcription factor-RNA interactions.
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Dexametasona , Receptores de Glucocorticoides , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional , Dexametasona/farmacologia , Dexametasona/metabolismo , Fatores de Transcrição/metabolismo , Glucocorticoides/farmacologia , Cromatina , DNA/metabolismo , RNA , Sítios de LigaçãoRESUMO
Aortic valve stenosis (AVS) is a progressive fibrotic disease that is caused by thickening and stiffening of valve leaflets. At the cellular level, quiescent valve interstitial cells (qVICs) activate to myofibroblasts (aVICs) that persist within the valve tissue. Given the persistence of myofibroblasts in AVS, epigenetic mechanisms have been implicated. Here, we studied changes that occur in VICs during myofibroblast activation by using a hydrogel matrix to recapitulate different stiffnesses in the valve leaflet during fibrosis. We first compared the chromatin landscape of qVICs cultured on soft hydrogels and aVICs cultured on stiff hydrogels, representing the native and diseased phenotypes respectively. Using assay for transposase-accessible chromatin sequencing (ATAC-Seq), we found that open chromatin regions in aVICs were enriched for transcription factor binding motifs associated with mechanosensing pathways compared to qVICs. Next, we used RNA-Seq to show that the open chromatin regions in aVICs correlated with pro-fibrotic gene expression, as aVICs expressed higher levels of contractile fiber genes, including myofibroblast markers such as alpha smooth muscle actin (αSMA), compared to qVICs. In contrast, chromatin remodeling genes were downregulated in aVICs compared to qVICs, indicating qVICs may be protected from myofibroblast activation through epigenetic mechanisms. Small molecule inhibition of one of these remodelers, CREB Binding Protein (CREBBP), prevented qVICs from activating to aVICs. Notably, CREBBP is more abundant in valves from healthy patients compared to fibrotic valves. Our findings reveal the role of mechanical regulation in chromatin remodeling during VIC activation and quiescence and highlight one potential therapeutic target for treating AVS.
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BACKGROUND: A variety of protocols exist for producing whole genome run-on transcription datasets. However, little is known about how differences between these protocols affect the signal within the resulting libraries. RESULTS: Using run-on transcription datasets generated from the same biological system, we show that a variety of GRO- and PRO-seq preparation methods leave identifiable signatures within each library. Specifically we show that the library preparation method results in differences in quality control metrics, as well as differences in the signal distribution at the 5 ' end of transcribed regions. These shifts lead to disparities in eRNA identification, but do not impact analyses aimed at inferring the key regulators involved in changes to transcription. CONCLUSIONS: Run-on sequencing protocol variations result in technical signatures that can be used to identify both the enrichment and library preparation method of a particular data set. These technical signatures are batch effects that limit detailed comparisons of pausing ratios and eRNAs identified across protocols. However, these batch effects have only limited impact on our ability to infer which regulators underlie the observed transcriptional changes.
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Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Controle de Qualidade , Transcrição GênicaRESUMO
Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron, increasing exposure to bactericidal copper, and altering zinc to restrict the growth of pathogens. Here, we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella enterica serovar Typhimurium. The 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations that plays an important role in modulating metal availability to the pathogen. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 h and 18 h postinfection and observed widespread changes in metal transport, metal-dependent genes, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 45-fold at 12 h postinfection. Further, manipulation of zinc in the medium alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing the transcriptomic changes to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response. Our results reveal that 129S6 macrophages represent a distinct model system compared to C57BL/6 macrophages. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. The 129S6 macrophages leverage intricate means of manipulating zinc availability and distribution to limit the pathogen's access to zinc, while simultaneously ensuring sufficient zinc to support the immune response.
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Macrófagos/imunologia , Metais/metabolismo , Salmonelose Animal/imunologia , Animais , Proteínas do Sistema Complemento/imunologia , Feminino , Expressão Gênica , Interações Hospedeiro-Patógeno , Camundongos , Camundongos Endogâmicos C57BL , Salmonella typhimurium , Zinco/metabolismoRESUMO
Fibrotic disease is caused by the continuous deposition of extracellular matrix by persistently activated fibroblasts (also known as myofibroblasts), even after the resolution of the injury. Using fibroblasts from porcine aortic valves cultured on hydrogels that can be softened via exposure to ultraviolet light, here we show that increased extracellular stiffness activates the fibroblasts, and that cumulative tension on the nuclear membrane and increases in the activity of histone deacetylases transform transiently activated fibroblasts into myofibroblasts displaying condensed chromatin with genome-wide alterations. The condensed structure of the myofibroblasts is associated with cytoskeletal stability, as indicated by the inhibition of chromatin condensation and myofibroblast persistence after detachment of the nucleus from the cytoskeleton via the displacement of endogenous nesprins from the nuclear envelope. We also show that the chromatin structure of myofibroblasts from patients with aortic valve stenosis is more condensed than that of myofibroblasts from healthy donors. Our findings suggest that nuclear mechanosensing drives distinct chromatin signatures in persistently activated fibroblasts.
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Montagem e Desmontagem da Cromatina , Fibroblastos , Animais , Diferenciação Celular , Matriz Extracelular , Humanos , Miofibroblastos , SuínosRESUMO
We studied mucosal immune responses in six HIV-1 vaccine trials investigating different envelope (Env)-containing immunogens. Regimens were classified into four categories: DNA/vector, DNA/vector plus protein, protein alone, and vector alone. We measured HIV-1-specific IgG and IgA in secretions from cervical (n = 111) and rectal swabs (n = 154), saliva (n = 141), and seminal plasma (n = 124) and compared to corresponding blood levels. Protein-containing regimens had up to 100% response rates and the highest Env-specific IgG response rates. DNA/vector groups elicited mucosal Env-specific IgG response rates of up to 67% that varied across specimen types. Little to no mucosal IgA responses were observed. Overall, gp41- and gp140-specific antibodies dominated gp120 mucosal responses. In one trial, prior vaccination with a protein-containing immunogen maintained durability of cervical and rectal IgG for up to 17 years. Mucosal IgG responses were boosted after revaccination. These findings highlight a role for protein immunization in eliciting HIV-1-specific mucosal antibodies and the ability of HIV-1 vaccines to elicit durable HIV-1-specific mucosal IgG.
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Flaviviruses such as dengue encode a protease that is essential for viral replication. The protease functions by cleaving well-conserved positions in the viral polyprotein. In addition to the viral polyprotein, the dengue protease cleaves at least one host protein involved in immune response. This raises the question, what other host proteins are targeted and cleaved? Here we present a new computational method for identifying putative host protein targets of the dengue virus protease. Our method relies on biochemical and secondary structure features at the known cleavage sites in the viral polyprotein in a two-stage classification process to identify putative cleavage targets. The accuracy of our predictions scaled inversely with evolutionary distance when we applied it to the known cleavage sites of several other flaviviruses-a good indication of the validity of our predictions. Ultimately, our classifier identified 257 human protein sites possessing both a similar target motif and accessible local structure. These proteins are promising candidates for further investigation. As the number of viral sequences expands, our method could be adopted to predict host targets of other flaviviruses.
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Dengue , Peptídeo Hidrolases , Biologia Computacional , Humanos , Proteínas não Estruturais ViraisRESUMO
Nascent transcription assays, such as global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), have uncovered a myriad of unstable RNAs being actively produced from numerous sites genome-wide. These transcripts provide a more complete and immediate picture of the impact of regulatory events. Transcription factors recruit RNA polymerase II, effectively initiating the process of transcription; repressors inhibit polymerase recruitment. Efficiency of recruitment is dictated by sequence elements in and around the RNA polymerase loading zone. A combination of sequence elements and RNA binding proteins subsequently influence the ultimate stability of the resulting transcript. Some of these transcripts are capable of providing feedback on the process, influencing subsequent transcription. By monitoring RNA polymerase activity, nascent assays provide insights into every step of the regulated process of transcription.