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Human and simian immunodeficiency viruses (HIV and SIV) are lentiviruses that reverse transcribe their RNA genome with subsequent integration into the genome of the target cell. How progressive infection and administration of antiretrovirals (ARVs) longitudinally influence the transcriptomic and epigenetic landscape of particular T cell subsets, and how these may influence the genetic location of integration are unclear. Here, we use RNAseq and ATACseq to study the transcriptomics and epigenetic landscape of longitudinally sampled naïve and memory CD4+ and CD8+ T cells in two species of non-human primates prior to SIV infection, during chronic SIV infection, and after administration of ARVs. We find that SIV infection leads to significant alteration to the transcriptomic profile of all T cell subsets that are only partially reversed by administration of ARVs. Epigenetic changes were more apparent in animals with longer periods of untreated SIV infection and correlated well with changes in corresponding gene expression. Known SIV integration sites did not vary due to SIV status but did contain more open chromatin in rhesus macaque memory T cells, and the expression of proteasome-related genes at the pre-SIV timepoint correlated with subsequent viremia.IMPORTANCEChronic inflammation during progressive human and simian immunodeficiency virus (HIV and SIV) infections leads to significant co-morbidities in infected individuals with significant consequences. Antiretroviral (ARV)-treated individuals also manifest increased levels of inflammation which are associated with increased mortalities. These data will help guide rational development of modalities to reduce inflammation observed in people living with HIV and suggest mechanisms underlying lentiviral integration site preferences.
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Antirretrovirais , Epigênese Genética , Células T de Memória , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Transcriptoma , Animais , Antirretrovirais/uso terapêutico , Antirretrovirais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Epigênese Genética/efeitos dos fármacos , Macaca mulatta/genética , Macaca mulatta/imunologia , Macaca mulatta/virologia , Macaca nemestrina/genética , Macaca nemestrina/imunologia , Macaca nemestrina/virologia , Células T de Memória/efeitos dos fármacos , Células T de Memória/imunologia , Células T de Memória/metabolismo , Células T de Memória/virologia , Complexo de Endopeptidases do Proteassoma/genética , RNA-Seq , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Viremia/tratamento farmacológico , Viremia/genética , Viremia/imunologia , Viremia/virologiaRESUMO
HBV is the most common risk factor for HCC development, accounting for almost 50% of cases worldwide. Despite significant advances in immunotherapy, there is limited information on the HBV-HCC tumor microenvironment (TME), which may influence the response to checkpoint inhibitors. Here, we characterize the TME in a unique series of liver specimens from HBV-HCC patients to identify who might benefit from immunotherapy. By combining an extensive immunohistochemistry analysis with the transcriptomic profile of paired liver samples (tumor vs. nontumorous tissue) from 12 well-characterized Caucasian patients with HBV-HCC, we identified two distinct tumor subtypes that we defined immune-high and immune-low. The immune-high subtype, seen in half of the patients, is characterized by a high number of infiltrating B and T cells in association with stromal activation and a transcriptomic profile featuring inhibition of antigen presentation and CTL activation. All the immune-high tumors expressed high levels of CTLA-4 and low levels of PD-1, while PD-L1 was present only in four of six cases. In contrast, the immune-low subtype shows significantly lower lymphocyte infiltration and stromal activation. By whole exome sequencing, we documented that four out of six individuals with the immune-low subtype had missense mutations in the CTNNB1 gene, while only one patient had mutations in this gene in the immune-high subtype. Outside the tumor, there were no differences between the two subtypes. This study identifies two distinctive immune subtypes in HBV-associated HCC, regardless of the microenvironment observed in the surrounding nontumorous tissue, providing new insights into pathogenesis. These findings may be instrumental in the identification of patients who might benefit from immunotherapy.
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ABSTRACT: Macrophages orchestrate tissue immunity from the initiation and resolution of antimicrobial immune responses to the repair of damaged tissue. Murine studies demonstrate that tissue-resident macrophages are a heterogenous mixture of yolk sac-derived cells that populate the tissue before birth, and bone marrow-derived replacements recruited in adult tissues at steady-state and in increased numbers in response to tissue damage or infection. How this translates to species that are constantly under immunologic challenge, such as humans, is unknown. To understand the ontogeny and longevity of tissue-resident macrophages in nonhuman primates (NHPs), we use a model of autologous hematopoietic stem progenitor cell (HSPC) transplantation with HSPCs genetically modified to be marked with clonal barcodes, allowing for subsequent analysis of clonal ontogeny. We study the contribution of HSPCs to tissue macrophages, their clonotypic profiles relative to leukocyte subsets in the peripheral blood, and their transcriptomic and epigenetic landscapes. We find that HSPCs contribute to tissue-resident macrophage populations in all anatomic sites studied. Macrophage clonotypic profiles are dynamic and overlap significantly with the clonal hierarchy of contemporaneous peripheral blood monocytes. Epigenetic and transcriptomic landscapes of HSPC-derived macrophages are similar to tissue macrophages isolated from NHPs that did not undergo transplantation. We also use in vivo bromodeoxyuridine infusions to monitor tissue macrophage turnover in NHPs that did not undergo transplantation and find evidence for macrophage turnover at steady state. These data demonstrate that the life span of most tissue-resident macrophages is limited and can be replenished continuously from HSPCs.
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Células-Tronco Hematopoéticas , Macaca , Humanos , Animais , Camundongos , Macrófagos , Monócitos , Medula ÓsseaRESUMO
Existing monitoring approaches in heart transplantation lack the sensitivity to provide deep molecular assessments to guide management, or require endomyocardial biopsy, an invasive and blind procedure that lacks the precision to reliably obtain biopsy samples from diseased sites. This study examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) as a noninvasive proxy to define molecular gene sets and sources of tissue injury in heart transplant patients. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of relevant immune and nonimmune molecular pathways that were predominantly down-regulated in immunosuppressed heart transplant patients compared with healthy controls. cfChIP-seq also identified cell-free DNA tissue sources. Compared with healthy controls, heart transplant patients demonstrated greater cell-free DNA from tissue types associated with heart transplant complications, including the heart, hematopoietic cells, lungs, liver, and vascular endothelium. cfChIP-seq may therefore be a reliable approach to profile dynamic assessments of molecular pathways and sources of tissue injury in heart transplant patients.
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Ácidos Nucleicos Livres , Transplante de Coração , Humanos , Imunoprecipitação da Cromatina , Coração , Sequenciamento de Cromatina por Imunoprecipitação , Ácidos Nucleicos Livres/genéticaRESUMO
Experimental simian immunodeficiency virus (SIV) infection of Asian macaques is an excellent model for HIV disease progression and therapeutic development. Recent coformulations of nucleoside analogs and an integrase inhibitor have been used for parenteral antiretroviral (ARV) administration in SIV-infected macaques, successfully resulting in undetectable plasma SIV RNA. In a cohort of SIVmac239-infected macaques, we recently observed that administration of coformulated ARVs resulted in an unexpected increase in plasma levels of soluble CD14 (sCD14), associated with stimulation of myeloid cells. We hypothesized that the coformulation solubilizing agent Kleptose (2-hydroxypropyl-ß-cyclodextrin [HPßCD]) may induce inflammation with myeloid cell activation and the release of sCD14. Herein, we stimulated peripheral blood mononuclear cells (PBMCs) from healthy macaques with HPßCD from different commercial sources and evaluated inflammatory cytokine production in vitro. Treatment of PBMCs resulted in increased sCD14 release and myeloid cell interleukin-1ß (IL-1ß) production-with stimulation varying significantly by HPßCD source-and destabilized lymphocyte CCR5 surface expression. We further treated healthy macaques with Kleptose alone. In vivo, we observed modestly increased myeloid cell activation in response to Kleptose treatment without significant perturbation of the immunological transcriptome or epigenome. Our results demonstrate a need for vehicle-only controls and highlight immunological perturbations that can occur when using HPßCD in pharmaceutical coformulations. IMPORTANCE SIV infection of nonhuman primates is the principal model system for assessing HIV disease progression and therapeutic development. HPßCD has recently been incorporated as a solubilizing agent in coformulations of ARVs in SIV-infected nonhuman primates. Although HPßCD has historically been considered inert, recent findings suggest that HPßCD may contribute to inflammation. Herein, we investigate the contribution of HPßCD to healthy macaque inflammation in vitro and in vivo. We observe that HPßCD causes an induction of sCD14 and IL-1ß from myeloid cells in vitro and demonstrate that HPßCD stimulatory capacity varies by commercial source. In vivo, we observe modest myeloid cell activation in blood and bronchoalveolar lavage specimens absent systemic immune activation. From our findings, it is unclear whether HPßCD stimulation may improve or diminish immune reconstitution in ARV-treated lentiviral infections. Our results demonstrate a need for vehicle-only controls and highlight immunological perturbations that can occur when using HPßCD in pharmaceutical coformulations.
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Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , 2-Hidroxipropil-beta-Ciclodextrina/farmacologia , 2-Hidroxipropil-beta-Ciclodextrina/uso terapêutico , Progressão da Doença , Inflamação , Leucócitos Mononucleares , Receptores de Lipopolissacarídeos , Macaca mulatta , Carga ViralRESUMO
Patient monitoring is a cornerstone in clinical practice to define disease phenotypes and guide clinical management. Unfortunately, this is often reliant on invasive and/or less sensitive methods that do not provide deep phenotype assessments of disease state to guide treatment. This paper examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) to define molecular gene sets in physiological and heart transplant patients taking immunosuppression medications. We show cfChIP-seq reliably detect gene signals that correlate with gene expression. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of the relevant immune and non-immune molecular pathways that were predominantly downregulated in immunosuppressed heart transplant patients compared to healthy controls. cfChIP-seq also identified tissue sources of cfDNA, detecting greater cell-free DNA from cardiac, hematopoietic, and other non-hematopoietic tissues such as the pulmonary, digestive, and neurological tissues in transplant patients than healthy controls. cfChIP-seq gene signals were reproducible between patient populations and blood collection methods. cfChIP-seq may therefore be a reliable approach to provide dynamic assessments of molecular pathways and tissue injury associated to disease.
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Biologically active small molecules can impart modulatory effects, in some cases providing extended long-term memory. In a screen of biologically active small molecules for regulators of tumor necrosis factor (TNF) induction, we identify several compounds with the ability to induce training effects on human macrophages. Rutaecarpine shows acute and long-term modulation, enhancing lipopolysaccharide (LPS)-induced pro-inflammatory cytokine secretion and relieving LPS tolerance in human macrophages. Rutaecarpine inhibits ß-glucan-induced H3K4Me3 marks at the promoters of several pro-inflammatory cytokines, highlighting the potential of this molecule to modulate chromosomal topology. Syk kinase inhibitor (SYKi IV), another screen hit, promotes an enhanced response to LPS similar to that previously reported for ß-glucan-induced training. Macrophages trained with SYKi IV show a high degree of resistance to influenza A, multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and OC43 coronavirus infection, highlighting a potential application of this molecule and other SYKis as prophylactic treatments for viral susceptibility.
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Tratamento Farmacológico da COVID-19 , beta-Glucanas , Citocinas , Humanos , Alcaloides Indólicos , Lipopolissacarídeos , Macrófagos , Quinazolinonas , SARS-CoV-2 , Quinase Syk , Fator de Necrose Tumoral alfaRESUMO
Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5'-CCCNNGG/AG-3', closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.
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Apoptose , Linfócitos T CD8-Positivos , Citotoxicidade Imunológica , Proteína Ligante Fas , Linfócitos T Citotóxicos , Fatores de Transcrição , Animais , Apoptose/fisiologia , Cromatina/metabolismo , Citotoxicidade Imunológica/genética , Proteína Ligante Fas/metabolismo , Granzimas/genética , Humanos , Camundongos , Perforina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease, including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLLs), otherwise known as "COVID toes," remains unclear. Studying multinational cohorts, we found that, in CLLs, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs after disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased NET levels when compared with other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.
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COVID-19 , Armadilhas Extracelulares , Actinas/metabolismo , Adulto , COVID-19/complicações , Criança , Desoxirribonuclease I , Humanos , Neutrófilos , SARS-CoV-2 , Síndrome de Resposta Inflamatória SistêmicaRESUMO
African green monkeys (AGMs), Chlorocebus pygerythrus, are a natural host for a lentivirus related to HIV, SIV. SIV-infected AGMs rarely progress to AIDS despite robust viral replication. Though multiple mechanisms are involved, a primary component is the animals' ability to downregulate CD4 expression on mature CD4+ Th cells, rendering these cells resistant to infection by SIV. These CD8αα+ T cells retain functional characteristics of CD4+ Th cells while simultaneously acquiring abilities of cytotoxic CD8αß+ T cells. To determine mechanisms underlying functional differences between T cell subsets in AGMs, chromatin accessibility in purified populations was determined by assay for transposase-accessible chromatin sequencing. Differences in chromatin accessibility alone were sufficient to cluster cells by subtype, and accessibility at the CD4 locus reflected changes in CD4 expression. DNA methylation at the CD4 locus also correlated with inaccessible chromatin. By associating accessible regions with nearby genes, gene expression was found to correlate with accessibility changes. T cell and immune system activation pathways were identified when comparing regions that changed accessibility from CD4+ T cells to CD8αα+ T cells. Different transcription factor binding sites are revealed as chromatin accessibility changes, and these differences may elicit downstream changes in differentiation. This comprehensive description of the epigenetic landscape of AGM T cells identified genes and pathways that could have translational value in therapeutic approaches recapitulating the protective effects CD4 downregulation.
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Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Linfócitos T CD4-Positivos , Chlorocebus aethiops , Cromatina/metabolismo , Regulação para Baixo , Epigênese Genética , Subpopulações de Linfócitos T , Linfócitos T Auxiliares-IndutoresRESUMO
Successful meiotic recombination, and thus fertility, depends on conserved axis proteins that organize chromosomes into arrays of anchored chromatin loops and provide a protected environment for DNA exchange. Here, we show that the stereotypic chromosomal distribution of axis proteins in Saccharomyces cerevisiae is the additive result of two independent pathways: a cohesin-dependent pathway, which was previously identified and mediates focal enrichment of axis proteins at gene ends, and a parallel cohesin-independent pathway that recruits axis proteins to broad genomic islands with high gene density. These islands exhibit elevated markers of crossover recombination as well as increased nucleosome density, which we show is a direct consequence of the underlying DNA sequence. A predicted PHD domain in the center of the axis factor Hop1 specifically mediates cohesin-independent axis recruitment. Intriguingly, other chromosome organizers, including cohesin, condensin, and topoisomerases, are differentially depleted from the same regions even in non-meiotic cells, indicating that these DNA sequence-defined chromatin islands exert a general influence on the patterning of chromosome structure.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Meiose/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease including MIS-C and chilblain-like lesions (CLL), otherwise known as "COVID toes", remains unclear. Studying multinational cohorts, we found that, in CLL, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs post-disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased levels of NETs when compared to other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients. Summary: NET formation and degradation are dysregulated in pediatric and symptomatic adult patients with various complications of COVID-19, in association with disease severity. NET degradation impairments are multifactorial and associated with natural inhibitors of DNase 1, G-actin and anti-DNase1L3 and anti-NET antibodies. Infection with the Omicron variant is associated with decreased levels of NETs when compared to other SARS-CoV-2 strains.
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INTRODUCTION: HCC is the third leading cause of cancer-related death worldwide, with chronic viral hepatitis accounting for more than 70% of the cases. Therapeutic options are limited and ineffective. The increasing use of immune-based therapies in solid tumors highlights the need to expand our knowledge on the immunologic microenvironment of HCC. METHODS: Access to liver samples from 20 well-characterized patients with HCC associated with HCV (n = 9) or HBV (n = 11) gave us the opportunity to study the immunologic landscape in these tumors. For each patient, RNA-sequencing was performed on the tumor and surrounding nontumorous tissue. RESULTS: We found that both HCV- and HBV-HCC are associated with a predominance of downregulated genes (74% and 67%, respectively). Analysis of the immune landscape using a curated gene list showed 216 of 2481 (9%) immune genes in HCV-HCC and 164 of 2560 (6%) in HBV-HCC. However, only 8 immune genes (4%) were upregulated in HCV-HCC and 27 (16.5%) in HBV-HCC. HCV-HCC was characterized by an enrichment of downregulated genes related to T-cell activation and oxidative stress. The dramatic downregulation of immune genes related to T-cell activation in HCV-HCC prompted us to perform an extensive immunohistochemistry analysis on paraffin-embedded liver specimen. Interestingly, we found a significant reduction of immune-cell infiltration (CD3, CD8 and CD20 positive cells) within the tumor. Moreover, we observed that HCV-HCC is characterized by an enrichment of M2-like CD68-positive cells. These data are consistent with the dramatic downregulation of immune-cell infiltration seen in HCV-HCC. Conversely, HBV-HCC was characterized by upregulation of genes related to monocyte/macrophage activation and cell cycle control, and downregulation of genes involved in various cell metabolisms. CONCLUSION: This study demonstrates a distinctive molecular signature and immune landscape in HCC of different viral etiology, which could provide new insights into pathogenesis and lead to the development of novel immune-based therapies.
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Oncogenic human papillomavirus (HPV) genomes are often integrated into host chromosomes in HPV-associated cancers. HPV genomes are integrated either as a single copy or as tandem repeats of viral DNA interspersed with, or without, host DNA. Integration occurs frequently in common fragile sites susceptible to tandem repeat formation and the flanking or interspersed host DNA often contains transcriptional enhancer elements. When co-amplified with the viral genome, these enhancers can form super-enhancer-like elements that drive high viral oncogene expression. Here we compiled highly curated datasets of HPV integration sites in cervical (CESC) and head and neck squamous cell carcinoma (HNSCC) cancers, and assessed the number of breakpoints, viral transcriptional activity, and host genome copy number at each insertion site. Tumors frequently contained multiple distinct HPV integration sites but often only one "driver" site that expressed viral RNA. As common fragile sites and active enhancer elements are cell-type-specific, we mapped these regions in cervical cell lines using FANCD2 and Brd4/H3K27ac ChIP-seq, respectively. Large enhancer clusters, or super-enhancers, were also defined using the Brd4/H3K27ac ChIP-seq dataset. HPV integration breakpoints were enriched at both FANCD2-associated fragile sites and enhancer-rich regions, and frequently showed adjacent focal DNA amplification in CESC samples. We identified recurrent integration "hotspots" that were enriched for super-enhancers, some of which function as regulatory hubs for cell-identity genes. We propose that during persistent infection, extrachromosomal HPV minichromosomes associate with these transcriptional epicenters and accidental integration could promote viral oncogene expression and carcinogenesis.
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The life cycle of human papillomavirus (HPV) depends on keratinocyte differentiation as the virus modulates and takes advantage of cellular pathways to replicate its genome and assemble viral particles in differentiated cells. Viral genomes are amplified in nuclear replication foci in differentiated keratinocytes, and DNA repair factors from the DNA damage response signaling pathway are recruited to replicate viral DNA. The HPV genome is associated with cellular histones at all stages of the infectious cycle, and here, we show that the histone variant macroH2A1 is bound to the HPV genome and enriched in viral replication foci in differentiated cells. macroH2A1 isoforms play important roles in cellular transcriptional repression, double-strand break repair, and replication stress. The viral E8^E2 protein also binds to the HPV genome and inhibits viral replication and gene expression by recruiting NCoR/SMRT complexes. We show here that E8^E2 and SMRT also localize within replication foci, though independently from macroH2A1. Conversely, transcription complexes containing RNA polymerase II and Brd4 are located on the surface of the foci. Foci generated with an HPV16 E8^E2 mutant genome are not enriched for SMRT or macroH2A1 but contain transcriptional complexes throughout the foci. We propose that both the cellular macroH2A1 protein and viral E8^E2 protein help to spatially separate replication and transcription activities within viral replication foci. IMPORTANCE Human papillomaviruses are small DNA viruses that cause chronic infection of cutaneous and mucosal epithelium. In some cases, persistent infection with HPV can result in cancer, and 5% of human cancers are the result of HPV infection. In differentiated cells, HPV amplifies viral DNA in nuclear replication factories and transcribes late mRNAs to produce capsid proteins. However, very little is known about the spatial organization of these activities in the nucleus. Here, we show that repressive viral and cellular factors localize within the foci to suppress viral transcription, while active transcription takes place on the surface. The cellular histone variant macroH2A1 is important for this spatial organization.
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Alphapapillomavirus/fisiologia , Genoma Viral , Infecções por Papillomavirus/virologia , Replicação Viral , Alphapapillomavirus/genética , Histonas/genética , Histonas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismoRESUMO
Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.
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Linfócitos B/fisiologia , Biomarcadores Tumorais/genética , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Linfoma Difuso de Grandes Células B/genética , Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação Neoplásica da Expressão Gênica , Testes Genéticos , Estudo de Associação Genômica Ampla , Antígenos HLA/metabolismo , Humanos , Vigilância Imunológica , Linfoma Difuso de Grandes Células B/metabolismo , Evasão Tumoral/genéticaRESUMO
Dysbiosis of the skin microbiota is increasingly implicated as a contributor to the pathogenesis of atopic dermatitis (AD). We previously reported first-in-human safety and clinical activity results from topical application of the commensal skin bacterium Roseomonas mucosa for the treatment of AD in 10 adults and 5 children older than 9 years of age. Here, we examined the potential mechanism of action of R. mucosa treatment and its impact on children with AD less than 7 years of age, the most common age group for children with AD. In 15 children with AD, R. mucosa treatment was associated with amelioration of disease severity, improvement in epithelial barrier function, reduced Staphylococcus aureus burden on the skin, and a reduction in topical steroid requirements without severe adverse events. Our observed response rates to R. mucosa treatment were greater than those seen in historical placebo control groups in prior AD studies. Skin improvements and colonization by R. mucosa persisted for up to 8 months after cessation of treatment. Analyses of cellular scratch assays and the MC903 mouse model of AD suggested that production of sphingolipids by R. mucosa, cholinergic signaling, and flagellin expression may have contributed to therapeutic impact through induction of a TNFR2-mediated epithelial-to-mesenchymal transition. These results suggest that a randomized, placebo-controlled trial of R. mucosa treatment in individuals with AD is warranted and implicate commensals in the maintenance of the skin epithelial barrier.
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Dermatite Atópica , Eczema , Methylobacteriaceae , Adulto , Criança , Dermatite Atópica/tratamento farmacológico , Humanos , Lipídeos , PeleRESUMO
During meiotic prophase, concurrent transcription, recombination, and chromosome synapsis place substantial topological strain on chromosomal DNA, but the role of topoisomerases in this context remains poorly defined. Here, we analyzed the roles of topoisomerases I and II (Top1 and Top2) during meiotic prophase in Saccharomyces cerevisiae We show that both topoisomerases accumulate primarily in promoter-containing intergenic regions of actively transcribing genes, including many meiotic double-strand break (DSB) hotspots. Despite the comparable binding patterns, top1 and top2 mutations have different effects on meiotic recombination. TOP1 disruption delays DSB induction and shortens the window of DSB accumulation by an unknown mechanism. By contrast, temperature-sensitive top2-1 mutants exhibit a marked delay in meiotic chromosome remodeling and elevated DSB signals on synapsed chromosomes. The problems in chromosome remodeling were linked to altered Top2 binding patterns rather than a loss of Top2 catalytic activity, and stemmed from a defect in recruiting the chromosome remodeler Pch2/TRIP13 to synapsed chromosomes. No chromosomal defects were observed in the absence of TOP1 Our results imply independent roles for Top1 and Top2 in modulating meiotic chromosome structure and recombination.
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Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/ultraestrutura , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo II/genética , Meiose , Ligação Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Faithful meiotic chromosome inheritance and fertility rely on the stimulation of meiotic crossover recombination by potentially genotoxic DNA double-strand breaks (DSBs). To avoid excessive damage, feedback mechanisms down-regulate DSBs, likely in response to initiation of crossover repair. In Saccharomyces cerevisiae, this regulation requires the removal of the conserved DSB-promoting protein Hop1/HORMAD during chromosome synapsis. Here, we identify privileged end-adjacent regions (EARs) spanning roughly 100 kb near all telomeres that escape DSB down-regulation. These regions retain Hop1 and continue to break in pachynema despite normal synaptonemal complex deposition. Differential retention of Hop1 requires the disassemblase Pch2/TRIP13, which preferentially removes Hop1 from telomere-distant sequences, and is modulated by the histone deacetylase Sir2 and the nucleoporin Nup2. Importantly, the uniform size of EARs among chromosomes contributes to disproportionately high DSB and repair signals on short chromosomes in pachynema, suggesting that EARs partially underlie the curiously high recombination rate of short chromosomes.
Assuntos
Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla , Meiose/genética , Saccharomyces cerevisiae/genética , Telômero/genética , Pareamento Cromossômico/genética , Cromossomos Fúngicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Telômero/metabolismoRESUMO
BACKGROUND: Chromatin-immunoprecipitation followed by sequencing (ChIP-seq) is the method of choice for mapping genome-wide binding of chromatin-associated factors. However, broadly applicable methods for between-sample comparisons are lacking. RESULTS: Here, we introduce SNP-ChIP, a method that leverages small-scale intra-species polymorphisms, mainly SNPs, for quantitative spike-in normalization of ChIP-seq results. Sourcing spike-in material from the same species ensures antibody cross-reactivity and physiological coherence, thereby eliminating two central limitations of traditional spike-in approaches. We show that SNP-ChIP is robust to changes in sequencing depth and spike-in proportions, and reliably identifies changes in overall protein levels, irrespective of changes in binding distribution. Application of SNP-ChIP to test cases from budding yeast meiosis allowed discovery of novel regulators of the chromosomal protein Red1 and quantitative analysis of the DNA-damage associated histone modification γ-H2AX. CONCLUSION: SNP-ChIP is fully compatible with the intra-species diversity of humans and most model organisms and thus offers a general method for normalizing ChIP-seq results.