RESUMO
ANCA vasculitis is an autoimmune disease with increased expression of the autoantigen genes, myeloperoxidase (MPO) and proteinase 3 (PRTN3), but the origin and significance of expression is less distinct. To clarify this, we measured MPO and PRTN3 messenger RNA in monocytes, normal-density neutrophils, and in enriched leukocytes from peripheral blood mononuclear cells. Increased autoantigen gene expression was detected in normal-density neutrophils and enriched leukocytes from patients during active disease compared to healthy individuals, with the largest difference in enriched leukocytes. RNA-seq of enriched leukocytes comparing active-remission pairs identified a gene signature for low-density neutrophils. Cell sorting revealed low-density neutrophils contained mature and immature neutrophils depending on the presence or absence of CD10. Both populations contributed to autoantigen expression but the frequency of immature cells in low-density neutrophils did not correlate with low-density neutrophil MPO or PRTN3 expression. Low-density neutrophils were refractory to MPO-ANCA induced oxidative burst, suggesting an alternative role for low-density neutrophils in ANCA vasculitis pathogenesis. In contrast, normal-density neutrophils were activated by MPO-ANCA and monoclonal anti-PR3 antibody. Normal-density neutrophil activation correlated with MPO and PRTN3 mRNA. Increased autoantigen gene expression originating from the mature low-density and normal-density neutrophils suggests transcriptional dysregulation is a hallmark of ANCA vasculitis. Thus, the correlation between autoantigen gene expression and antibody-mediated normal-density neutrophil activation connects autoantigen gene expression with disease pathogenesis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos , Neutrófilos , Autoantígenos/genética , Expressão Gênica , Humanos , Leucócitos Mononucleares , Mieloblastina , Ativação de Neutrófilo , Peroxidase/genéticaRESUMO
Long noncoding RNAs (lncRNAs) cause Polycomb repressive complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Airn and Kcnq1ot1 lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, the extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, preexisting genome architecture, and the abundance of Airn itself. Specific CpG islands (CGIs) displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance and that within lncRNA-targeted domains, PRCs are recruited to CGIs via lncRNA-independent mechanisms. We propose that CGIs that autonomously recruit PRCs interact with lncRNAs and their associated proteins through three-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.
Assuntos
RNA Longo não Codificante/genética , Animais , Cromatina/genética , Montagem e Desmontagem da Cromatina , Ilhas de CpG/genética , Genoma/genética , Impressão Genômica/genética , Humanos , Camundongos , Complexo Repressor Polycomb 1/genética , Regiões Promotoras Genéticas , Células-Tronco/metabolismo , Trofoblastos/metabolismoRESUMO
Obesity in humans is associated with poorer health outcomes after infections compared with non-obese individuals. Here, we examined the effects of white adipose tissue and obesity on T cell responses to viral infection in mice. We show that lymphocytic choriomeningitis virus (LCMV) grows to high titer in adipose tissue. Virus-specific T cells enter the adipose tissue to resolve infection but then remain as a memory population distinct from memory T cells in lymphoid tissues. Memory T cells in adipose tissue are abundant in lean mice, and diet-induced obesity further increases memory T cell number in adipose tissue and spleen. Upon re-challenge infection, memory T cells rapidly cause severe pathogenesis, leading to increases in lipase levels, calcification of adipose tissue, pancreatitis, and reduced survival in obese mice but not lean mice. Thus, obesity leads to a unique form of viral pathogenesis involving memory T cell-dependent adipocyte destruction and damage to other tissues.
Assuntos
Tecido Adiposo/fisiologia , Obesidade/genética , Linfócitos T/metabolismo , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/patologiaRESUMO
Long noncoding RNAs (lncRNAs) constitute a significant fraction of mammalian transcriptomes and they have emerged as intricate regulators of many biological processes. Their broad capacity to adopt diverse structures facilitates their involvement in the transcriptional, translational and signaling processes that are central to embryonic stem (ES) cell self-renewal and pluripotency. While lncRNAs have been implicated in ES cell maintenance, detailed analyses of those that show significant expression in ES cells is largely absent. Moreover, cooperative molecular relationships that facilitate lncRNA action are poorly understood. Cyrano is a developmentally important lncRNA, and in ES cells, it supports gene expression network maintenance, cell adhesion and cell survival. We have interrogated the interactome of Cyrano to identify protein partners and find that Cyrano is involved in multiple protein networks. We identify a developmentally important cell-signaling hub and find STAT3 as a candidate through which Cyrano can function to reinforce self-renewal of ES cells. Based on commonalities between ES cells and cancer cells, we postulate such functional interactions may support cell proliferation, cell identity and adhesion characteristics in rapidly proliferating cell types. The interactome data will therefore provide a resource for further investigations into interactions that regulate Cyrano or mediate its function.
Assuntos
Células-Tronco Embrionárias/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Transcriptoma/genética , Animais , Adesão Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Autorrenovação Celular/genética , Células-Tronco Embrionárias/citologia , Redes Reguladoras de Genes , Humanos , CamundongosRESUMO
BACKGROUND: Polycomb repressive complex 2 (PRC2) is responsible for establishing and maintaining histone H3K27 methylation during cell differentiation and proliferation. H3K27 can be mono-, di-, or trimethylated, resulting in differential gene regulation. However, it remains unknown how PRC2 specifies the degree and biological effects of H3K27 methylation within a given cellular context. One way to determine PRC2 specificity may be through alternative splicing of Ezh2, PRC2's catalytic subunit, during cell differentiation and tissue maturation. RESULTS: We fully characterized the alternative splicing of Ezh2 in somatic cells and male germ cells and found that Ezh's exon 14 was differentially regulated during mitosis and meiosis. The Ezh2 isoform containing exon 14 (ex14-Ezh2) is upregulated during cell cycle progression, consistent with a role in maintaining H3K27 methylation during chromatin replication. In contrast, the isoform lacking exon 14 (ex14D-Ezh2) was almost exclusively present in spermatocytes when new H3K27me2 is established during meiotic differentiation. Moreover, Ezh2's transcript is normally controlled by E2F transcription activators, but in spermatocytes, Ezh2's transcription is controlled by the meiotic regulator MYBL1. Compared to ex14-EZH2, ex14D-EZH2 has a diminished efficiency for catalyzing H3K27me3 and promotes embryonic stem cell differentiation. CONCLUSIONS: Ezh2's expression is regulated at transcriptional and post-transcriptional levels in a cellular context-dependent manner. EZH2 variants determine functional specificity of PRC2 in histone methylation during cell proliferation and differentiation.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Processamento Alternativo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Variação Genética , Histonas/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Metilação , Camundongos , Complexo Repressor Polycomb 2/genética , Processamento de Proteína Pós-Traducional , Espermatócitos/citologia , Espermatócitos/metabolismoRESUMO
Chronic inflammatory demyelinating polyneuropathy (CIDP) and Guillain-Barre syndrome (GBS) are inflammatory neuropathies that affect humans and are characterized by peripheral nerve myelin destruction and macrophage-containing immune infiltrates. In contrast to the traditional view that the peripheral nerve is simply the target of autoimmunity, we report here that peripheral nerve Schwann cells exacerbate the autoimmune process through extracellular matrix (ECM) protein induction. In a spontaneous autoimmune peripheral polyneuropathy (SAPP) mouse model of inflammatory neuropathy and CIDP nerve biopsies, the ECM protein periostin (POSTN) was upregulated in affected sciatic nerves and was primarily expressed by Schwann cells. Postn deficiency delayed the onset and reduced the extent of neuropathy, as well as decreased the number of macrophages infiltrating the sciatic nerve. In an in vitro assay, POSTN promoted macrophage chemotaxis in an integrin-AM (ITGAM) and ITGAV-dependent manner. The PNS-infiltrating macrophages in SAPP-affected nerves were pathogenic, since depletion of macrophages protected against the development of neuropathy. Our findings show that Schwann cells promote macrophage infiltration by upregulating Postn and suggest that POSTN is a novel target for the treatment of macrophage-associated inflammatory neuropathies.
Assuntos
Moléculas de Adesão Celular/imunologia , Macrófagos/imunologia , Células de Schwann/imunologia , Animais , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Moléculas de Adesão Celular/genética , Humanos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/genética , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/patologia , Células de Schwann/patologiaRESUMO
BACKGROUND & AIMS: The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce postmitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages requires the transcription factor Atoh1, which regulates differentiation of the secretory cell lineage. However, less is known about the origin of tuft cells, which participate in type II immune responses to parasite infections and appear to differentiate independently of Atoh1. We investigated the role of Sox4 in ISC differentiation. METHODS: We performed experiments in mice with intestinal epithelial-specific disruption of Sox4 (Sox4fl/fl:vilCre; SOX4 conditional knockout [cKO]) and mice without disruption of Sox4 (control mice). Crypt- and single-cell-derived organoids were used in assays to measure proliferation and ISC potency. Lineage allocation and gene expression changes were studied by immunofluorescence, real-time quantitative polymerase chain reaction, and RNA-seq analyses. Intestinal organoids were incubated with the type 2 cytokine interleukin 13 and gene expression was analyzed. Mice were infected with the helminth Nippostrongylus brasiliensis and intestinal tissues were collected 7 days later for analysis. Intestinal tissues collected from mice that express green fluorescent protein regulated by the Atoh1 promoter (Atoh1GFP mice) and single-cell RNA-seq analysis were used to identify cells that coexpress Sox4 and Atoh1. We generated SOX4-inducible intestinal organoids derived from Atoh1fl/fl:vilCreER (ATOH1 inducible knockout) mice and assessed differentiation. RESULTS: Sox4cKO mice had impaired ISC function and secretory differentiation, resulting in decreased numbers of tuft and enteroendocrine cells. In control mice, numbers of SOX4+ cells increased significantly after helminth infection, coincident with tuft cell hyperplasia. Sox4 was activated by interleukin 13 in control organoids; SOX4cKO mice had impaired tuft cell hyperplasia and parasite clearance after infection with helminths. In single-cell RNA-seq analysis, Sox4+/Atoh1- cells were enriched for ISC, progenitor, and tuft cell genes; 12.5% of Sox4-expressing cells coexpressed Atoh1 and were enriched for enteroendocrine genes. In organoids, overexpression of Sox4 was sufficient to induce differentiation of tuft and enteroendocrine cells-even in the absence of Atoh1. CONCLUSIONS: We found Sox4 promoted tuft and enteroendocrine cell lineage allocation independently of Atoh1. These results challenge the longstanding model in which Atoh1 is the sole regulator of secretory differentiation in the intestine and are relevant for understanding epithelial responses to parasitic infection.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Células Enteroendócrinas/citologia , Células Caliciformes/citologia , Mucosa Intestinal/citologia , Fatores de Transcrição SOXC/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Receptores de Hialuronatos/análise , Camundongos , Fatores de Transcrição SOXC/análiseRESUMO
Kabuki syndrome, a congenital craniofacial disorder, manifests from mutations in an X-linked histone H3 lysine 27 demethylase (UTX/KDM6A) or a H3 lysine 4 methylase (KMT2D). However, the cellular and molecular etiology of histone-modifying enzymes in craniofacial disorders is unknown. We now establish Kabuki syndrome as a neurocristopathy, whereby the majority of clinical features are modeled in mice carrying neural crest (NC) deletion of UTX, including craniofacial dysmorphism, cardiac defects, and postnatal growth retardation. Female UTX NC knockout (FKO) demonstrates enhanced phenotypic severity over males (MKOs), due to partial redundancy with UTY, a Y-chromosome demethylase-dead homolog. Thus, NC cells may require demethylase-independent UTX activity. Consistently, Kabuki causative point mutations upstream of the JmjC domain do not disrupt UTX demethylation. We have isolated primary NC cells at a phenocritical postmigratory timepoint in both FKO and MKO mice, and genome-wide expression and histone profiling have revealed UTX molecular function in establishing appropriate chromatin structure to regulate crucial NC stem-cell signaling pathways. However, the majority of UTX regulated genes do not experience aberrations in H3K27me3 or H3K4me3, implicating alternative roles for UTX in transcriptional control. These findings are substantiated through demethylase-dead knockin mutation of UTX, which supports appropriate facial development.
Assuntos
Anormalidades Múltiplas/etiologia , Face/anormalidades , Doenças Hematológicas/etiologia , Histona Desmetilases/metabolismo , Crista Neural/fisiopatologia , Doenças Vestibulares/etiologia , Animais , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Histona Desmetilases/genética , Humanos , Lisina/metabolismo , Masculino , Camundongos Knockout , Camundongos Transgênicos , Mutação , Crista Neural/metabolismo , Proteínas Nucleares/genética , Crânio/embriologiaRESUMO
Blockade of immune checkpoint proteins (e.g., CTLA-4, PD-1) improves overall survival in advanced melanoma; however, therapeutic benefit is limited to only a subset of patients. Because checkpoint blockade acts by "removing the brakes" on effector T cells, the efficacy of checkpoint blockade may be constrained by the limited pool of melanoma-reactive T cells in the periphery. In the thymus, autoimmune regulator (Aire) promotes deletion of T cells reactive against self-antigens that are also expressed by tumors. Thus, while protecting against autoimmunity, Aire also limits the generation of melanoma-reactive T cells. Here, we show that Aire deficiency in mice expands the pool of CD4+ T cells capable of melanoma cell eradication and has additive effects with anti-CTLA-4 antibody in slowing melanoma tumor growth and increasing survival. Moreover, pharmacologic blockade of central T cell tolerance and peripheral checkpoint blockade in combination enhanced antimelanoma immunity in a synergistic manner. In melanoma patients treated with anti-CTLA-4 antibody, clinical response to therapy was associated with a human Aire polymorphism. Together, these findings suggest that Aire-mediated central tolerance constrains the efficacy of peripheral checkpoint inhibition and point to simultaneous blockade of Aire and checkpoint inhibitors as a novel strategy to enhance antimelanoma immunity.
Assuntos
Antígeno CTLA-4/antagonistas & inibidores , Tolerância Central , Melanoma/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo , Xenoenxertos , Humanos , Imunoterapia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Fatores de Transcrição/genética , Proteína AIRERESUMO
Of the thousands of long noncoding RNAs expressed in embryonic stem cells (ESCs), few have known roles and fewer have been functionally implicated in the regulation of self-renewal and pluripotency, or the reprogramming of somatic cells to the pluripotent state. In ESCs, Cyrano is a stably expressed long intergenic noncoding RNA with no previously assigned role. We demonstrate that Cyrano contributes to ESC maintenance, as its depletion results in the loss of hallmarks of self-renewal. Delineation of Cyrano's network through transcriptomics revealed widespread effects on signaling pathways and gene expression networks that contribute to ESC maintenance. Cyrano shares unique sequence complementarity with the differentiation-associated microRNA, mir-7, and mir-7 overexpression reduces expression of a key self-renewal factor to a similar extent as Cyrano knockdown. This suggests that Cyrano functions to restrain the action of mir-7. Altogether, we provide a view into the multifaceted function of Cyrano in ESC maintenance.
Assuntos
Autorrenovação Celular , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/citologia , RNA Longo não Codificante/genética , Animais , Linhagem Celular , Sobrevivência Celular , Redes Reguladoras de Genes , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , TranscriptomaRESUMO
We previously reported the requirement of Polycomb Repressive Complex 2 (PRC2) for spermatogenesis through transcriptional repression of somatic genes and meiosis-specific genes. To characterize how PRC2's two methyltransferase subunits, EZH1 and EZH2, regulate histone H3 lysine 27 (H3K27) methylation during germ cell development, we generated mouse models with a germline ablation of EZH1 and/or EHZ2. Only the combined loss of EZH1 and EZH2 caused a depletion of global H3K27me3 marks and meiotic arrest in spermatocytes. Genome-wide analysis of H3K27me3 in spermatogenic cells revealed that a noncanonical EZH1-PRC2 could establish and maintain this histone mark on somatic genes and certain meiotic genes. Consistent with it having active enhancers in testis, Ezh1 was not only abundant in highly differentiated spermatocytes but also in actively proliferating progenitor and stem germ cells. Taken together, our findings suggest that the expression level of Ezh1 determines the restoration of H3K27 methylation in the absence of the canonical EZH2-PRC2.
Assuntos
Complexo Repressor Polycomb 2/metabolismo , Espermatogênese , Espermatozoides/metabolismo , Animais , Sequência de Bases , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fertilidade , Deleção de Genes , Genoma , Histonas/metabolismo , Lisina/metabolismo , Masculino , Metilação , Camundongos Knockout , Mitose , Modelos Biológicos , Testículo/metabolismoRESUMO
ANCA-associated vasculitis is an autoimmune condition characterized by vascular inflammation and organ damage. Pharmacologically induced remission of this condition is complicated by relapses. Potential triggers of relapse are immunologic challenges and environmental insults, both of which associate with changes in epigenetic silencing modifications. Altered histone modifications implicated in gene silencing associate with aberrant autoantigen expression. To establish a link between DNA methylation, a model epigenetic gene silencing modification, and autoantigen gene expression and disease status in ANCA-associated vasculitis, we measured gene-specific DNA methylation of the autoantigen genes myeloperoxidase (MPO) and proteinase 3 (PRTN3) in leukocytes of patients with ANCA-associated vasculitis observed longitudinally (n=82) and of healthy controls (n=32). Patients with active disease demonstrated hypomethylation of MPO and PRTN3 and increased expression of the autoantigens; in remission, DNA methylation generally increased. Longitudinal analysis revealed that patients with ANCA-associated vasculitis could be divided into two groups, on the basis of whether DNA methylation increased or decreased from active disease to remission. In patients with increased DNA methylation, MPO and PRTN3 expression correlated with DNA methylation. Kaplan-Meier estimate of relapse revealed patients with increased DNA methylation at the PRTN3 promoter had a significantly greater probability of a relapse-free period (P<0.001), independent of ANCA serotype. Patients with decreased DNA methylation at the PRTN3 promoter had a greater risk of relapse (hazard ratio, 4.55; 95% confidence interval, 2.09 to 9.91). Thus, changes in the DNA methylation status of the PRTN3 promoter may predict the likelihood of stable remission and explain autoantigen gene regulation.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Autoantígenos/genética , Metilação de DNA , Mieloblastina/genética , Peroxidase/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Indução de RemissãoRESUMO
Male gender is protective against multiple sclerosis and other T-cell-mediated autoimmune diseases. This protection may be due, in part, to higher androgen levels in males. Androgen binds to the androgen receptor (AR) to regulate gene expression, but how androgen protects against autoimmunity is not well understood. Autoimmune regulator (Aire) prevents autoimmunity by promoting self-antigen expression in medullary thymic epithelial cells, such that developing T cells that recognize these self-antigens within the thymus undergo clonal deletion. Here we show that androgen upregulates Aire-mediated thymic tolerance to protect against autoimmunity. Androgen recruits AR to Aire promoter regions, with consequent enhancement of Aire transcription. In mice and humans, thymic Aire expression is higher in males compared with females. Androgen administration and male gender protect against autoimmunity in a multiple sclerosis mouse model in an Aire-dependent manner. Thus, androgen control of an intrathymic Aire-mediated tolerance mechanism contributes to gender differences in autoimmunity.
Assuntos
Androgênios/farmacologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Sistema Nervoso Central/patologia , Sexismo , Fatores de Transcrição/metabolismo , Animais , Antígenos/metabolismo , Di-Hidrotestosterona/farmacologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/prevenção & controle , Feminino , Imunofluorescência , Humanos , Masculino , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/metabolismo , Timo/efeitos dos fármacos , Timo/metabolismo , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Proteína AIRERESUMO
BACKGROUND: Correctly identifying genomic regions enriched with histone modifications and transcription factors is key to understanding their regulatory and developmental roles. Conceptually, these regions are divided into two categories, narrow peaks and broad domains, and different algorithms are used to identify each one. Datasets that span these two categories are often analyzed with a single program for peak calling combined with an ad hoc method for domains. RESULTS: We developed hiddenDomains, which identifies both peaks and domains, and compare it to the leading algorithms using H3K27me3, H3K36me3, GABP, ESR1 and FOXA ChIP-seq datasets. The output from the programs was compared to qPCR-validated enriched and depleted sites, predicted transcription factor binding sites, and highly-transcribed gene bodies. With every method, hiddenDomains, performed as well as, if not better than algorithms dedicated to a specific type of analysis. CONCLUSIONS: hiddenDomains performs as well as the best domain and peak calling algorithms, making it ideal for analyzing ChIP-seq datasets, especially those that contain a mixture of peaks and domains.
Assuntos
Algoritmos , Imunoprecipitação da Cromatina , Receptor alfa de Estrogênio/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Histonas/metabolismo , Humanos , Cadeias de MarkovRESUMO
Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection.
Assuntos
Histona Desmetilases/deficiência , Histona Desmetilases/fisiologia , Vírus da Coriomeningite Linfocítica/imunologia , Proteínas Nucleares/deficiência , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Viremia/imunologia , Animais , Anticorpos Antivirais/biossíntese , Diferenciação Celular , Feminino , Dosagem de Genes , Regulação da Expressão Gênica/imunologia , Predisposição Genética para Doença , Histonas/metabolismo , Humanos , Memória Imunológica , Subunidade alfa de Receptor de Interleucina-6/biossíntese , Subunidade alfa de Receptor de Interleucina-6/genética , Cooperação Linfocítica , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Metilação , Camundongos , Modelos Imunológicos , Otite Média/etiologia , Processamento de Proteína Pós-Traducional , Receptores CXCR5/análise , Especificidade da Espécie , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/virologia , Linfócitos T Auxiliares-Indutores/enzimologia , Linfócitos T Auxiliares-Indutores/virologia , Transcrição Gênica , Síndrome de Turner/complicações , Síndrome de Turner/enzimologia , Virulência , Inativação do Cromossomo XRESUMO
Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans and has been implicated in many important biological processes, including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components or in H3.3 encoding genes have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3-null mouse models through classical genetic approaches. We found that H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres, and pericentromeric regions of chromosomes, leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Instabilidade Genômica/genética , Crescimento e Desenvolvimento/genética , Histonas/metabolismo , Animais , Morte Celular/genética , Linhagem Celular , Proliferação de Células/genética , Células Cultivadas , Fertilidade/genética , Genes Letais/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Camundongos , MutaçãoRESUMO
BACKGROUND: Whole-exome sequencing (WES) is a popular next-generation sequencing technology used by numerous laboratories with various levels of statistical and analytical expertise. Centralized databases, such as the Sequence Read Archive and the European Nucleotide Archive, allow data to be reanalyzed by independent labs to confirm results and derive additional insights. Access to new and shared data highlights the necessity for software that both lowers the statistical and analytical expertise required to generate results and promotes reproducible methodology among laboratories. FINDINGS: We have developed fastq2vcf, a pipeline that automates the genomic variant calling process using multiple callers. Fastq2vcf offers improved flexibility, efficiency, and reproducibility by seamlessly integrating several leading sequencing analysis tools. It outputs not only the annotated variant call set for each caller, but also the consensus variant call set shared by different callers. Furthermore, it can be customized and extended easily. CONCLUSIONS: Our software tool automatically generates executable command lines for a variety of tools required for analyzing WES data. It is also highly configurable and provides users with complete control of the processing procedure, making it easy to submit and track jobs in both single workstation and parallelized computing environments. By using this pipeline, WES analysis can be easily reproduced.
Assuntos
Exoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Software , Benchmarking , Humanos , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
Several hundred mammalian genes are expressed preferentially from one parental allele as the result of a process called genomic imprinting. Genomic imprinting is prevalent in extra-embryonic tissue, where it plays an essential role during development. Here, we profiled imprinted gene expression via RNA-Seq in a panel of six mouse trophoblast stem lines, which are ex vivo derivatives of a progenitor population that gives rise to the placental tissue of the mouse. We found evidence of imprinted expression for 48 genes, 31 of which had been described previously as imprinted and 17 of which we suggest as candidate imprinted genes. An equal number of maternally and paternally biased genes were detected. On average, candidate imprinted genes were more lowly expressed and had weaker parent-of-origin biases than known imprinted genes. Several known and candidate imprinted genes showed variability in parent-of-origin expression bias between the six trophoblast stem cell lines. Sixteen of the 48 known and candidate imprinted genes were previously or newly annotated noncoding RNAs and six encoded for a total of 60 annotated microRNAs. Pyrosequencing across our panel of trophoblast stem cell lines returned levels of imprinted expression that were concordant with RNA-Seq measurements for all eight genes examined. Our results solidify trophoblast stem cells as a cell culture-based experimental model to study genomic imprinting, and provide a quantitative foundation upon which to delineate mechanisms by which the process is maintained in the mouse.
Assuntos
Expressão Gênica , Impressão Genômica , Células-Tronco/metabolismo , Trofoblastos/metabolismo , Alelos , Animais , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , MicroRNAs , RNA não Traduzido/genética , Reprodutibilidade dos TestesRESUMO
Ovarian clear-cell carcinoma (OCCC) is an aggressive form of ovarian cancer with high ARID1A mutation rates. Here we present a mutant mouse model of OCCC. We find that ARID1A inactivation is not sufficient for tumour formation, but requires concurrent activation of the phosphoinositide 3-kinase catalytic subunit, PIK3CA. Remarkably, the mice develop highly penetrant tumours with OCCC-like histopathology, culminating in haemorrhagic ascites and a median survival period of 7.5 weeks. Therapeutic treatment with the pan-PI3K inhibitor, BKM120, prolongs mouse survival by inhibiting the tumour cell growth. Cross-species gene expression comparisons support a role for IL-6 inflammatory cytokine signalling in OCCC pathogenesis. We further show that ARID1A and PIK3CA mutations cooperate to promote tumour growth through sustained IL-6 overproduction. Our findings establish an epistatic relationship between SWI/SNF chromatin remodelling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signalling. We propose that ARID1A protects against inflammation-driven tumorigenesis.
Assuntos
Adenocarcinoma de Células Claras/genética , Carcinogênese/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Inflamação/metabolismo , Mutação/genética , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Fosfatidilinositol 3-Quinases/genética , Adenocarcinoma de Células Claras/tratamento farmacológico , Adenocarcinoma de Células Claras/patologia , Alelos , Animais , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Genes Supressores de Tumor , Haploinsuficiência/efeitos dos fármacos , Inflamação/patologia , Interleucina-6/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Fatores de TranscriçãoRESUMO
Polycomb-repressive complex 2 (PRC2) catalyzes the methylation of histone H3 Lys27 (H3K27) and functions as a critical epigenetic regulator of both stem cell pluripotency and somatic differentiation, but its role in male germ cell development is unknown. Using conditional mutagenesis to remove the core PRC2 subunits EED and SUZ12 during male germ cell development, we identified a requirement for PRC2 in both mitotic and meiotic germ cells. We observed a paucity of mutant spermatogonial stem cells (SSCs), which appears independent of repression of the known cell cycle inhibitors Ink4a/Ink4b/Arf. Moreover, mutant spermatocytes exhibited ectopic expression of somatic lamins and an abnormal distribution of SUN1 proteins on the nuclear envelope. These defects were coincident with abnormal chromosome dynamics, affecting homologous chromosome pairing and synapsis. We observed acquisition of H3K27me3 on stage-specific genes during meiotic progression, indicating a requirement for PRC2 in regulating the meiotic transcriptional program. Together, these data demonstrate that transcriptional repression of soma-specific genes by PRC2 facilitates homeostasis and differentiation during mammalian spermatogenesis.