RESUMO
Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neurofibromatose 1 , Inibidores de Proteínas Quinases , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Camundongos , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/patologia , Neoplasias de Bainha Neural/tratamento farmacológico , Linhagem Celular Tumoral , Transdução de Sinais , Linhagem da Célula/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Neurofibrossarcoma/genética , Neurofibrossarcoma/patologia , Neurofibrossarcoma/tratamento farmacológico , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/genéticaRESUMO
Endometrial epithelia are known to harbor cancer driver mutations in the absence of any pathologies, including mutations in PIK3CA. Insulin plays an important role in regulating uterine metabolism during pregnancy, and hyperinsulinemia is associated with conditions impacting fertility. Hyperinsulinemia also promotes cancer, but the direct action of insulin on mutated endometrial epithelial cells is unknown. Here, we treated 12Z endometriotic epithelial cells carrying the PIK3CAH1047R oncogene with insulin and examined transcriptomes by RNA-seq. While cells naively responded to insulin, the magnitude of differential gene expression (DGE) was nine times greater in PIK3CAH1047R cells, representing a synergistic effect between insulin signaling and PIK3CAH1047R expression. Interferon signaling and the unfolded protein response (UPR) were enriched pathways among affected genes. Insulin treatment in wild-type cells activated normal endoplasmic reticulum stress (ERS) response programs, while PIK3CAH1047R cells activated programs necessary to avoid ERS-induced apoptosis. PIK3CAH1047R expression alone resulted in overexpression (OE) of Viperin (RSAD2), which is involved in viral response and upregulated in the endometrium during early pregnancy. The transcriptional changes induced by insulin in PIK3CAH1047R cells were rescued by knockdown of Viperin, while Viperin OE alone was insufficient to induce a DGE response to insulin, suggesting that Viperin is necessary but not sufficient for the synergistic effect of PIK3CAH1047R and insulin treatment. We identified interferon signaling, viral response, and protein targeting pathways that are induced by insulin but dependent on Viperin in PIK3CAH1047R mutant cells. These results suggest that response to insulin signaling is altered in mutated endometriotic epithelial cells.
Assuntos
Hiperinsulinismo , Neoplasias , Feminino , Humanos , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Células Epiteliais/metabolismo , Insulina/farmacologia , Insulina/genética , Interferons/genética , Mutação , Endométrio/metabolismoRESUMO
Obesity impacts fertility and is positively correlated with endometrial hyperplasia and endometrial cancer occurrence. Endometrial epithelia often harbor disease driver-mutations, while endometrial stroma are highly regulative of neighboring epithelia. Here, we sought to determine distinct transcriptome changes occurring in individual cell types in the obese mouse uterus. Outbred CD-1 mice were fed high-fat or control diets for 18 weeks, estrous cycle staged, and endometrial epithelia, macrophages, and stroma isolated for transcriptomic analysis. High-fat diet mice displayed increased body mass and developed glucose intolerance, hyperinsulinemia, and fatty liver. Obese mouse epithelia displayed differential gene expression for genes related to innate immunity and leukocyte chemotaxis. The obese mouse stroma differentially expressed factors related to circadian rhythm, and expression of these genes correlated with glucose tolerance or body mass. We observed correlations between F4/80 + macrophage numbers, Cleaved Caspase 3 (CC3) apoptosis marker staining and glucose intolerance among obese mice, including a subgroup of obese mice with high CC3 + luminal epithelia. This subgroup displayed differential gene expression among all cell types, with pathways related to immune escape in epithelia and macrophages, while the stroma dysregulated pathways related to regulation of epithelia. These results suggest an important role for differential response of both the epithelia and stroma in their response to obesity, while macrophages are dysregulated in the context of apoptotic epithelia. The obesity-related gene expression programs in cells within the uterine microenvironment may influence the ability of the endometrium to function during pregnancy and influence disease pathogenesis.
Assuntos
Intolerância à Glucose , Transcriptoma , Gravidez , Feminino , Camundongos , Animais , Camundongos Obesos , Obesidade/genética , Obesidade/metabolismo , Dieta Hiperlipídica/efeitos adversosRESUMO
BACKGROUND: SWI/SNF (BAF) chromatin remodeling complexes regulate lineage-specific enhancer activity by promoting accessibility for diverse DNA-binding factors and chromatin regulators. Additionally, they are known to modulate the function of the epigenome through regulation of histone post-translational modifications and nucleosome composition, although the way SWI/SNF complexes govern the epigenome remains poorly understood. Here, we investigate the function of ARID1A, a subunit of certain mammalian SWI/SNF chromatin remodeling complexes associated with malignancies and benign diseases originating from the uterine endometrium. RESULTS: Through genome-wide analysis of human endometriotic epithelial cells, we show that more than half of ARID1A binding sites are marked by the variant histone H3.3, including active regulatory elements such as super-enhancers. ARID1A knockdown leads to H3.3 depletion and gain of canonical H3.1/3.2 at ARID1A-bound active regulatory elements, and a concomitant redistribution of H3.3 toward genic elements. ARID1A interactions with the repressive chromatin remodeler CHD4 (NuRD) are associated with H3.3, and ARID1A is required for CHD4 recruitment to H3.3. ZMYND8 interacts with CHD4 to suppress a subset of ARID1A, CHD4, and ZMYND8 co-bound, H3.3+ H4K16ac+ super-enhancers near genes governing extracellular matrix, motility, adhesion, and epithelial-to-mesenchymal transition. Moreover, these gene expression alterations are observed in human endometriomas. CONCLUSIONS: These studies demonstrate that ARID1A-containing BAF complexes are required for maintenance of the histone variant H3.3 at active regulatory elements, such as super-enhancers, and this function is required for the physiologically relevant activities of alternative chromatin remodelers.
Assuntos
Cromatina , Proteínas de Ligação a DNA , Histonas , Fatores de Transcrição , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Histonas/genética , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Nucleossomos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We have developed a mouse DNA methylation array that contains 296,070 probes representing the diversity of mouse DNA methylation biology. We present a mouse methylation atlas as a rich reference resource of 1,239 DNA samples encompassing distinct tissues, strains, ages, sexes, and pathologies. We describe applications for comparative epigenomics, genomic imprinting, epigenetic inhibitors, patient-derived xenograft assessment, backcross tracing, and epigenetic clocks. We dissect DNA methylation processes associated with differentiation, aging, and tumorigenesis. Notably, we find that tissue-specific methylation signatures localize to binding sites for transcription factors controlling the corresponding tissue development. Age-associated hypermethylation is enriched at regions of Polycomb repression, while hypomethylation is enhanced at regions bound by cohesin complex members. Apc Min/+ polyp-associated hypermethylation affects enhancers regulating intestinal differentiation, while hypomethylation targets AP-1 binding sites. This Infinium Mouse Methylation BeadChip (version MM285) is widely accessible to the research community and will accelerate high-sample-throughput studies in this important model organism.
RESUMO
Desmoplastic small round cell tumor (DSRCT) is a rare pediatric sarcoma with poor overall survival. This tumor is absolutely dependent on the continued expression and activity of its defining molecular lesion, the EWS-WT1 transcription factor. Unfortunately, the therapeutic targeting of transcription factors is challenging, and there is a critical need to identify compounds that inhibit EWS-WT1. Here we show that the compound lurbinectedin inhibits EWS-WT1 by redistributing the protein within the nucleus to the nucleolus. This nucleolar redistribution interferes with the activity of EWS-WT1 to reverse the expression of over 70% of the transcriptome. In addition, the compound blocks the expression of the EWS-WT1 fusion protein to inhibit cell proliferation at the lowest GI50 ever reported for this compound in any cell type. The effects occur at concentrations that are easily achievable in the clinic and translate to the in vivo setting to cause tumor regressions in multiple mice in a xenograft and PDX model of DSRCT. Importantly, this mechanism of nucleolar redistribution is also seen with wild-type EWSR1 and the related fusion protein EWS-FLI1. This provides evidence for a "class effect" for the more than 18 tumors driven by EWSR1 fusion proteins. More importantly, the data establish lurbinectedin as a promising clinical candidate for DSRCT.
Assuntos
Carbolinas , Tumor Desmoplásico de Pequenas Células Redondas , Compostos Heterocíclicos de 4 ou mais Anéis , Proteínas de Fusão Oncogênica , Sarcoma , Animais , Carbolinas/farmacologia , Tumor Desmoplásico de Pequenas Células Redondas/tratamento farmacológico , Tumor Desmoplásico de Pequenas Células Redondas/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismoRESUMO
Endometrial cancer (EC) is characterized by high estrogen levels unopposed by progesterone. Treatment with progestins is standard for early EC, but the response to progestins is dependent on progesterone receptor (PGR) expression. Here, we show that the expression of PGR in endometrial epithelial cells is dependent on ARID1A, a DNA-binding subunit of the SWI/SNF chromatin-remodeling complex that is commonly mutated in EC. In endometrial epithelial cells with estrogen receptor overexpression, we find that ARID1A promotes estrogen signaling and regulates common gene expression programs. Normally, endometrial epithelial cells expressing estrogen receptors respond to estrogen by upregulating the PGR. However, when ARID1A expression is lost, upregulation of PGR expression is significantly reduced. This phenomenon can also occur following the loss of the SWI/SNF subunit BRG1, suggesting a role for ARID1A- and BRG1-containing complexes in PGR regulation. We find that PGR is regulated by a bivalent promoter, which harbors both H3K4me3 and H3K27me3 histone tail modifications. H3K27me3 is deposited by EZH2, and inhibition of EZH2 in the context of ARID1A loss results in restoration of estrogen-induced PGR expression. Our results suggest a role for ARID1A deficiency in the loss of PGR in late-stage EC and a therapeutic utility for EZH2 inhibitors in this disease.
Assuntos
Histonas , Proteínas Nucleares , Estrogênios/farmacologia , Feminino , Humanos , Proteínas Nucleares/metabolismo , Progestinas/farmacologia , Receptores de Progesterona/metabolismoRESUMO
TP53 and ARID1A are frequently mutated across cancer but rarely in the same primary tumor. Endometrial cancer has the highest TP53-ARID1A mutual exclusivity rate. However, the functional relationship between TP53 and ARID1A mutations in the endometrium has not been elucidated. We used genetically engineered mice and in vivo genomic approaches to discern both unique and overlapping roles of TP53 and ARID1A in the endometrium. TP53 loss with oncogenic PIK3CAH1047R in the endometrial epithelium results in features of endometrial hyperplasia, adenocarcinoma, and intraepithelial carcinoma. Mutant endometrial epithelial cells were transcriptome profiled and compared to control cells and ARID1A/PIK3CA mutant endometrium. In the context of either TP53 or ARID1A loss, PIK3CA mutant endometrium exhibited inflammatory pathway activation, but other gene expression programs differed based on TP53 or ARID1A status, such as epithelial-to-mesenchymal transition. Gene expression patterns observed in the genetic mouse models are reflective of human tumors with each respective genetic alteration. Consistent with TP53-ARID1A mutual exclusivity, the p53 pathway is activated following ARID1A loss in the endometrial epithelium, where ARID1A normally directly represses p53 pathway genes in vivo, including the stress-inducible transcription factor, ATF3. However, co-existing TP53-ARID1A mutations led to invasive adenocarcinoma associated with mutant ARID1A-driven ATF3 induction, reduced apoptosis, TP63+ squamous differentiation and invasion. These data suggest TP53 and ARID1A mutations drive shared and distinct tumorigenic programs in the endometrium and promote invasive endometrial cancer when existing simultaneously. Hence, TP53 and ARID1A mutations may co-occur in a subset of aggressive or metastatic endometrial cancers, with ARID1A loss promoting squamous differentiation and the acquisition of invasive properties.
Assuntos
Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Carcinogênese/genética , Carcinoma in Situ/genética , Carcinoma in Situ/patologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Hiperplasia Endometrial/genética , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/patologia , Endométrio/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Mutação/genéticaRESUMO
Genome editing in pigs has been made efficient, practical, and economically viable by the CRISPR/Cas9 platform, representing a promising new era in translational modeling of human disease for research and preclinical development of therapies and devices. Porcine embryo microinjection provides a universally available, efficient option over somatic-cell nuclear transfer, but requires that critical considerations be made in genotypic validation of the models that routinely go unaddressed. Accurate validation of genotypes is especially important when modeling genetic disorders, such as neurofibromatosis type 1 (NF1) that exhibits complex genotype-phenotypic relationships. NF1, an autosomal dominant disorder, is particularly hard to model as it manifests very differently across patients, and even within families, with over 3,000 disease-associated mutations of the neurofibromin 1 (NF1) gene identified. The precise nature of the mutations plays a role in the complex phenotypic presentation of the disorder that includes benign and malignant peripheral and central nervous system tumors, a variety of motor deficits and debilitating cognitive impairments and musculoskeletal, cardiovascular, and gastrointestinal disorders. NF1 can also often involve mutations in passenger genes such as TP53. In this manuscript, we describe the creation of three novel porcine models of NF1 and a model additionally harboring a mutation in TP53 by embryo microinjection of CRISPR/Cas9. We present the challenges encountered in validation of genotypes and the methodological strategies developed to counter the hurdles. We present simple options for quantifying level of mosaicism: a quantitative method (targeted amplicon sequencing) for small edits such as SNPs and indels and a semiquantitative method (competitive PCR) for large edits. Characterization of mosaicism allowed for strategic selection of founder pigs for rapid, economical expansion of genetically defined lines. We also present commonly observed unexpected DNA repair products (i.e., structural variants or cryptic alleles) that are refractory to PCR amplification and thus evade detection. We present the use of copy number variance assays to overcome hurdles in detecting cryptic alleles. The report provides a framework for genotypic validation of porcine models created by embryo microinjection and the expansion of lines in an efficient manner.
RESUMO
Benign peripheral nerve sheath tumors are the clinical hallmark of Neurofibromatosis Type 1. They account for substantial morbidity and mortality in NF1. Cutaneous (CNF) and plexiform neurofibromas (PNF) share nearly identical histology, but maintain different growth rates and risk of malignant conversion. The reasons for this disparate clinical behavior are not well explained by recent genome or transcriptome profiling studies. We hypothesized that CNFs and PNFs are epigenetically distinct tumor types that exhibit differential signaling due to genome-wide and site-specific methylation events. We interrogated the methylation profiles of 45 CNFs and 17 PNFs from NF1 subjects with the Illumina EPIC 850K methylation array. Based on these profiles, we confirm that CNFs and PNFs are epigenetically distinct tumors with broad differences in higher-order chromatin states and specific methylation events altering genes involved in key biological and cellular processes, such as inflammation, RAS/MAPK signaling, actin cytoskeleton rearrangement, and oxytocin signaling. Based on our identification of two separate DMRs associated with alternative leading exons in MAP2K3, we demonstrate differential RAS/MKK3/p38 signaling between CNFs and PNFs. Epigenetic reinforcement of RAS/MKK/p38 was a defining characteristic of CNFs leading to pro-inflammatory signaling and chromatin conformational changes, whereas PNFs signaled predominantly through RAS/MEK. Tumor size also correlated with specific CpG methylation events. Taken together, these findings confirm that NF1 deficiency influences the epigenetic regulation of RAS signaling fates, accounting for observed differences in CNF and PNF clinical behavior. The extension of these findings is that CNFs may respond differently than PNFs to RAS-targeted therapeutics raising the possibility of targeting p38-mediated inflammation for CNF treatment.
Assuntos
Neurofibroma Plexiforme , Neurofibromatose 1 , Epigênese Genética , Epigenômica , Humanos , Neurofibroma Plexiforme/genética , Neurofibromatose 1/genética , Transdução de SinaisRESUMO
Endometriosis affects 1 in 10 women and is characterized by the presence of abnormal endometrium at ectopic sites. ARID1A mutations are observed in deeply invasive forms of the disease, often correlating with malignancy. To identify epigenetic dependencies driving invasion, we use an unbiased approach to map chromatin state transitions accompanying ARID1A loss in the endometrium. We show that super-enhancers marked by high H3K27 acetylation are strongly associated with ARID1A binding. ARID1A loss leads to H3K27 hyperacetylation and increased chromatin accessibility and enhancer RNA transcription at super-enhancers, but not typical enhancers, indicating that ARID1A normally prevents super-enhancer hyperactivation. ARID1A co-localizes with P300 at super-enhancers, and genetic or pharmacological inhibition of P300 in ARID1A mutant endometrial epithelia suppresses invasion and induces anoikis through the rescue of super-enhancer hyperacetylation. Among hyperactivated super-enhancers, SERPINE1 (PAI-1) is identified as an essential target gene driving ARID1A mutant endometrial invasion. Broadly, our findings provide rationale for therapeutic strategies targeting super-enhancers in ARID1A mutant endometrium.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endometriose/metabolismo , Endométrio/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Endometriose/patologia , Endométrio/patologia , Feminino , Humanos , Camundongos , Mutação , Coelhos , RatosRESUMO
Although ARID1A mutations are a hallmark feature, mutations in other SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling subunits are also observed in endometrial neoplasms. Here, we interrogated the roles of Brahma/SWI2-related gene 1 (BRG1, SMARCA4), the SWI/SNF catalytic subunit, in the endometrial epithelium. BRG1 loss affects more than one-third of all active genes and highly overlaps with the ARID1A gene regulatory network. Chromatin immunoprecipitation studies revealed widespread subunit-specific differences in transcriptional regulation, as BRG1 promoter interactions are associated with gene activation, while ARID1A binding is associated with gene repression. However, we identified a physiologically relevant subset of BRG1 and ARID1A co-regulated epithelial identity genes. Mice were genetically engineered to inactivate BRG1 specifically in the endometrial epithelium. Endometrial glands were observed embedded in uterine myometrium, indicating adenomyosis-like phenotypes. Molecular similarities were observed between BRG1 and ARID1A mutant endometrial cells in vivo, including loss of epithelial cell adhesion and junction genes. Collectively, these studies illustrate overlapping contributions of multiple SWI/SNF subunit mutations in the translocation of endometrium to distal sites, with loss of cell integrity being a common feature in SWI/SNF mutant endometrial epithelia.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Endométrio/patologia , Epitélio/patologia , Regulação da Expressão Gênica , Mutação , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Endométrio/metabolismo , Epitélio/metabolismo , Feminino , Camundongos , Camundongos KnockoutRESUMO
There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor-targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1-targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic "class" effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1-targeted combination therapy for Ewing sarcoma.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Plicamicina/farmacologia , Sarcoma de Ewing/tratamento farmacológico , Animais , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Nus , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Uterine fibroids are benign myometrial smooth muscle tumors of unknown etiology that, when symptomatic, are the most common indication for hysterectomy in the United States. Unsupervised clustering of results from DNA methylation analyses segregates normal myometrium from fibroids and further segregates the fibroids into subtypes characterized by MED12 mutation or activation of either HMGA2 or HMGA1 expression. Upregulation of HMGA2 expression does not always appear to be dependent on translocation but is associated with hypomethylation in the HMGA2 gene body. HOXA13 expression is upregulated in fibroids and correlates with expression of typical uterine fibroid genes. Significant overlap of differentially expressed genes is observed between cervical stroma and uterine fibroids compared with normal myometrium. These analyses show a possible role of DNA methylation in fibroid biology and suggest that homeotic transformation of myometrial cells to a more cervical stroma phenotype could be an important mechanism for etiology of the disease.
Assuntos
Epigenoma/genética , Exoma/genética , Leiomioma/genética , Leiomioma/metabolismo , Transcriptoma/genética , Metilação de DNA/genética , Metilação de DNA/fisiologia , Feminino , Perfilação da Expressão Gênica/métodos , Proteína HMGA1a/genética , Proteína HMGA2/genética , Proteínas de Homeodomínio/genética , Humanos , Mutação/genética , Miométrio/metabolismoRESUMO
ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. Here we show that monoallelic loss of ARID1A in the mouse endometrial epithelium is sufficient for vaginal bleeding when combined with PI3K activation. Sorted mutant epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT). We further show that ARID1A is bound to promoters with open chromatin, but ARID1A loss leads to increased promoter chromatin accessibility and the expression of EMT genes. PI3K activation partially rescues the mesenchymal phenotypes driven by ARID1A loss through antagonism of ARID1A target gene expression, resulting in partial EMT and invasion. We propose that ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates, and that coexistent ARID1A and PI3K mutations promote epithelial transdifferentiation and collective invasion. Broadly, our findings support a role for collective epithelial invasion in the spread of abnormal endometrial tissue.
Assuntos
Transformação Celular Neoplásica/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/genética , Transição Epitelial-Mesenquimal/genética , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular , Movimento Celular/genética , Cromatina/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Neoplasias do Endométrio/patologia , Endométrio/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Haploinsuficiência , Humanos , Mutação com Perda de Função , Camundongos , Camundongos Transgênicos , Miométrio/patologia , Invasividade Neoplásica/genética , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismoRESUMO
Oral papillomatosis was diagnosed in a gray wolf ( Canis lupus ) with sarcoptic mange from Minnesota, US found dead in February 2015. Intranuclear inclusion bodies were evident histologically, and papillomaviral antigens were confirmed using immunohistochemistry. Sequencing of the L1 papillomavirus gene showed closest similarity to Lambdapapillomavirus 2.
Assuntos
Lambdapapillomavirus/classificação , Infecções por Papillomavirus/veterinária , Escabiose/veterinária , Lobos , Determinação da Idade pelos Dentes/veterinária , Animais , DNA Viral/química , DNA Viral/genética , DNA Viral/isolamento & purificação , Evolução Fatal , Imuno-Histoquímica/veterinária , Lambdapapillomavirus/genética , Lambdapapillomavirus/imunologia , Lábio/patologia , Lábio/virologia , Masculino , Minnesota , Boca/patologia , Boca/virologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Sarcoptes scabiei/classificação , Escabiose/complicações , Escabiose/diagnóstico , Pele/parasitologia , Pele/patologiaRESUMO
BACKGROUND: Methionine is an important nutrient in animal feed and several approaches have been developed to increase methionine concentration in maize (Zea mays L.) grain. One approach is through traditional breeding using recurrent selection. Using divergent selection, genetically related populations with extreme differences in grain methionine content were produced. In order to better understand the molecular mechanisms controlling grain methionine content, we examined seed proteins, transcript levels of candidate genes, and genotypes of these populations. RESULTS: Two populations were selected for high or low methionine concentration for eight generations and 40 and 56% differences between the high and low populations in grain methionine concentration were observed. Mean values between the high and low methionine populations differed by greater than 1.5 standard deviations in some cycles of selection. Other amino acids and total protein concentration exhibited much smaller changes. In an effort to understand the molecular mechanisms that contribute to these differences, we compared transcript levels of candidate genes encoding high methionine seed storage proteins involved in sulfur assimilation or methionine biosynthesis. In combination, we also explored the genetic mechanisms at the SNP level through implementation of an association analysis. Significant differences in methionine-rich seed storage protein genes were observed in comparisons of high and low methionine populations, while transcripts of seed storage proteins lacking high levels of methionine were unchanged. Seed storage protein levels were consistent with transcript levels. Two genes involved in sulfur assimilation, Cys2 and CgS1 showed substantial differences in allele frequencies when two selected populations were compared to the starting populations. Major genes identified across cycles of selection by a high-stringency association analysis included dzs18, wx, dzs10, and zp27. CONCLUSIONS: We hypothesize that transcriptional changes alter sink strength by altering the levels of methionine-rich seed storage proteins. To meet the altered need for sulfur, a cysteine-rich seed storage protein is altered while sulfur assimilation and methionine biosynthesis throughput is changed by selection for certain alleles of Cys2 and CgS1.
Assuntos
Metionina/metabolismo , Zea mays/metabolismo , Regulação da Expressão Gênica de Plantas , Polimorfismo de Nucleotídeo Único/genética , Zea mays/genéticaRESUMO
Epstein-Barr virus (EBV) reactivation involves the ordered induction of approximately 90 viral genes that participate in the generation of infectious virions. Using strand-specific RNA-seq to assess the EBV transcriptome during reactivation, we found extensive bidirectional transcription extending across nearly the entire genome. In contrast, only 4% of the EBV genome is currently bidirectionally annotated. Most of the newly identified transcribed regions show little evidence of coding potential, supporting noncoding roles for most of these RNAs. Based on previous cellular long noncoding RNA size calculations, we estimate that there are likely hundreds more EBV genes expressed during reactivation than was previously known. Limited 5' and 3' rapid amplification of cDNA ends (RACE) experiments and findings of novel splicing events by RNA-seq suggest that the complexity of the viral genome during reactivation may be even greater. Further analysis of antisense transcripts at some of the EBV latency gene loci showed that they are "late" genes, they are nuclear, and they tend to localize in areas of the nucleus where others find newly synthesized viral genomes. This raises the possibility that these transcripts perform functions such as new genome processing, stabilization, organization, etc. The finding of a significantly more complex EBV transcriptome during reactivation changes our view of the viral production process from one that is facilitated and regulated almost entirely by previously identified viral proteins to a process that also involves the contribution of a wide array of virus encoded noncoding RNAs. Epstein-Barr virus (EBV) is a herpesvirus that infects the majority of the world's population, in rare cases causing serious disease such as lymphoma and gastric carcinoma. Using strand-specific RNA-seq, we have studied viral gene expression during EBV reactivation and have discovered hundreds more viral transcripts than were previously known. The finding of alternative splicing and the prevalence of overlapping transcripts indicate additional complexity. Most newly identified transcribed regions do not encode proteins but instead likely function as noncoding RNA molecules which could participate in regulating gene expression, gene splicing or even activities such as viral genome processing. These findings broaden the scope of what we need to consider to understand the viral manufacturing process. As more detailed studies are undertaken they will likely change the way we view this process as a whole.
Assuntos
Infecções por Vírus Epstein-Barr/virologia , Genoma Viral , Herpesvirus Humano 4/genética , Transcrição Gênica , Ativação Viral , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Humanos , Splicing de RNA , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/genética , Latência ViralRESUMO
PURPOSE: Little is known about the role of sequence variation in the pathology of HSV-1 keratitis virus. The goal was to show that a multiplex, high-throughput genome-sequencing approach is feasible for simultaneously sequencing seven HSV-1 ocular strains. METHODS: A genome sequencer was used to sequence the HSV-1 ocular isolates TFT401, 134, CJ311, CJ360, CJ394, CJ970, and OD4, in a single lane. Reads were mapped to the HSV-1 strain 17 reference genome by high-speed sequencing. ClustalW was used for alignment, and the Mega 4 package was used for phylogenetic analysis (www.megasoftware.net). Simplot was used to compare genetic variability and high-speed sequencing was used to identify SNPs (developed by Stuart Ray, Johns Hopkins University School of Medicine, Baltimore, MD, http://sray.med.som.jhml.edu/SCRoftware/simplot). RESULTS: Approximately 95% to 99% of the seven genomes were sequenced in a single lane with average coverage ranging from 224 to 1345. Phylogenetic analysis of the sequenced genome regions revealed at least three clades. Each strain had approximately 200 coding SNPs compared to strain 17, and these were evenly spaced along the genomes. Four genes were highly conserved, and six were more variable. Reduced coverage was obtained in the highly GC-rich terminal repeat regions. CONCLUSIONS: Multiplex sequencing is a cost-effective way to obtain the genomic sequences of ocular HSV-1 isolates with sufficient coverage of the unique regions for genomic analysis. The number of SNPs and their distribution will be useful for analyzing the genetics of virulence, and the sequence data will be useful for studying HSV-1 evolution and for the design of structure-function studies.