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1.
BMC Bioinformatics ; 25(1): 21, 2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38216886

RESUMO

BACKGROUND: Metagene plots provide a visualization of biological signal trends over subsections of the genome and are used to perform high-level analysis of experimental data by aggregating genome-level data to create an average profile. The generation of metagene plots is useful for summarizing the results of many sequencing-based applications. Despite their prevalence and utility, the standard metagene plot is blind to conflicting signals within data. If multiple distinct trends occur, they can interact destructively, creating a plot that does not accurately represent any of the underlying trends. RESULTS: We present MetageneCluster, a Python tool to generate a collection of representative metagene plots based on k-means clustering of genomic regions of interest. Clustering the data by similarity allows us to identify patterns within the features of interest. We are then able to summarize each pattern present in the data, rather than averaging across the entire feature space. We show that our method performs well when used to identify conflicting signals in real-world genome-level data. CONCLUSIONS: Overall, MetageneCluster is a user-friendly tool for the creation of metagene plots that capture distinct patterns in underlying sequence data.


Assuntos
Genoma , Genômica , Genômica/métodos , Software
3.
Nature ; 606(7913): 396-405, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35650435

RESUMO

Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.


Assuntos
Envelhecimento , Pulmão , Melanoma , Metástase Neoplásica , Células Estromais , Microambiente Tumoral , Idoso , Envelhecimento/patologia , Fibroblastos/patologia , Humanos , Pulmão/patologia , Melanoma/patologia , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Neoplasia Residual , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Pele/patologia , Células Estromais/patologia , Proteína Wnt-5a , c-Mer Tirosina Quinase , Receptor Tirosina Quinase Axl
4.
JID Innov ; 2(2): 100076, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35146482

RESUMO

Murine cancer cell lines are powerful research tools to complement studies in genetically engineered mouse models. We have established 21 melanoma cell lines from embryonic stem cell-genetically engineered mouse models driven by alleles that model the most frequent genetic alterations in human melanoma. In addition, these cell lines harbor regulatory alleles for the genomic integration of transgenes and the regulation of expression of such transgenes. In this study, we report a comprehensive characterization of these cell lines. Specifically, we validated melanocytic origin, driver allele recombination and expression, and activation of the oncogenic MAPK and protein kinase B pathways. We further tested tumor formation in syngeneic immunocompetent recipients as well as the functionality of the integrated Tet-ON system and recombination-mediated cassette exchange homing cassette. Finally, by deleting the transcription factor MAFG with an inducible CRISPR/Cas9 approach, we show the utility of the regulatory alleles for candidate gene modulation. These cell lines will be a valuable resource for studying melanoma biology and therapy.

5.
J Clin Invest ; 131(6)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33720046

RESUMO

Continued thinning of the atmospheric ozone, which protects the earth from damaging ultraviolet radiation (UVR), will result in elevated levels of UVR reaching the earth's surface, leading to a drastic increase in the incidence of skin cancer. In addition to promoting carcinogenesis in skin cells, UVR is a potent extrinsic driver of age-related changes in the skin known as "photoaging." We are in the preliminary stages of understanding of the role of intrinsic aging in melanoma, and the tumor-permissive effects of photoaging on the skin microenvironment remain largely unexplored. In this Review, we provide an overview of the impact of UVR on the skin microenvironment, addressing changes that converge or diverge with those observed in intrinsic aging. Intrinsic and extrinsic aging promote phenotypic changes to skin cell populations that alter fundamental processes such as melanogenesis, extracellular matrix deposition, inflammation, and immune response. Given the relevance of these processes in cancer, we discuss how photoaging might render the skin microenvironment permissive to melanoma progression.


Assuntos
Melanoma/etiologia , Envelhecimento da Pele/efeitos da radiação , Neoplasias Cutâneas/etiologia , Microambiente Tumoral/efeitos da radiação , Envelhecimento/imunologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Progressão da Doença , Matriz Extracelular/efeitos da radiação , Humanos , Tolerância Imunológica/efeitos da radiação , Melaninas/biossíntese , Melanoma/imunologia , Melanoma/metabolismo , Camundongos , Receptores de Hidrocarboneto Arílico/metabolismo , Pele/imunologia , Pele/metabolismo , Pele/efeitos da radiação , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Raios Ultravioleta/efeitos adversos , Ácido Urocânico/metabolismo , Vitamina D/metabolismo
6.
Cancer Res ; 81(3): 658-670, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262126

RESUMO

Metastatic dissemination remains a significant barrier to successful therapy for melanoma. Wnt5A is a potent driver of invasion in melanoma and is believed to be secreted from the tumor microenvironment (TME). Our data suggest that myeloid-derived suppressor cells (MDSC) in the TME are a major source of Wnt5A and are reliant upon Wnt5A for multiple actions. Knockdown of Wnt5A specifically in the myeloid cells demonstrated a clear decrease in Wnt5A expression within the TME in vivo as well as a decrease in intratumoral MDSC and regulatory T cell (Treg). Wnt5A knockdown also decreased the immunosuppressive nature of MDSC and decreased expression of TGFß1 and arginase 1. In the presence of Wnt5A-depleted MDSC, tumor-infiltrating lymphocytes expressed decreased PD-1 and LAG3, suggesting a less exhausted phenotype. Myeloid-specific Wnt5A knockdown also led to decreased lung metastasis. Tumor-infiltrating MDSC from control animals showed a strong positive correlation with Treg, which was completely ablated in animals with Wnt5A-negative MDSC. Overall, our data suggest that while MDSC contribute to an immunosuppressive and less immunogenic environment, they exhibit an additional function as the major source of Wnt5A in the TME. SIGNIFICANCE: These findings demonstrate that myeloid cells provide a major source of Wnt5A to facilitate metastatic potential in melanoma cells and rely on Wnt5A for their immunosuppressive function.


Assuntos
Melanoma/metabolismo , Células Supressoras Mieloides/metabolismo , Microambiente Tumoral , Proteína Wnt-5a/metabolismo , Animais , Antígenos CD/metabolismo , Arginase/metabolismo , Linhagem Celular Tumoral , Feminino , Neoplasias Pulmonares/secundário , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Melanoma/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Supressoras Mieloides/imunologia , Invasividade Neoplásica , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteína do Gene 3 de Ativação de Linfócitos
7.
Clin Cancer Res ; 26(21): 5709-5719, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33097493

RESUMO

PURPOSE: Angiogenesis is thought to be critical for tumor metastasis. However, inhibiting angiogenesis using antibodies such as bevacizumab (Avastin), has had little impact on melanoma patient survival. We have demonstrated that both angiogenesis and metastasis are increased in older individuals, and therefore sought to investigate whether there was an age-related difference in response to bevacizumab, and if so, what the underlying mechanism could be. EXPERIMENTAL DESIGN: We analyzed data from the AVAST-M trial of 1,343 patients with melanoma treated with bevacizumab to determine whether there is an age-dependent response to bevacizumab. We also examined the age-dependent expression of VEGF and its cognate receptors in patients with melanoma, while using syngeneic melanoma animal models to target VEGF in young versus old mice. We also examined the age-related proangiogenic factor secreted frizzled-related protein 2 (sFRP2) and whether it could modulate response to anti-VEGF therapy. RESULTS: We show that older patients respond poorly to bevacizumab, whereas younger patients show improvement in both disease-free survival and overall survival. We find that targeting VEGF does not ablate angiogenesis in an aged mouse model, while sFRP2 promotes angiogenesis in vitro and in young mice. Targeting sFRP2 in aged mice successfully ablates angiogenesis, while the effects of targeting VEGF in young mice can be overcome by increasing sFRP2. CONCLUSIONS: VEGF is decreased during aging, thereby reducing response to bevacizumab. Despite the decrease in VEGF, angiogenesis is increased because of an increase in sFRP2 in the aged tumor microenvironment. These results stress the importance of considering age as a factor for designing targeted therapies.


Assuntos
Melanoma/genética , Proteínas de Membrana/genética , Neovascularização Patológica/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Animais , Bevacizumab/administração & dosagem , Linhagem Celular Tumoral , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Microambiente Tumoral/efeitos dos fármacos
8.
Cancer Discov ; 10(9): 1282-1295, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32499221

RESUMO

Older patients with melanoma (>50 years old) have poorer prognoses and response rates to targeted therapy compared with young patients (<50 years old), which can be driven, in part, by the aged microenvironment. Here, we show that aged dermal fibroblasts increase the secretion of neutral lipids, especially ceramides. When melanoma cells are exposed to the aged fibroblast lipid secretome, or cocultured with aged fibroblasts, they increase the uptake of lipids via the fatty acid transporter FATP2, which is upregulated in melanoma cells in the aged microenvironment and known to play roles in lipid synthesis and accumulation. We show that blocking FATP2 in melanoma cells in an aged microenvironment inhibits their accumulation of lipids and disrupts their mitochondrial metabolism. Inhibiting FATP2 overcomes age-related resistance to BRAF/MEK inhibition in animal models, ablates tumor relapse, and significantly extends survival time in older animals. SIGNIFICANCE: These data show that melanoma cells take up lipids from aged fibroblasts, via FATP2, and use them to resist targeted therapy. The response to targeted therapy is altered in aged individuals because of the influences of the aged microenvironment, and these data suggest FATP2 as a target to overcome resistance.See related commentary by Montal and White, p. 1255.This article is highlighted in the In This Issue feature, p. 1241.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Coenzima A Ligases/metabolismo , Fibroblastos/metabolismo , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Senescência Celular , Técnicas de Cocultura , Coenzima A Ligases/antagonistas & inibidores , Derme/citologia , Derme/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Queratinócitos/metabolismo , Metabolismo dos Lipídeos , Melanoma/patologia , Terapia de Alvo Molecular/métodos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Cutâneas/patologia , Microambiente Tumoral
10.
Mol Cell ; 77(3): 633-644.e5, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31836388

RESUMO

Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.


Assuntos
Melanoma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Wnt-5a/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , MAP Quinase Quinase Quinases/metabolismo , Melanoma/genética , Melanoma/patologia , Terapia de Alvo Molecular , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/fisiologia
11.
Cell Rep ; 27(13): 3760-3769.e4, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242410

RESUMO

In the eukaryotic cell, spliceosomes assemble onto pre-mRNA cotranscriptionally. Spliceosome assembly takes place in the context of the chromatin environment, suggesting that the state of the chromatin may affect splicing. The molecular details and mechanisms through which chromatin affects splicing, however, are still unclear. Here, we show a role for the histone methyltransferase Set2 and its histone modification, H3K36 methylation, in pre-mRNA splicing through high-throughput sequencing. Moreover, the effect of H3K36 methylation on pre-mRNA splicing is mediated through the chromodomain protein Eaf3. We find that Eaf3 is recruited to intron-containing genes and that Eaf3 interacts with the splicing factor Prp45. Eaf3 acts with Prp45 and Prp19 after formation of the precatalytic B complex around the time of splicing activation, thus revealing the step in splicing that is regulated by H3K36 methylation. These studies support a model whereby H3K36 facilitates recruitment of an "adapter protein" to support efficient, constitutive splicing.


Assuntos
Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Transcrição Gênica , Acetiltransferases/genética , Histonas/genética , Metilação , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética
12.
Pigment Cell Melanoma Res ; 32(2): 237-247, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30216694

RESUMO

This review will focus on the role of the tumor microenvironment (TME) in the development of drug resistance in melanoma. Resistance to mitogen-activated protein kinase inhibitors (MAPKi) in melanoma is observed months after treatment, a phenomenon that is often attributed to the incredible plasticity of melanoma cells but may also depend on the TME. The TME is unique in its cellular composition-it contains fibroblasts, immune cells, endothelial cells, adipocytes, and among others. In addition, the TME provides "non-homeostatic" levels of oxygen, nutrients (hypoxia and metabolic stress), and extracellular matrix proteins, creating a pro-tumorigenic niche that drives resistance to MAPKi treatment. In this review, we will focus on how changes in the tumor microenvironment regulate MAPKi resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma/tratamento farmacológico , Melanoma/patologia , Terapia de Alvo Molecular , Microambiente Tumoral , Animais , Humanos , Imunidade , Melanoma/imunologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo
13.
Cancer Discov ; 9(1): 82-95, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30279172

RESUMO

Older patients with melanoma have lower rates of sentinel lymph node (LN) metastases yet paradoxically have inferior survival. Patient age correlated with an inability to retain Technetium radiotracer during sentinel LN biopsy in more than 1,000 patients, and high Technetium counts correlated to better survival. We hypothesized that loss of integrity in the lymphatic vasculature due to extracellular matrix (ECM) degradation might play a role. We have implicated HAPLN1 in age-dependent ECM degradation in the dermis. Here, we queried whether HAPLN1 could be altered in the lymphatic ECM. Lymphatic HAPLN1 expression was prognostic of long-term patient survival. Adding recombinant HAPLN1 to aged fibroblast ECMs in vitro reduced endothelial permeability via modulation of VE-cadherin junctions, whereas endothelial permeability was increased following HAPLN1 knockdown in young fibroblasts. In vivo, reconstitution of HAPLN1 in aged mice increased the number of LN metastases, but reduced visceral metastases. These data suggest that age-related changes in ECM can contribute to impaired lymphatics. SIGNIFICANCE: Our studies reveal that changes in the stroma during aging may influence the way tumor cells traffic through the lymphatic vasculature. Aging may dictate the route of metastatic dissemination of tumor cells, and understanding these changes may help to reveal targetable moieties in the aging tumor microenvironment.See related commentary by Marie and Merlino, p. 19.This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Envelhecimento , Proteínas da Matriz Extracelular/metabolismo , Melanoma/metabolismo , Proteoglicanas/metabolismo , Pele/metabolismo , Adulto , Animais , Células Cultivadas , Humanos , Sistema Imunitário , Metástase Linfática , Melanoma/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Pele/fisiopatologia , Microambiente Tumoral
14.
Cancer Discov ; 9(1): 64-81, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30279173

RESUMO

Physical changes in skin are among the most visible signs of aging. We found that young dermal fibroblasts secrete high levels of extracellular matrix (ECM) constituents, including proteoglycans, glycoproteins, and cartilage-linking proteins. The most abundantly secreted was HAPLN1, a hyaluronic and proteoglycan link protein. HAPLN1 was lost in aged fibroblasts, resulting in a more aligned ECM that promoted metastasis of melanoma cells. Reconstituting HAPLN1 inhibited metastasis in an aged microenvironment, in 3-D skin reconstruction models, and in vivo. Intriguingly, aged fibroblast-derived matrices had the opposite effect on the migration of T cells, inhibiting their motility. HAPLN1 treatment of aged fibroblasts restored motility of mononuclear immune cells, while impeding that of polymorphonuclear immune cells, which in turn affected regulatory T-cell recruitment. These data suggest that although age-related physical changes in the ECM can promote tumor cell motility, they may adversely affect the motility of some immune cells, resulting in an overall change in the immune microenvironment. Understanding the physical changes in aging skin may provide avenues for more effective therapy for older patients with melanoma. SIGNIFICANCE: These data shed light on the mechanochemical interactions that occur between aged skin, tumor, and immune cell populations, which may affect tumor metastasis and immune cell infiltration, with implications for the efficacy of current therapies for melanoma.See related commentary by Marie and Merlino, p. 19.This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Envelhecimento , Colágeno/metabolismo , Melanoma/metabolismo , Pele/metabolismo , Animais , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Sistema Imunitário , Melanoma/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Proteoglicanas/metabolismo , Pele/fisiopatologia , Microambiente Tumoral
15.
Clin Cancer Res ; 24(21): 5347-5356, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29898988

RESUMO

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1.Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies.Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice.Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347-56. ©2018 AACR See related commentary by Pawelec, p. 5193.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Imunomodulação/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fatores Etários , Animais , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Melanoma/tratamento farmacológico , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Transgênicos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Oncol Lett ; 14(5): 5497-5504, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29113177

RESUMO

Indoleamine 2,3-dioxygenase (IDO)-induced immunosuppression can be clinically beneficial for autoimmune diseases. Primary biliary cirrhosis (PBC) is characterized by autoimmune lesions of intrahepatic bile duct epithelial cells that may lead to irreversible cirrhosis or hepatocellular carcinoma. The present study assessed the expression and function of IDO in a cell culture model and in PBC patients. IDO expression was monitored in a human immortalized but non-malignant biliary epithelial cell (iBEC) line. Increased expression of IDO1/2 was observed in the iBECs following stimulation with interferon-γ (IFN-γ). The induction of IDO was IFN-γ-dependent, but was independent of the transforming growth factor-ß (TGF-ß) pathway. IDO enzymatic activity was observed in the supernatant of iBECs following stimulation with IFN-γ using colorimetric assays. A total of 47 serum samples from PBC patients were used to examine IDO activity by high-performance liquid chromatography, with samples from 24 healthy volunteers used as controls. Patients with PBC exhibited an increased rate of tryptophan to kynurenine conversion (P>0.01). Liver sections from patients with PBC (n=5) and those of healthy controls (n=5) were used for immunohistochemical studies. IDO expression was observed in biliary epithelial cells and in hepatocytes of PBC patients. Finally, the effect of tryptophan metabolites on human cluster of differentiation (CD) 4+ T cells in inducing polarization towards a regulatory T cell phenotype was examined. 3-Hydroxykynurenine significantly upregulated the fraction of CD4+ cells expressing forkhead box p3 (Foxp3). The results of the present study suggest a therapeutic opportunity for the management of PBC and indicate that tryptophan catabolism could serve as a potential biomarker to monitor disease progression.

17.
J Biol Chem ; 292(36): 14851-14866, 2017 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-28739803

RESUMO

Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae Here, we report a role for the chromatin remodeler SWI/SNF in respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q6 (ubiquinone or CoQ6) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism.


Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ubiquinona/biossíntese , Proteína Fosfatase 2/genética , Splicing de RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
18.
Nucleic Acids Res ; 45(13): 7708-7721, 2017 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-28637241

RESUMO

Despite its relatively streamlined genome, there are important examples of regulated RNA splicing in Saccharomyces cerevisiae, such as splicing of meiotic transcripts. Like other eukaryotes, S. cerevisiae undergoes a dramatic reprogramming of gene expression during meiosis, including regulated splicing of a number of crucial meiosis-specific RNAs. Splicing of a subset of these is dependent upon the splicing activator Mer1. Here we show a crucial role for the chromatin remodeler Swi/Snf in regulation of splicing of meiotic genes and find that the complex affects meiotic splicing in two ways. First, we show that Swi/Snf regulates nutrient-dependent downregulation of ribosomal protein encoding RNAs, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs (the ribosomal protein genes) to Mer1-regulated transcripts. We also demonstrate that Mer1 expression is dependent on Snf2, its acetylation state and histone H3 lysine 9 acetylation at the MER1 locus. Hence, Snf2 exerts systems level control of meiotic gene expression through two temporally distinct mechanisms, demonstrating that it is a key regulator of meiotic splicing in S. cerevisiae. We also reveal an evolutionarily conserved mechanism whereby the cell redirects its energy from maintaining its translational capacity to the process of meiosis.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Genes Fúngicos , Modelos Biológicos , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/citologia , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Genes Dev ; 31(7): 702-717, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28446598

RESUMO

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from premessenger RNA (pre-mRNA). Recent studies show the processes of RNA synthesis and RNA processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae, H2A.Z is deposited into chromatin by the SWR-C complex, is found near the 5' ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP (small nuclear RNA [snRNA] plus associated proteins), as H2A.Z shows extensive genetic interactions with U2 snRNP-associated proteins, and RNA sequencing (RNA-seq) reveals that introns with nonconsensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z's role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled, and unspliced RNAs are released. Together, these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.


Assuntos
Regulação Fúngica da Expressão Gênica , Histonas/genética , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Íntrons/genética , Nucleossomos/genética , Regiões Promotoras Genéticas , Precursores de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Spliceossomos/genética
20.
Plant J ; 86(6): 481-92, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27061965

RESUMO

MicroRNAs (miRNAs) are important regulatory molecules in eukaryotic organisms. Existing methods for the identification of mature miRNA sequences in plants rely extensively on the search for stem-loop structures, leading to high false negative rates. Here, we describe a probabilistic method for ranking putative plant miRNAs using a naïve Bayes classifier and its publicly available implementation. We use a number of properties to construct the classifier, including sequence length, number of observations, existence of detectable predicted miRNA* sequences, the distribution of nearby reads and mapping multiplicity. We apply the method to small RNA sequence data from soybean, peach, Arabidopsis and rice and provide experimental validation of several predictions in soybean. The approach performs well overall and strongly enriches for known miRNAs over other types of sequences. By utilizing a Bayesian approach to rank putative miRNAs, our method is able to score miRNAs that would be eliminated by other methods, such as those that have low counts or lack detectable miRNA* sequences. As a result, we are able to detect several soybean miRNA candidates, including some that are 24 nucleotides long, a class that is almost universally eliminated by other methods.


Assuntos
Teorema de Bayes , Biologia Computacional/métodos , MicroRNAs/genética , RNA de Plantas/genética , Sequência de Bases , Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/classificação , RNA de Plantas/classificação
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