RESUMO
Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.
Assuntos
Neoplasias da Mama/patologia , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes/patologia , Animais , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologia , Análise de Sequência de RNA , Análise de Célula Única , gama Catenina/metabolismoRESUMO
Sterol regulatory element-binding proteins (SREBPs) activate genes involved in the synthesis and trafficking of cholesterol and other lipids and are critical for maintaining lipid homeostasis. Aberrant SREBP activity, however, can contribute to obesity, fatty liver disease, and insulin resistance, hallmarks of metabolic syndrome. Our studies identify a conserved regulatory circuit in which SREBP-1 controls genes in the one-carbon cycle, which produces the methyl donor S-adenosylmethionine (SAMe). Methylation is critical for the synthesis of phosphatidylcholine (PC), a major membrane component, and we find that blocking SAMe or PC synthesis in C. elegans, mouse liver, and human cells causes elevated SREBP-1-dependent transcription and lipid droplet accumulation. Distinct from negative regulation of SREBP-2 by cholesterol, our data suggest a feedback mechanism whereby maturation of nuclear, transcriptionally active SREBP-1 is controlled by levels of PC. Thus, nutritional or genetic conditions limiting SAMe or PC production may activate SREBP-1, contributing to human metabolic disorders.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Humanos , Lipogênese , Camundongos , Modelos Animais , Fosfatidilcolinas/biossíntese , Interferência de RNA , S-Adenosilmetionina/biossínteseRESUMO
Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Animais , Blastocisto , Instabilidade Cromossômica , Metilação de DNA , Feminino , Impressão Genômica , Cariotipagem , Masculino , CamundongosRESUMO
Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Mutação , Regiões Promotoras Genéticas/genética , Estudos de Coortes , Fatores de Transcrição E2F/metabolismo , Exoma/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica/genética , RNA Longo não Codificante/genética , Receptores de Estrogênio/antagonistas & inibidoresRESUMO
Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.
Assuntos
Carcinogênese/metabolismo , Prolactina/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de Sinais/fisiologia , Células Estromais/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Celecoxib/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/metabolismo , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Receptores X de Retinoides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here, we show in Xenopus that RARγ is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets upregulated by RA receptor signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the genes encoding the RA-synthesizing enzyme Aldh1a2 and the RA-degrading enzyme Cyp26a1, RARγ1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RARγ1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of ncam1 mRNA in animal caps harvested from RARγ1 knockdown embryos. RARγ1 knockdown obliterates somite boundaries, and causes loss of Myod protein in the presomitic mesoderm, but ectopic, persistent expression of Myod protein in the trunk. Thus, RARγ1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal skeletal muscle differentiation.
Assuntos
Padronização Corporal/genética , Mesoderma/embriologia , Músculo Esquelético/embriologia , Receptores do Ácido Retinoico/genética , Xenopus laevis/embriologia , Família Aldeído Desidrogenase 1 , Aldeído Oxidase/biossíntese , Aldeído Oxidase/genética , Animais , Antígeno CD56/metabolismo , Adesão Celular/genética , Gastrulação/genética , Proteína MyoD/metabolismo , Receptores do Ácido Retinoico/metabolismo , Retinal Desidrogenase , Ácido Retinoico 4 Hidroxilase/biossíntese , Ácido Retinoico 4 Hidroxilase/genética , Transdução de Sinais/genética , Ativação Transcricional/genética , Tretinoína/metabolismo , Proteínas de Xenopus/biossíntese , Proteínas de Xenopus/genética , Xenopus laevis/genética , Receptor gama de Ácido RetinoicoRESUMO
Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.
Assuntos
Células-Tronco Embrionárias/metabolismo , Instabilidade Genômica , MAP Quinase Quinase Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Metilação de DNA , Células-Tronco Embrionárias/enzimologia , Humanos , Proteoma , TranscriptomaRESUMO
Pluripotent stem cell-derived human primordial germ cell-like cells (hPGCLCs) provide important opportunities to study primordial germ cells (PGCs). We robustly produced CD38+ hPGCLCs [â¼43% of FACS-sorted embryoid body (EB) cells] from primed-state induced pluripotent stem cells (iPSCs) after a 72-hour transient incubation in the four chemical inhibitors (4i)-naïve reprogramming medium and showed transcriptional consistency of our hPGCLCs with hPGCLCs generated in previous studies using various and distinct protocols. Both CD38+ hPGCLCs and CD38- EB cells significantly expressed PRDM1 and TFAP2C, although PRDM1 mRNA in CD38- cells lacked the 3'-UTR harboring miRNA binding sites regulating mRNA stability. Genes up-regulated in hPGCLCs were enriched for cell migration genes, and their promoters were enriched for the binding motifs of TFAP2 (which was identified in promoters of T, NANOS3, and SOX17) and the RREB-1 cell adhesion regulator. In EBs, hPGCLCs were identified exclusively in the outermost surface monolayer as dispersed cells or cell aggregates with strong and specific expression of POU5F1/OCT4 protein. Time-lapse live cell imaging revealed active migration of hPGCLCs on Matrigel. Whereas all hPGCLCs strongly expressed the CXCR4 chemotaxis receptor, its ligand CXCL12/SDF1 was not significantly expressed in the whole EBs. Exposure of hPGCLCs to CXCL12/SDF1 induced cell migration genes and antiapoptosis genes. Thus, our study shows that transcriptionally consistent hPGCLCs can be readily produced from hiPSCs after transition of their pluripotency from the primed state using various methods and that hPGCLCs resemble the early-stage PGCs randomly migrating in the midline region of human embryos before initiation of the CXCL12/SDF1-guided chemotaxis.
Assuntos
Movimento Celular/fisiologia , Corpos Embrioides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Agregação Celular , Diferenciação Celular , Movimento Celular/genética , Quimiocina CXCL12/metabolismo , Quimiocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Corpos Embrioides/citologia , Perfilação da Expressão Gênica , Genes Homeobox , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores CXCR4/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXF/metabolismo , Fator de Transcrição AP-2 , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.
Assuntos
Desmetilação do DNA , Epigênese Genética , Genoma , Impressão Genômica , Células Germinativas/metabolismo , Células-Tronco Pluripotentes/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Metilação de DNA , Embrião de Mamíferos , Feminino , Células Germinativas/citologia , Sequenciamento de Nucleotídeos em Larga Escala , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Masculino , Camundongos , Mutação , Células-Tronco Pluripotentes/citologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30-40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.
Assuntos
Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Masculino , Neoplasias da Próstata/patologia , Análise de Sequência de RNARESUMO
The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD(+)-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.
Assuntos
Regulação para Baixo , Jejum/fisiologia , Sirtuína 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Acetilação , Animais , Benzamidas/farmacologia , Caenorhabditis elegans , Linhagem Celular , Colesterol/biossíntese , Regulação para Baixo/efeitos dos fármacos , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Lipídeos/biossíntese , Camundongos , Naftóis/farmacologia , Niacinamida/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Sirtuínas/antagonistas & inibidoresRESUMO
Induced pluripotent stem cells (iPSCs) have been generated by enforced expression of defined sets of transcription factors in somatic cells. It remains controversial whether iPSCs are molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells. By comparing genetically identical mouse ES cells and iPSCs, we show here that their overall messenger RNA and microRNA expression patterns are indistinguishable with the exception of a few transcripts encoded within the imprinted Dlk1-Dio3 gene cluster on chromosome 12qF1, which were aberrantly silenced in most of the iPSC clones. Consistent with a developmental role of the Dlk1-Dio3 gene cluster, these iPSC clones contributed poorly to chimaeras and failed to support the development of entirely iPSC-derived animals ('all-iPSC mice'). In contrast, iPSC clones with normal expression of the Dlk1-Dio3 cluster contributed to high-grade chimaeras and generated viable all-iPSC mice. Notably, treatment of an iPSC clone that had silenced Dlk1-Dio3 with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of all-iPSC mice. Thus, the expression state of a single imprinted gene cluster seems to distinguish most murine iPSCs from ES cells and allows for the prospective identification of iPSC clones that have the full development potential of ES cells.
Assuntos
Cromossomos de Mamíferos/genética , Perfilação da Expressão Gênica , Inativação Gênica , Impressão Genômica/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Epigênese Genética/genética , Feminino , Fibroblastos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Família Multigênica/genética , Proteínas Nucleares/genética , Células-Tronco Pluripotentes/citologia , Proteínas/genética , RNA Longo não Codificante , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/genéticaRESUMO
Although biological effects of endocrine disrupting chemicals (EDCs) are often observed at unexpectedly low doses with occasional nonmonotonic dose-response characteristics, transcriptome-wide profiles of sensitivities or dose-dependent behaviors of the EDC responsive genes have remained unexplored. Here, we describe expressome analysis for the comprehensive examination of dose-dependent gene responses and its applications to characterize estrogen responsive genes in MCF-7 cells. Transcriptomes of MCF-7 cells exposed to varying concentrations of representative natural and xenobiotic estrogens for 48 h were determined by microarray and used for computational calculation of interpolated approximations of estimated transcriptomes for 300 doses uniformly distributed in log space for each chemical. The entire collection of these estimated transcriptomes, designated as the expressome, has provided unique opportunities to profile chemical-specific distributions of ligand sensitivities for large numbers of estrogen responsive genes, revealing that at low concentrations estrogens generally tended to suppress rather than to activate transcription. Gene ontology analysis demonstrated distinct functional enrichment between high- and low-sensitivity estrogen responsive genes, supporting the notion that a single EDC chemical can cause qualitatively distinct biological responses at different doses. Expressomal heatmap visualization of dose-dependent induction of Bisphenol A inducible genes showed a weak gene activation peak at a very low concentration range (ca. 0.1 nM) in addition to the main, strong gene activation peak at and above 100 nM. Thus, expressome analysis is a powerful approach to understanding the EDC dose-dependent dynamic changes in gene expression at the transcriptomal level, providing important information on the overall profiles of ligand sensitivities and nonmonotonic responses.
Assuntos
Disruptores Endócrinos/toxicidade , Estrogênios/toxicidade , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Compostos Benzidrílicos , Relação Dose-Resposta a Droga , Ontologia Genética , Humanos , Células MCF-7 , Análise em Microsséries , FenóisRESUMO
In Huntington's disease (HD), the size of the expanded HTT CAG repeat mutation is the primary driver of the processes that determine age at onset of motor symptoms. However, correlation of cellular biochemical parameters also extends across the normal repeat range, supporting the view that the CAG repeat represents a functional polymorphism with dominant effects determined by the longer allele. A central challenge to defining the functional consequences of this single polymorphism is the difficulty of distinguishing its subtle effects from the multitude of other sources of biological variation. We demonstrate that an analytical approach based upon continuous correlation with CAG size was able to capture the modest (â¼21%) contribution of the repeat to the variation in genome-wide gene expression in 107 lymphoblastoid cell lines, with alleles ranging from 15 to 92 CAGs. Furthermore, a mathematical model from an iterative strategy yielded predicted CAG repeat lengths that were significantly positively correlated with true CAG allele size and negatively correlated with age at onset of motor symptoms. Genes negatively correlated with repeat size were also enriched in a set of genes whose expression were CAG-correlated in human HD cerebellum. These findings both reveal the relatively small, but detectable impact of variation in the CAG allele in global data in these peripheral cells and provide a strategy for building multi-dimensional data-driven models of the biological network that drives the HD disease process by continuous analysis across allelic panels of neuronal cells vulnerable to the dominant effects of the HTT CAG repeat.
Assuntos
Expressão Gênica , Doença de Huntington/genética , Proteínas do Tecido Nervoso/genética , Repetições de Trinucleotídeos/genética , Idade de Início , Alelos , Linhagem Celular , Cerebelo/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Proteína Huntingtina , Doença de Huntington/diagnóstico , Doença de Huntington/metabolismo , Masculino , Modelos Genéticos , Polimorfismo Genético , Reprodutibilidade dos Testes , TranscriptomaRESUMO
An in vitro model of human meiosis would accelerate research into this important reproductive process and development of therapies for infertility. We have developed a method to induce meiosis starting from male or female human pluripotent stem cells. We demonstrate that DNMT1 inhibition, retinoid signaling activation, and overexpression of regulatory factors (anti-apoptotic BCL2, and pro-meiotic HOXB5, BOLL, or MEIOC) rapidly activates meiosis, with leptonema beginning at 6 days, zygonema at 9 days, and pachynema at 12 days. Immunofluorescence microscopy shows key aspects of meiosis, including chromosome synapsis and sex body formation. The meiotic cells express genes similar to meiotic oogonia in vivo, including all synaptonemal complex components and machinery for meiotic recombination. These findings establish an accessible system for inducing human meiosis in vitro.
RESUMO
Huntington's disease is initiated by the expression of a CAG repeat-encoded polyglutamine region in full-length huntingtin, with dominant effects that vary continuously with CAG size. The mechanism could involve a simple gain of function or a more complex gain of function coupled to a loss of function (e.g. dominant negative-graded loss of function). To distinguish these alternatives, we compared genome-wide gene expression changes correlated with CAG size across an allelic series of heterozygous CAG knock-in mouse embryonic stem (ES) cell lines (Hdh(Q20/7), Hdh(Q50/7), Hdh(Q91/7), Hdh(Q111/7)), to genes differentially expressed between Hdh(ex4/5/ex4/5) huntingtin null and wild-type (Hdh(Q7/7)) parental ES cells. The set of 73 genes whose expression varied continuously with CAG length had minimal overlap with the 754-member huntingtin-null gene set but the two were not completely unconnected. Rather, the 172 CAG length-correlated pathways and 238 huntingtin-null significant pathways clustered into 13 shared categories at the network level. A closer examination of the energy metabolism and the lipid/sterol/lipoprotein metabolism categories revealed that CAG length-correlated genes and huntingtin-null-altered genes either were different members of the same pathways or were in unique, but interconnected pathways. Thus, varying the polyglutamine size in full-length huntingtin produced gene expression changes that were distinct from, but related to, the effects of lack of huntingtin. These findings support a simple gain-of-function mechanism acting through a property of the full-length huntingtin protein and point to CAG-correlative approaches to discover its effects. Moreover, for therapeutic strategies based on huntingtin suppression, our data highlight processes that may be more sensitive to the disease trigger than to decreased huntingtin levels.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas Nucleares/biossíntese , Peptídeos/metabolismo , Expansão das Repetições de Trinucleotídeos , Alelos , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Estudo de Associação Genômica Ampla , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/terapia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genéticaRESUMO
Huntington's disease (HD) involves marked early neurodegeneration in the striatum, whereas the cerebellum is relatively spared despite the ubiquitous expression of full-length mutant huntingtin, implying that inherent tissue-specific differences determine susceptibility to the HD CAG mutation. To understand this tissue specificity, we compared early mutant huntingtin-induced gene expression changes in striatum to those in cerebellum in young Hdh CAG knock-in mice, prior to onset of evident pathological alterations. Endogenous levels of full-length mutant huntingtin caused qualitatively similar, but quantitatively different gene expression changes in the two brain regions. Importantly, the quantitatively different responses in the striatum and cerebellum in mutant mice were well accounted for by the intrinsic molecular differences in gene expression between the striatum and cerebellum in wild-type animals. Tissue-specific gene expression changes in response to the HD mutation, therefore, appear to reflect the different inherent capacities of these tissues to buffer qualitatively similar effects of mutant huntingtin. These findings highlight a role for intrinsic quantitative tissue differences in contributing to HD pathogenesis, and likely to other neurodegenerative disorders exhibiting tissue-specificity, thereby guiding the search for effective therapeutic interventions.
Assuntos
Cerebelo/patologia , Doença de Huntington/genética , Neostriado/patologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Ataxina-1 , Ataxinas , Biomarcadores/metabolismo , Cerebelo/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Doença de Huntington/patologia , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Neostriado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Transdução de Sinais/genéticaRESUMO
TOX3 is a nuclear protein containing a high mobility group (HMG)-box domain, which regulates Ca(2+)-dependent transcription in neurons through interaction with the cAMP-response-element-binding protein (CREB). TOX3 appears to be associated with breast cancer susceptibility and was previously shown to be expressed downstream of a cytoprotective cascade together with CITED1, a transcriptional regulator that does not bind directly to DNA. In the present study we show that TOX3 is predominantly expressed in the brain, forms homodimers and interacts with CITED1. TOX3 overexpression protects neuronal cells from cell death caused by endoplasmic reticulum stress or BAX overexpression through the induction of anti-apoptotic transcripts and repression of pro-apoptotic transcripts, which correlates with enhanced transcription involving isolated estrogen-responsive elements and estrogen-responsive promoters. However, both functions cannot be inhibited with the anti-estrogen fulvestrant and are only attenuated by mutation of estrogen-responsive elements. TOX3 also interacts with native CREB and induces the CREB-responsive BCL-2 promoter, which can be inhibited by coexpression of CITED1. Coexpression of CREB, by contrast, abolishes TOX3-mediated transcription from the estrogen-responsive complement C3 promoter. Our results suggest that TOX3 can enhance transcriptional activation from different cytoprotective promoters and that this is dependent on the predominance of either phosphorylated CREB or CITED1 within the transcriptionally active complex.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Progesterona/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Proteínas Reguladoras de Apoptose , Células COS , Sobrevivência Celular , Chlorocebus aethiops , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Grupo de Alta Mobilidade , Humanos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Progesterona/genética , Transativadores , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different "poor-outcome" cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by "poor-prognosis" signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Neoplasias da Mama/genética , Movimento Celular/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , PrognósticoRESUMO
Primordial germ cells (PGCs) are the earliest form of mammalian germ lineage. In humans, PGCs are present during a very early and limited window in development, limiting the ability to study fundamental developmental steps in human reproductive biology. However, recent advancements in generating in-vitro models of gametogenesis have allowed the field to generate human primordial germ cell-like cells (hPGCLCs). In this chapter, we will review the generation of hPGCLCs using the incipient mesoderm-like cell (iMeLC) protocol and the subsequent expansion of hPGCLCs in a long-term culture system.