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1.
iScience ; 25(4): 104137, 2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35402882

RESUMO

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.

2.
Mol Cell ; 77(5): 937-950.e6, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31926851

RESUMO

Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits T cell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in T cells remains enigmatic. Here we investigated how PD-1 affects transcriptome changes induced by T cell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of T cell populations.


Assuntos
Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/metabolismo , Transcriptoma , Animais , Apoptose , Sítios de Ligação , Proliferação de Células , Técnicas de Cocultura , Ilhas de CpG , Citocinas/genética , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Codificadores dos Receptores de Linfócitos T , Células HEK293 , Humanos , Células Jurkat , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Receptor de Morte Celular Programada 1/deficiência , Receptor de Morte Celular Programada 1/genética , Regiões Promotoras Genéticas , Transdução de Sinais , Linfócitos T/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional
3.
Nat Genet ; 51(9): 1369-1379, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477927

RESUMO

Promoters and enhancers are key cis-regulatory elements, but how they operate to generate cell type-specific transcriptomes is not fully understood. We developed a simple and robust method, native elongating transcript-cap analysis of gene expression (NET-CAGE), to sensitively detect 5' ends of nascent RNAs in diverse cells and tissues, including unstable transcripts such as enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, characterizing the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without the influence of RNA turnover, and show that enhancer-promoter pairs are generally activated simultaneously on stimulation. By integrating NET-CAGE data with chromatin interaction maps, we show that cis-regulatory elements are topologically connected according to their cell type specificity. We identified new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our NET-CAGE dataset derived from human and mouse cells expands the FANTOM5 atlas of transcribed enhancers, with broad applicability to biomedical research.


Assuntos
Regiões 5' não Traduzidas/genética , Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA/genética , Transcrição Gênica , Perfilação da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Sítio de Iniciação de Transcrição , Transcriptoma
4.
Proc Natl Acad Sci U S A ; 116(31): 15635-15644, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31300537

RESUMO

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-ß (TGF-ß) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.


Assuntos
Sistemas CRISPR-Cas , Neoplasias Colorretais , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas de Neoplasias , Organoides , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Organoides/metabolismo , Organoides/patologia
5.
Oncotarget ; 8(8): 13872-13885, 2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28099924

RESUMO

We established an in vitro cell culture system to determine novel activities of the retinoblastoma (Rb) protein during tumor progression. Rb depletion in p53-null mouse-derived soft tissue sarcoma cells induced a spherogenic phenotype. Cells retrieved from Rb-depleted spheres exhibited slower proliferation and less efficient BrdU incorporation, however, much higher spherogenic activity and aggressive behavior. We discovered six miRNAs, including mmu-miR-18a, -25, -29b, -140, -337, and -1839, whose expression levels correlated tightly with the Rb status and spherogenic activity. Among these, mmu-miR-140 appeared to be positively controlled by Rb and to antagonize the effect of Rb depletion on spherogenesis and tumorigenesis. Furthermore, among genes potentially targeted by mmu-miR-140, Il-6 was upregulated by Rb depletion and downregulated by mmu-mir-140 overexpression. Altogether, we demonstrate the possibility that mmu-mir-140 mediates the Rb function to downregulate Il-6 by targeting its 3'-untranslated region. Finally, we detected the same relationship among RB, hsa-miR-140 and IL-6 in a human breast cancer cell line MCF-7. Because IL-6 is a critical modulator of malignant features of cancer cells and the RB pathway is impaired in the majority of cancers, hsa-miR-140 might be a promising therapeutic tool that disrupts linkage between tumor suppressor inactivation and pro-inflammatory cytokine response.


Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Interleucina-6/metabolismo , MicroRNAs/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Proteína do Retinoblastoma/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , MicroRNAs/genética , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteína do Retinoblastoma/genética , Transcriptoma
6.
Mol Cancer Res ; 8(5): 665-76, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20407016

RESUMO

Vascular endothelial cells produce considerable amounts of matrix metalloproteinases (MMP), including MMP-2, MMP-9, and membrane type 1 (MT1)-MMP. However, little is known about the regulatory mechanisms of these protease activities exhibited during vascular development. A glycosylphosphatidylinositol-anchored glycoprotein, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), has been shown to attenuate MMP-2 maturation by directly interacting with MT1-MMP. Here, we show that an angiogenic factor angiopoietin-1 induces RECK expression in human umbilical vein endothelial cells (HUVEC), and RECK depletion in these cells results in defective vascular tube formation and cellular senescence. We further observed that RECK depletion downregulates beta1-integrin activation, which was associated with decreased autophosphorylation of focal adhesion kinase and increased expression of a cyclin-dependent kinase inhibitor p21(CIP1). In agreement, significant downregulation of beta1-integrin activity was observed in vascular endothelial cells in Reck-/- mouse embryos. In HUVECs, specific inhibition of MMP-2 significantly antagonized the effect of RECK depletion on beta1-integrin signaling, cell proliferation, and tube elongation. Furthermore, we observed that hypervascular tumor-derived cell lines can induce high RECK expression in convoluted vascular endothelial cells, and this in turn supports tumor growth. Targeting RECK specifically in tumor-associated vascular endothelial cells resulted in tumor regression. Therefore, we propose that RECK in tumor vascular endothelial cells can be an interesting target of cancer treatment via abortion of tumor angiogenesis.


Assuntos
Integrina beta1/fisiologia , Glicoproteínas de Membrana/fisiologia , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Senescência Celular/genética , Técnicas de Cocultura , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Proteínas Ligadas por GPI , Humanos , Integrina beta1/metabolismo , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Neoplasias/genética , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/fisiopatologia , Interferência de RNA
9.
Cancer Cell ; 15(4): 255-69, 2009 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-19345325

RESUMO

Oncogene-induced cellular senescence is well documented, but little is known about how infinite cell proliferation induced by loss of tumor suppressor genes is antagonized by cellular functions. Rb heterozygous mice generate Rb-deficient C cell adenomas that progress to adenocarcinomas following biallelic loss of N-ras. Here, we demonstrate that pRb inactivation induces aberrant expression of farnesyl diphosphate synthase, many prenyltransferases, and their upstream regulators sterol regulatory element-binding proteins (SREBPs) in an E2F-dependent manner, leading to enhanced isoprenylation and activation of N-Ras. Consequently, elevated N-Ras activity induces DNA damage response and p130-dependent cellular senescence in Rb-deficient cells. Furthermore, Rb heterozygous mice additionally lacking any of Ink4a, Arf, or Suv39h1 generated C cell adenocarcinomas, suggesting that cellular senescence antagonizes Rb-deficient carcinogenesis.


Assuntos
Senescência Celular , Dano ao DNA , Fatores de Transcrição E2F/metabolismo , Genes ras/fisiologia , Prenilação de Proteína , Proteína do Retinoblastoma/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Animais , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/metabolismo , Neoplasias do Tronco Encefálico/patologia , Membrana Celular , Imunoprecipitação da Cromatina , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Reparo do DNA , Fatores de Transcrição E2F/genética , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Knockout , Prenilação , Transporte Proteico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia
10.
Nat Neurosci ; 10(7): 838-45, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17558399

RESUMO

We report that during cortical development in the mouse embryo, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) critically regulates Notch signaling by antagonizing the ectodomain shedding of Notch ligands, which is mediated by a disintegrin and metalloproteinase domain 10 (ADAM10). In the embryonic brain, RECK is specifically expressed in Nestin-positive neural precursor cells (NPCs). Reck-deficient NPCs undergo precocious differentiation that is associated with downregulated Nestin expression, impaired Notch signaling and defective self-renewal. These phenotypes were substantially rescued either by enhancing Notch signaling or by suppressing endogenous ADAM10 activity. Consequently, we found that RECK regulates the ectodomain shedding of Notch ligands by directly inhibiting the proteolytic activity of ADAM10. This mechanism appeared to be essential for Notch ligands to properly induce Notch signaling in neighboring cells. These findings indicate that RECK is a physiological inhibitor of ADAM10, an upstream regulator of Notch signaling and a critical modulator of brain development.


Assuntos
Proteínas ADAM/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Neurônios/fisiologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Proteína ADAM10 , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Regulação para Baixo/fisiologia , Feminino , Imunofluorescência , Proteínas Ligadas por GPI , Immunoblotting , Imunoprecipitação , Ligantes , Luciferases/biossíntese , Luciferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Plasmídeos/genética , Gravidez , Interferência de RNA , Proteínas Recombinantes/genética , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
J Biol Chem ; 282(16): 12341-52, 2007 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-17329256

RESUMO

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is anchored to the cell surface via glycosylphosphatidylinositol. This molecule antagonizes the function of membrane type 1 matrix metalloproteinase (MT1-MMP) to promote proMMP-2 maturation. Here, we attempt to clarify the mechanism underlying RECK functions. First, we found that RECK forms a complex with MT1-MMP and inhibits its proteolytic activity. Notably, RECK increases the amount of MT1-MMP that associates with detergent-resistant membranes during sucrose gradient ultracentrifugation. Furthermore, perturbation of membrane cholesterol significantly affected the function of RECK in suppressing MT1-MMP function. These findings indicate that RECK possibly regulates MT1-MMP function by modulating its behavior on the cell surface as well as by enzymatic action; this prompted us to find another molecule whose behavior in detergent-resistant membranes is influenced by RECK. Subsequently, we found that RECK interacts with CD13/aminopeptidase N. Further, we found that RECK inhibits the proteolytic activity of CD13 in a cholesterol perturbation-sensitive manner. Finally, we examined whether RECK influences the behavior of MT1-MMP and CD13 during their internalization from the cell surface. In the absence of RECK, MT1-MMP and CD13 were internalized along with the markers of clathrin- or caveolae-dependent endocytosis. However, interestingly, in the presence of RECK these molecules were internalized preferentially with an endocytic marker that is neither clathrinnor caveolae-dependent, indicating that RECK modulates endocytic pathways of MT1-MMP and CD13. This modulation was correlated with the accelerated internalization and decay of MT1-MMP and CD13. This study unveils the novel function and target molecules of RECK.


Assuntos
Antígenos CD13/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Glicoproteínas de Membrana/fisiologia , Aminopeptidases/química , Biotinilação , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , Clatrina/química , Endocitose , Proteínas Ligadas por GPI , Humanos , Cinética , Glicoproteínas de Membrana/química , Microscopia de Fluorescência , Proteínas Recombinantes/química , Sacarose/farmacologia , Transferrina/química
12.
Nat Genet ; 38(1): 118-23, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16369533

RESUMO

Mutations in the gene encoding the retinoblastoma tumor suppressor predispose humans and mice to tumor development. Here we have assessed the effect of Nras loss on tumor development in Rb1 heterozygous mice. Loss of one or two Nras alleles is shown to significantly reduce the severity of pituitary tumors arising in Rb1(+/-) animals by enhancing their differentiation. By contrast, C-cell thyroid adenomas occurring in Rb1(+/-) mice progress to metastatic medullary carcinomas after loss of Nras. In Rb1(+/-)Nras(+/-) animals, distant medullary thyroid carcinoma metastases are associated with loss of the remaining wild-type Nras allele. Loss of Nras in Rb1-deficient C cells results in elevated Ras homolog family A (RhoA) activity, and this is causally linked to the invasiveness and metastatic behavior of these cells. These findings suggest that the loss of the proto-oncogene Nras in certain cellular contexts can promote malignant tumor progression.


Assuntos
Genes ras/genética , Tumores Neuroendócrinos/patologia , Proteína do Retinoblastoma/deficiência , Neoplasias da Glândula Tireoide/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/patologia , Hormônio Adrenocorticotrópico/metabolismo , Amidas/farmacologia , Animais , Carcinoma Medular/genética , Carcinoma Medular/patologia , Inibidores Enzimáticos/farmacologia , Heterozigoto , Camundongos , Camundongos Mutantes , Camundongos Nus , Metástase Neoplásica , Tumores Neuroendócrinos/genética , Proto-Oncogene Mas , Piridinas/farmacologia , Proteína do Retinoblastoma/efeitos dos fármacos , Proteína do Retinoblastoma/genética , Transdução de Sinais , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/secundário , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
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