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1.
Proc Natl Acad Sci U S A ; 120(31): e2308750120, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37487068

RESUMO

Adipose tissue is central to regulation of energy homeostasis. Adaptive thermogenesis, which relies on mitochondrial oxidative phosphorylation (Ox-Phos), dissipates energy to counteract obesity. On the other hand, chronic inflammation in adipose tissue is linked to type 2 diabetes and obesity. Here, we show that nuclear factor I-A (NFIA), a transcriptional regulator of brown and beige adipocytes, improves glucose homeostasis by upregulation of Ox-Phos and reciprocal downregulation of inflammation. Mice with transgenic expression of NFIA in adipocytes exhibited improved glucose tolerance and limited weight gain. NFIA up-regulates Ox-Phos and brown-fat-specific genes by enhancer activation that involves facilitated genomic binding of PPARγ. In contrast, NFIA in adipocytes, but not in macrophages, down-regulates proinflammatory cytokine genes to ameliorate adipose tissue inflammation. NFIA binds to regulatory region of the Ccl2 gene, which encodes proinflammatory cytokine MCP-1 (monocyte chemoattractant protein-1), to down-regulate its transcription. CCL2 expression was negatively correlated with NFIA expression in human adipose tissue. These results reveal the beneficial effect of NFIA on glucose and body weight homeostasis and also highlight previously unappreciated role of NFIA in suppressing adipose tissue inflammation.


Assuntos
Diabetes Mellitus Tipo 2 , Fatores de Transcrição NFI , Humanos , Animais , Camundongos , Adipócitos , Homeostase , Inflamação , Tecido Adiposo Marrom , Citocinas
2.
EMBO J ; 39(7): e103949, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32125007

RESUMO

Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs.


Assuntos
Células Endoteliais/imunologia , Histona Desmetilases/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , MicroRNAs/genética , Animais , Adesão Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Histonas/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Lisina/metabolismo , Masculino , Metilação , Camundongos , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
3.
Cell ; 136(3): 535-50, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19203586

RESUMO

p53 And Akt are critical players regulating tumorigenesis with opposite effects: whereas p53 transactivates target genes to exert its function as a tumor suppressor, Akt phosphorylates its substrates and transduces downstream survival signals. In addition, p53 and Akt negatively regulate each other to balance survival and death signals within a cell. We now identify PHLDA3 as a p53 target gene that encodes a PH domain-only protein. We find that PHLDA3 competes with the PH domain of Akt for binding of membrane lipids, thereby inhibiting Akt translocation to the cellular membrane and activation. Ablation of endogenous PHLDA3 results in enhanced Akt activity and decrease of p53-dependent apoptosis. We also demonstrate the suppression of anchorage-independent cell growth by PHLDA3. Loss of the PHLDA3 genomic locus was frequently observed in primary lung cancers, suggesting a role of PHLDA3 in tumor suppression. Our results reveal a new mode of coordination between the p53 and Akt pathways.


Assuntos
Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Proteína Oncogênica v-akt/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Transdução de Sinais
4.
Blood ; 137(1): 75-88, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730594

RESUMO

The pseudokinase Trib1 functions as a myeloid oncogene that recruits the E3 ubiquitin ligase COP1 to C/EBPα and interacts with MEK1 to enhance extracellular signal-regulated kinase (ERK) phosphorylation. A close genetic effect of Trib1 on Hoxa9 has been observed in myeloid leukemogenesis, where Trib1 overexpression significantly accelerates Hoxa9-induced leukemia onset. However, the mechanism underlying how Trib1 functionally modulates Hoxa9 transcription activity is unclear. Herein, we provide evidence that Trib1 modulates Hoxa9-associated super-enhancers. Chromatin immunoprecipitation sequencing analysis identified increased histone H3K27Ac signals at super-enhancers of the Erg, Spns2, Rgl1, and Pik3cd loci, as well as increased messenger RNA expression of these genes. Modification of super-enhancer activity was mostly achieved via the degradation of C/EBPα p42 by Trib1, with a slight contribution from the MEK/ERK pathway. Silencing of Erg abrogated the growth advantage acquired by Trib1 overexpression, indicating that Erg is a critical downstream target of the Trib1/Hoxa9 axis. Moreover, treatment of acute myeloid leukemia (AML) cells with the BRD4 inhibitor JQ1 showed growth inhibition in a Trib1/Erg-dependent manner both in vitro and in vivo. Upregulation of ERG by TRIB1 was also observed in human AML cell lines, suggesting that Trib1 is a potential therapeutic target of Hoxa9-associated AML. Taken together, our study demonstrates a novel mechanism by which Trib1 modulates chromatin and Hoxa9-driven transcription in myeloid leukemogenesis.


Assuntos
Regulação Leucêmica da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Progressão da Doença , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica
5.
Mol Cell ; 60(4): 584-96, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26590716

RESUMO

Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPß binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.


Assuntos
Adipócitos/citologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Metilação de DNA , Histonas/genética , PPAR gama/metabolismo , Células 3T3 , Adipócitos/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Cromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Estrutura Terciária de Proteína
6.
PLoS Genet ; 16(9): e1009044, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991581

RESUMO

The transcription factor nuclear factor I-A (NFIA) is a regulator of brown adipocyte differentiation. Here we show that the C-terminal 17 amino acid residues of NFIA (which we call pro#3 domain) are required for the transcriptional activity of NFIA. Full-length NFIA-but not deletion mutant lacking pro#3 domain-rescued impaired expression of PPARγ, the master transcriptional regulator of adipogenesis and impaired adipocyte differentiation in NFIA-knockout cells. Mechanistically, the ability of NFIA to penetrate chromatin and bind to the crucial Pparg enhancer is mediated through pro#3 domain. However, the deletion mutant still binds to Myod1 enhancer to repress expression of MyoD, the master transcriptional regulator of myogenesis as well as proximally transcribed non-coding RNA called DRReRNA, via competition with KLF5 in terms of enhancer binding, leading to suppression of myogenic gene program. Therefore, the negative effect of NFIA on the myogenic gene program is, at least partly, independent of the positive effect on PPARγ expression and its downstream adipogenic gene program. These results uncover multiple ways of action of NFIA to ensure optimal regulation of brown and beige adipocyte differentiation.


Assuntos
Adipócitos Bege/citologia , Adipócitos Marrons/citologia , Adipogenia/fisiologia , Desenvolvimento Muscular/fisiologia , Fatores de Transcrição NFI/metabolismo , Adipócitos Bege/fisiologia , Adipócitos Marrons/fisiologia , Adipogenia/genética , Animais , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular/genética , Proteína MyoD/genética , Miogenina/genética , Fatores de Transcrição NFI/genética , PPAR gama/genética , PPAR gama/metabolismo , Prolina , Domínios Proteicos
7.
Cancer Sci ; 113(3): 940-949, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34897916

RESUMO

The activation of RIG-I-like receptor (RLR) signaling in cancer cells is widely recognized as a critical cancer therapy method. The expected mechanism of RLR ligand-mediated cancer therapy involves the promotion of cancer cell death and strong induction of interferon (IFN)-ß that affects the tumor microenvironment. We have recently shown that activation of RLR signaling in triple-negative breast cancer cells (TNBC) attenuates transforming growth factor-ß (TGF-ß) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF-ß signaling by RLR ligands with respect to IFN-ß-mediated tumor suppression are not well characterized. This study showed that transfection of a typical RLR ligand polyI:C in cancer cells produces significant levels of IFN-ß, which inhibits the growth of the surrounding cancer cells. In addition, IFN-ß-induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN-ß expression through the alleviation of IFN regulatory factor 3 binding to the canonical target genes, as suggested by ChIP sequencing analysis. Based on these findings, a new facet of the protumorigenic function of TGF-ß that suppresses IFN-ß expression is suggested when RLR-mediated cancer treatment is used in TNBC.


Assuntos
Interferon beta/metabolismo , Poli I-C/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/metabolismo , Poli I-C/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad3/genética , Proteína Smad3/metabolismo , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/efeitos dos fármacos
8.
Cancer Sci ; 112(7): 2855-2869, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33970549

RESUMO

Ten-eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1-upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast-like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5-hmC) profiling and found that 5-hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4-monomethylated, where the H3K27-acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast-like HCC.


Assuntos
Proteína HMGA2/genética , Neoplasias Hepáticas/genética , Oxigenases de Função Mista/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Citosina/metabolismo , Metilação de DNA , Dioxigenases/metabolismo , Epigênese Genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína HMGA2/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Oxigenases de Função Mista/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regulação para Cima
9.
J Biol Chem ; 294(42): 15466-15479, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31481467

RESUMO

Smad proteins are transcriptional regulators activated by TGF-ß. They are known to bind to two distinct Smad-responsive motifs, namely the Smad-binding element (SBE) (5'-GTCTAGAC-3') and CAGA motifs (5'-AGCCAGACA-3' or 5'-TGTCTGGCT-3'). However, the mechanisms by which these motifs promote Smad activity are not fully elucidated. In this study, we performed DNA CASTing, binding assays, ChIP sequencing, and quantitative RT-PCR to dissect the details of Smad binding and function of the SBE and CAGA motifs. We observed a preference for Smad3 to bind CAGA motifs and Smad4 to bind SBE, and that either one SBE or a triple-CAGA motif forms a cis-acting functional half-unit for Smad-dependent transcription activation; combining two half-units allows efficient activation. Unexpectedly, the extent of Smad binding did not directly correlate with the abilities of Smad-binding sequences to induce gene expression. We found that Smad proteins are more tolerant of single bp mutations in the context of the CAGA motifs, with any mutation in the SBE disrupting function. CAGA and CAGA-like motifs but not SBE are widely distributed among stimulus-dependent Smad2/3-binding sites in normal murine mammary gland epithelial cells, and the number of CAGA and CAGA-like motifs correlates with fold-induction of target gene expression by TGF-ß. These data, demonstrating Smad responsiveness can be tuned by both sequence and number of repeats, provide a compelling explanation for why CAGA motifs are predominantly used for Smad-dependent transcription activation in vivo.


Assuntos
Proteína Smad3/química , Proteína Smad3/metabolismo , Proteína Smad4/química , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Humanos , Ligação Proteica , Elementos de Resposta , Proteína Smad2/química , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad4/genética , Ativação Transcricional
10.
Cancer Sci ; 111(2): 451-466, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31834974

RESUMO

The tumor suppressor gene p53 encodes a transcriptional activator that has two transactivation domains (TAD) located in its amino terminus. These two TAD can transactivate genes independently, and at least one TAD is required for p53 transactivation function. The 1st TAD (a.a. 1-40) is essential for the induction of numerous classical p53 target genes, while the second TAD (a.a. 41-61) suffices for tumor suppression, although its precise molecular function remains unclear. In this study, we comprehensively identified the sites to which p53 lacking the 1st TAD (Δ1stTAD-p53) binds, as well as its potential target genes. We found that the binding sequences for Δ1stTAD-p53 are divergent and include not only the canonical p53 consensus binding sequences but also sequences similar to those recognized by a number of other known transcription factors. We identified and analyzed the functions of three Δ1stTAD-p53 target genes, PTP4A1, PLK2 and RPS27L. All three genes were induced by both full-length p53 and Δ1stTAD-p53, and were dependent on the transactivation activity of the 2nd TAD. We also found that two of these, PTP4A1 and PLK2, are endoplasmic reticulum (ER) stress-inducible genes. We found that upon ER stress, PTP4A1 suppresses apoptosis while PLK2 induces apoptosis. These results reveal a novel Δ1stTAD-p53 downstream pathway that is dependent on the transcription activation activity of the 2nd TAD.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Membrana/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Ribossômicas/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Sítios de Ligação , Estresse do Retículo Endoplasmático , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Domínios Proteicos , Ativação Transcricional , Proteína Supressora de Tumor p53/genética
11.
Cancer Sci ; 109(9): 2907-2918, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29945296

RESUMO

EWS-FLI1 constitutes an oncogenic transcription factor that plays key roles in Ewing sarcoma development and maintenance. We have recently succeeded in generating an ex vivo mouse model for Ewing sarcoma by introducing EWS-FLI1 into embryonic osteochondrogenic progenitors. The model well recapitulates the biological characteristics, small round cell morphology, and gene expression profiles of human Ewing sarcoma. Here, we clarified the global DNA binding properties of EWS-FLI1 in mouse Ewing sarcoma. GGAA microsatellites were found to serve as binding sites of EWS-FLI1 albeit with less frequency than that in human Ewing sarcoma; moreover, genomic distribution was not conserved between human and mouse. Nevertheless, EWS-FLI1 binding sites within GGAA microsatellites were frequently associated with the histone H3K27Ac enhancer mark, suggesting that EWS-FLI1 could affect global gene expression by binding its target sites. In particular, the Fox transcription factor binding motif was frequently observed within EWS-FLI1 peaks and Foxq1 was identified as the cooperative partner that interacts with the EWS portion of EWS-FLI1. Trib1 and Nrg1 were demonstrated as target genes that are co-regulated by EWS-FLI1 and Foxq1, and are important for cell proliferation and survival of Ewing sarcoma. Collectively, our findings present novel aspects of EWS-FLI1 function as well as the importance of GGAA microsatellites.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Repetições de Microssatélites/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/patologia , Animais , Apoptose/genética , Sítios de Ligação/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Condrogênese/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Células-Tronco Embrionárias/citologia , Transição Epitelial-Mesenquimal/genética , Fatores de Transcrição Forkhead/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Neuregulina-1/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/genética
12.
Cancer Sci ; 109(11): 3532-3542, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30207029

RESUMO

The PHLDA family (pleckstrin homology-like domain family) of genes consists of 3 members: PHLDA1, 2, and 3. Both PHLDA3 and PHLDA2 are phosphatidylinositol (PIP) binding proteins and function as repressors of Akt. They have tumor suppressive functions, mainly through Akt inhibition. Several reports suggest that PHLDA1 also has a tumor suppressive function; however, the precise molecular functions of PHLDA1 remain to be elucidated. Through a comprehensive screen for p53 target genes, we identified PHLDA1 as a novel p53 target, and we show that PHLDA1 has the ability to repress Akt in a manner similar to that of PHLDA3 and PHLDA2. PHLDA1 has a so-called split PH domain in which the PH domain is divided into an N-terminal (ß sheets 1-3) and a C-terminal (ß sheets 4-7 and an α-helix) portions. We show that the PH domain of PHLDA1 is responsible for its localization to the plasma membrane and binding to phosphatidylinositol. We also show that the function of the PH domain is essential for Akt repression. In addition, PHLDA1 expression analysis suggests that PHLDA1 has a tumor suppressive function in breast and ovarian cancers.


Assuntos
Neoplasias da Mama/genética , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Transplante de Neoplasias , Fosfatidilinositóis/metabolismo , Ligação Proteica , Fatores de Transcrição/química
13.
EMBO J ; 32(12): 1665-80, 2013 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-23644382

RESUMO

High-throughput techniques have identified numerous antisense (AS) transcripts and long non-coding RNAs (ncRNAs). However, their significance in cancer biology remains largely unknown. Here, we report an androgen-responsive long ncRNA, CTBP1-AS, located in the AS region of C-terminal binding protein 1 (CTBP1), which is a corepressor for androgen receptor. CTBP1-AS is predominantly localized in the nucleus and its expression is generally upregulated in prostate cancer. CTBP1-AS promotes both hormone-dependent and castration-resistant tumour growth. Mechanistically, CTBP1-AS directly represses CTBP1 expression by recruiting the RNA-binding transcriptional repressor PSF together with histone deacetylases. CTBP1-AS also exhibits global androgen-dependent functions by inhibiting tumour-suppressor genes via the PSF-dependent mechanism thus promoting cell cycle progression. Our findings provide new insights into the functions of ncRNAs that directly contribute to prostate cancer progression.


Assuntos
Núcleo Celular/metabolismo , Neoplasias da Próstata/metabolismo , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Idoso , Oxirredutases do Álcool/biossíntese , Oxirredutases do Álcool/genética , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/patologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fator de Processamento Associado a PTB , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo
14.
Proc Natl Acad Sci U S A ; 111(4): 1604-9, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24474786

RESUMO

Neural stem/progenitor cell (NSPC) multipotency is highly regulated so that specific neural networks form during development. NSPCs cannot respond to gliogenic signals without acquiring gliogenic competence and decreasing their neurogenic competence as development proceeds. Coup-tfI and Coup-tfII are triggers of these temporal NSPC competence changes. However, the downstream effectors of Coup-tfs that mediate the neurogenic-to-gliogenic competence transition remain unknown. Here, we identified the microRNA-17/106 (miR-17/106)-p38 axis as a critical regulator of this transition. Overexpression of miR-17 inhibited the acquisition of gliogenic competence and forced stage-progressed NSPCs to regain neurogenic competence without altering the methylation status of a glial gene promoter. We also identified Mapk14 (also known as p38) as a target of miR-17/106 and found that Mapk14 inhibition restored neurogenic competence after the neurogenic phase. These results demonstrate that the miR-17/106-p38 axis is a key regulator of the neurogenic-to-gliogenic NSPC competence transition and that manipulation of this axis permits bidirectional control of NSPC multipotency.


Assuntos
Diferenciação Celular/fisiologia , MicroRNAs/fisiologia , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Sequência de Bases , Proteína Glial Fibrilar Ácida/genética , Camundongos , Camundongos Endogâmicos ICR , MicroRNAs/química , Células-Tronco Neurais/metabolismo , Regiões Promotoras Genéticas , Homologia de Sequência de Aminoácidos
15.
Cancer Sci ; 107(6): 734-45, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26998741

RESUMO

The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and ChIP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, α-l-1 (FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression.


Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Proteína Supressora de Tumor p53/metabolismo , alfa-L-Fucosidase/genética , alfa-L-Fucosidase/metabolismo , Morte Celular , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fucose/metabolismo , Humanos , Proteínas Mutantes/biossíntese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neoplasias/enzimologia , Neoplasias/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , alfa-L-Fucosidase/biossíntese
16.
EMBO J ; 31(23): 4404-14, 2012 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23103767

RESUMO

Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired-end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete 'NFκB factories'. Some factories further specialize in transcribing responsive genes encoding micro-RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Cromossomos/ultraestrutura , Citocinas/biossíntese , Citoplasma/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Células Endoteliais/citologia , Humanos , Hibridização In Situ , Hibridização in Situ Fluorescente , N-Acetilglucosaminiltransferases/metabolismo , NF-kappa B/metabolismo , Conformação Proteica , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Fatores de Tempo , Transcrição Gênica , Fator de Crescimento Transformador beta/metabolismo
17.
EMBO J ; 30(13): 2582-95, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21666600

RESUMO

GATA2 is well recognized as a key transcription factor and regulator of cell-type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell-specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2-associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial-specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial-expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.


Assuntos
Endotélio Vascular/metabolismo , Epigênese Genética/fisiologia , Fator de Transcrição GATA2/metabolismo , Sialoglicoproteínas/genética , Animais , Sequência de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Células K562 , Análise em Microsséries , Modelos Biológicos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , RNA Interferente Pequeno/farmacologia , Sialoglicoproteínas/metabolismo
18.
Genome Res ; 22(2): 208-19, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22156295

RESUMO

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Exoma , Instabilidade Genômica , Haploinsuficiência , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Alelos , Linhagem Celular Tumoral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Perda de Heterozigosidade , Proteína 1 Homóloga a MutL , Mutação , Taxa de Mutação , Reprodutibilidade dos Testes
19.
Proc Natl Acad Sci U S A ; 109(50): 20584-9, 2012 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23112162

RESUMO

Intestinal metaplasia of the stomach, a mucosal change characterized by the conversion of gastric epithelium into an intestinal phenotype, is a precancerous lesion from which intestinal-type gastric adenocarcinoma arises. Chronic infection with Helicobacter pylori is a major cause of gastric intestinal metaplasia, and aberrant induction by H. pylori of the intestine-specific caudal-related homeobox (CDX) transcription factors, CDX1 and CDX2, plays a key role in this metaplastic change. As such, a critical issue arises as to how these factors govern the cell- and tissue-type switching. In this study, we explored genes directly activated by CDX1 in gastric epithelial cells and identified stemness-associated reprogramming factors SALL4 and KLF5. Indeed, SALL4 and KLF5 were aberrantly expressed in the CDX1(+) intestinal metaplasia of the stomach in both humans and mice. In cultured gastric epithelial cells, sustained expression of CDX1 gave rise to the induction of early intestinal-stemness markers, followed by the expression of intestinal-differentiation markers. Furthermore, the induction of these markers was suppressed by inhibiting either SALL4 or KLF5 expression, indicating that CDX1-induced SALL4 and KLF5 converted gastric epithelial cells into tissue stem-like progenitor cells, which then transdifferentiated into intestinal epithelial cells. Our study places the stemness-related reprogramming factors as critical components of CDX1-directed transcriptional circuitries that promote intestinal metaplasia. Requirement of a transit through dedifferentiated stem/progenitor-like cells, which share properties in common with cancer stem cells, may underlie predisposition of intestinal metaplasia to neoplastic transformation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Animais , Sequência de Bases , Linhagem Celular , Transdiferenciação Celular/genética , Transdiferenciação Celular/fisiologia , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Marcadores Genéticos , Helicobacter pylori/patogenicidade , Proteínas de Homeodomínio/genética , Humanos , Mucosa Intestinal/patologia , Fatores de Transcrição Kruppel-Like/genética , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Fenótipo , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética
20.
Cancer Sci ; 105(8): 974-82, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24863656

RESUMO

Transforming growth factor (TGF)-ß exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner. The anti-apoptotic function of TGF-ß is mediated by several downstream regulatory mechanisms, and has been implicated in the tumor-progressive phenotype of breast cancer cells. We conducted RNA sequencing of mouse mammary gland epithelial (NMuMG) cells and identified a long non-coding RNA, termed lncRNA-Smad7, which has anti-apoptotic functions, as a target of TGF-ß. lncRNA-Smad7 was located adjacent to the mouse Smad7 gene, and its expression was induced by TGF-ß in all of the mouse mammary gland epithelial cell lines and breast cancer cell lines that we evaluated. Suppression of lncRNA-Smad7 expression cancelled the anti-apoptotic function of TGF-ß. In contrast, forced expression of lncRNA-Smad7 rescued apoptosis induced by a TGF-ß type I receptor kinase inhibitor in the mouse breast cancer cell line JygMC(A). The anti-apoptotic effect of lncRNA-Smad7 appeared to occur independently of the transcriptional regulation by TGF-ß of anti-apoptotic DEC1 and pro-apoptotic Bim proteins. Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-ß-induced epithelial-mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-ß functions may be restricted to apoptosis. Our findings suggest a complex mechanism for regulating the anti-apoptotic and tumor-progressive aspects of TGF-ß signaling.


Assuntos
Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Linfotoxina-alfa/metabolismo , RNA Longo não Codificante/metabolismo , Proteína Smad7/metabolismo , Animais , Sequência de Bases , Northern Blotting , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad7/genética
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