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1.
Nucleic Acids Res ; 48(14): 7728-7747, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609811

RESUMO

UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.


Assuntos
Processamento Alternativo , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Histonas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Regulação Alostérica , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Código das Histonas , Humanos , Camundongos , Ligação Proteica , Domínio Tudor , Ubiquitina-Proteína Ligases/genética
2.
J Gen Virol ; 101(4): 385-398, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32553055

RESUMO

The influenza A virus (IAV) ribonucleoprotein (vRNP) complex consists of polymerase subunits, nucleoprotein (NP) and viral RNA and is responsible for RNA transcription and replication. Interactions between the vRNP complex and host factors play important roles in virus replication, pathogenicity and species tropism. In this study, Strep-tag affinity purification coupled with mass spectrometry was used to identify host factors that interact with IAV vRNP complex in infected human cells. We purified vRNP complex from HEK 293T cells infected with a recombinant mouse-adapted IAV (A/Chicken/Hubei/489/2004) containing a Strep-tag PB2 subunit and identified Y-box-binding protein 3 (YBX3) as a negative regulator of IAV replication. Overexpression of YBX3 inhibited the virus replication, viral protein expression and vRNA synthesis. Conversely, RNAi knockdown of YBX3 resulted in significantly increased virus growth rate. Furthermore, knockdown of YBX3 augmented the nuclear accumulation of NP and viral primary transcription in infected cells. Our results suggest that YBX3 restricts IAV replication by interacting with vRNP complex and subsequently imparing its function.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Choque Térmico/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Replicação Viral , Células A549 , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Cães , Células HEK293 , Proteínas de Choque Térmico/genética , Interações entre Hospedeiro e Microrganismos , Humanos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/crescimento & desenvolvimento , Células Madin Darby de Rim Canino , Espectrometria de Massas , Camundongos , Ligação Proteica , RNA Interferente Pequeno , RNA Viral/metabolismo , Transcrição Genética , Regulação para Cima , Proteínas do Core Viral/genética , Replicação Viral/fisiologia
3.
Proc Natl Acad Sci U S A ; 117(24): 13670-13679, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32471953

RESUMO

Acute myeloid leukemia (AML) is a deadly hematologic malignancy with poor prognosis, particularly in the elderly. Even among individuals with favorable-risk disease, approximately half will relapse with conventional therapy. In this clinical circumstance, the determinants of relapse are unclear, and there are no therapeutic interventions that can prevent recurrent disease. Mutations in the transcription factor CEBPA are associated with favorable risk in AML. However, mutations in the growth factor receptor CSF3R are commonly co-occurrent in CEBPA mutant AML and are associated with an increased risk of relapse. To develop therapeutic strategies for this disease subset, we performed medium-throughput drug screening on CEBPA/CSF3R mutant leukemia cells and identified sensitivity to inhibitors of lysine-specific demethylase 1 (LSD1). Treatment of CSF3R/CEBPA mutant leukemia cells with LSD1 inhibitors reactivates differentiation-associated enhancers driving immunophenotypic and morphologic differentiation. LSD1 inhibition is ineffective as monotherapy but demonstrates synergy with inhibitors of JAK/STAT signaling, doubling median survival in vivo. These results demonstrate that combined inhibition of JAK/STAT signaling and LSD1 is a promising therapeutic strategy for CEBPA/CSF3R mutant AML.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Inibidores Enzimáticos/administração & dosagem , Histona Desmetilases/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Receptores de Fator Estimulador de Colônias/genética , Fatores de Transcrição STAT/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Feminino , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Fator Estimulador de Colônias/metabolismo , Fatores de Transcrição STAT/antagonistas & inibidores , Fatores de Transcrição STAT/genética , Transdução de Sinais/efeitos dos fármacos
4.
Nat Commun ; 11(1): 1222, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144273

RESUMO

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/genética , Ubiquitinação , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Humanos , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Espermatozoides/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Xenopus laevis
5.
Cancer Invest ; 38(4): 240-249, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32212938

RESUMO

We evaluated the value of UHRF1, a regulator of methylation, as a biomarker for lung cancer. UHRF1 is expressed at higher levels in both lung adenocarcinoma (AD) and squamous cell carcinoma (SQ); however, a meta-analysis showed that UHRF1 expression is correlated with worse survival in patients with AD but not in those with SQ. UHRF1 knockdown suppressed the growth of lung cancer cell lines through G1 cell cycle arrest in some cell lines. These results suggest that UHRF1 may server as a diagnostic marker for AD and SQ and as a prognostic marker for AD in lung cancer.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Biomarcadores Tumorais/análise , Proteínas Estimuladoras de Ligação a CCAAT/análise , Carcinoma de Células Escamosas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Ubiquitina-Proteína Ligases/análise , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Biologia Computacional , Metilação de DNA , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Prognóstico , Interferência de RNA , Análise de Sobrevida , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
Biochem J ; 477(4): 817-831, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32016357

RESUMO

Inorganic phosphate (Pi) homeostasis is regulated by intestinal absorption via type II sodium-dependent co-transporter (Npt2b) and by renal reabsorption via Npt2a and Npt2c. Although we previously reported that vitamin A-deficient (VAD) rats had increased urine Pi excretion through the decreased renal expression of Npt2a and Npt2c, the effect of vitamin A on the intestinal Npt2b expression remains unclear. In this study, we investigated the effects of treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A, on the Pi absorption and the Npt2b expression in the intestine of VAD rats, as well as and the underlying molecular mechanisms. In VAD rats, the intestinal Pi uptake activity and the expression of Npt2b were increased, but were reduced by the administration of ATRA. The transcriptional activity of reporter plasmid containing the promoter region of the rat Npt2b gene was reduced by ATRA in NIH3T3 cells overexpressing retinoic acid receptor (RAR) and retinoid X receptor (RXR). On the other hand, CCAAT/enhancer-binding proteins (C/EBP) induced transcriptional activity of the Npt2b gene. Knockdown of the C/EBP gene and a mutation analysis of the C/EBP responsible element in the Npt2b gene promoter indicated that C/EBP plays a pivotal role in the regulation of Npt2b gene transcriptional activity by ATRA. EMSA revealed that the RAR/RXR complex inhibits binding of C/EBP to Npt2b gene promoter. Together, these results suggest that ATRA may reduce the intestinal Pi uptake by preventing C/EBP activation of the intestinal Npt2b gene.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Intestino Delgado/metabolismo , Rim/metabolismo , Regiões Promotoras Genéticas , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Transcrição Genética/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Antineoplásicos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Hipofosfatemia Familiar/metabolismo , Hipofosfatemia Familiar/patologia , Hipofosfatemia Familiar/prevenção & controle , Intestino Delgado/efeitos dos fármacos , Rim/efeitos dos fármacos , Masculino , Camundongos , Células NIH 3T3 , Ratos , Ratos Wistar , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismo
7.
Am J Respir Cell Mol Biol ; 63(1): 67-78, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32101459

RESUMO

Epithelial dysfunction in the small airways may cause the development of emphysema in chronic obstructive pulmonary disease. C/EBPα (CCAAT/enhancer binding protein-α), a transcription factor, is required for lung maturation during development, and is also important for lung homeostasis after birth, including the maintenance of serine protease/antiprotease balance in the bronchiolar epithelium. This study aimed to show the roles of C/EBPα in the distal airway during chronic cigarette smoke exposure in mice and in the small airways in smokers. In a model of chronic smoke exposure using epithelial cell-specific C/EBPα-knockout mice, significant pathological phenotypes, such as higher protease activity, impaired ciliated cell regeneration, epithelial cell barrier dysfunction via reduced zonula occludens-1 (Zo-1), and decreased alveolar attachments, were found in C/EBPα-knockout mice compared with control mice. We found that Spink5 (serine protease inhibitor kazal-type 5) gene (encoding lymphoepithelial Kazal-type-related inhibitor [LEKTI], an anti-serine protease) expression in the small airways is a key regulator of protease activity in this model. Finally, we showed that daily antiprotease treatment counteracted the phenotypes of C/EBPα-knockout mice. In human studies, CEBPA (CCAAT/enhancer binding protein-α) gene expression in the lung was downregulated in patients with emphysema, and six smokers with centrilobular emphysema (CLE) showed a significant reduction in LEKTI in the small airways compared with 22 smokers without CLE. LEKTI downregulation in the small airways was associated with disease development during murine small airway injury and CLE in humans, suggesting that LEKTI might be a key factor linking small airway injury to the development of emphysema.


Assuntos
Pulmão/metabolismo , Pulmão/patologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Serina Proteases/metabolismo , Animais , Bronquíolos/metabolismo , Bronquíolos/patologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Inibidor de Serinopeptidase do Tipo Kazal 5/metabolismo , Fumar/metabolismo
8.
Nat Commun ; 11(1): 785, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034145

RESUMO

Extracellular signals such as TGF-ß can induce epithelial-to-mesenchymal transition (EMT) in cancers of epithelial origin, promoting molecular and phenotypical changes resulting in pro-metastatic characteristics. We identified C/EBPα as one of the most TGF-ß-mediated downregulated transcription factors in human mammary epithelial cells. C/EBPα expression prevents TGF-ß-driven EMT by inhibiting expression of known EMT factors. Depletion of C/EBPα is sufficient to induce mesenchymal-like morphology and molecular features, while cells that had undergone TGF-ß-induced EMT reverted to an epithelial-like state upon C/EBPα re-expression. In vivo, mice injected with C/EBPα-expressing breast tumor organoids display a dramatic reduction of metastatic lesions. Collectively, our results show that C/EBPα is required for maintaining epithelial homeostasis by repressing the expression of key mesenchymal markers, thereby preventing EMT-mediated tumorigenesis. These data suggest that C/EBPα is a master epithelial "gatekeeper" whose expression is required to prevent unwarranted mesenchymal transition, supporting an important role for EMT in mediating breast cancer metastasis.


Assuntos
Neoplasias da Mama/patologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Glândulas Mamárias Humanas/patologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Glândulas Mamárias Humanas/metabolismo , Camundongos SCID , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
PLoS One ; 15(2): e0229144, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084194

RESUMO

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein's NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation.


Assuntos
5-Metilcitosina/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ilhas de CpG/genética , Citosina/análogos & derivados , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT/química , Citosina/metabolismo , Epigênese Genética , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato , Ubiquitina-Proteína Ligases/química
10.
BMB Rep ; 53(2): 112-117, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31964471

RESUMO

A recent study suggested that methylation of ubiquitin-like with PHD and RING finger domain 1 (UHRF1) is regulated by SET7 and lysine-specific histone demethylase 1A (LSD1) and is essential for homologous recombination (HR). The study demonstrated that SET7-mediated methylation of UHRF1 promotes polyubiquitination of proliferating cell nuclear antigen (PCNA), inducing HR. However, studies on mediators that interact with and recruit UHRF1 to damaged lesions are needed to elucidate the mechanism of UHRF1 methylationinduced HR. Here, we identified that poly [ADP-ribose] polymerase 1 (PARP1) interacts with damage-induced methylated UHRF1 specifically and mediates UHRF1 to induce HR progression. Furthermore, cooperation of UHRF1-PARP1 is essential for cell viability, suggesting the importance of the interaction of UHRF1-PARP1 for damage tolerance in response to damage. Our data revealed that PARP1 mediates the HR mechanism, which is regulated by UHRF1 methylation. The data also indicated the significant role of PARP1 as a mediator of UHRF1 methylation-correlated HR pathway. [BMB Reports 2020; 53(2): 112-117].


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Dano ao DNA/genética , Recombinação Homóloga/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , Sobrevivência Celular/genética , Dano ao DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Células HCT116 , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Ligação Proteica , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
11.
Proc Natl Acad Sci U S A ; 117(2): 1223-1232, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31892538

RESUMO

The LEAFY COTYLEDON1 (LEC1) transcription factor is a central regulator of seed development, because it controls diverse biological programs during seed development, such as embryo morphogenesis, photosynthesis, and seed maturation. To understand how LEC1 regulates different gene sets during development, we explored the possibility that LEC1 acts in combination with other transcription factors. We identified and compared genes that are directly transcriptionally regulated by ABA-RESPONSIVE ELEMENT BINDING PROTEIN3 (AREB3), BASIC LEUCINE ZIPPER67 (bZIP67), and ABA INSENSITIVE3 (ABI3) with those regulated by LEC1. We showed that LEC1 operates with specific sets of transcription factors to regulate different gene sets and, therefore, distinct developmental processes. Thus, LEC1 controls diverse processes through its combinatorial interactions with other transcription factors. DNA binding sites for the transcription factors are closely clustered in genomic regions upstream of target genes, defining cis-regulatory modules that are enriched for DNA sequence motifs that resemble sequences known to be bound by these transcription factors. Moreover, cis-regulatory modules for genes regulated by distinct transcription factor combinations are enriched for different sets of DNA motifs. Expression assays with embryo cells indicate that the enriched DNA motifs are functional cis elements that regulate transcription. Together, the results suggest that combinatorial interactions between LEC1 and other transcription factors are mediated by cis-regulatory modules containing clustered cis elements and by physical interactions that are documented to occur between the transcription factors.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Sementes/crescimento & desenvolvimento , Soja/crescimento & desenvolvimento , Soja/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Ligação a DNA , Regulação da Expressão Gênica de Plantas , Motivos de Nucleotídeos , Desenvolvimento Vegetal/genética , Desenvolvimento Vegetal/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro , Soja/embriologia , Soja/genética , Fatores de Transcrição/genética
12.
PLoS One ; 15(1): e0227279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999703

RESUMO

Fibrous dysplasia (FD) of bone is a complex disease of the skeleton caused by dominant activating mutations of the GNAS locus encoding for the α subunit of the G protein-coupled receptor complex (Gsα). The mutation involves a substitution of arginine at position 201 by histidine or cysteine (GsαR201H or R201C), which leads to overproduction of cAMP. Several signaling pathways are implicated downstream of excess cAMP in the manifestation of disease. However, the pathogenesis of FD remains largely unknown. The overall FD phenotype can be attributed to alterations of skeletal stem/progenitor cells which normally develop into osteogenic or adipogenic cells (in cis), and are also known to provide support to angiogenesis, hematopoiesis, and osteoclastogenesis (in trans). In order to dissect the molecular pathways rooted in skeletal stem/progenitor cells by FD mutations, we engineered human skeletal stem/progenitor cells with the GsαR201C mutation and performed transcriptomic analysis. Our data suggest that this FD mutation profoundly alters the properties of skeletal stem/progenitor cells by pushing them towards formation of disorganized bone with a concomitant alteration of adipogenic differentiation. In addition, the mutation creates an altered in trans environment that induces neovascularization, cytokine/chemokine changes and osteoclastogenesis. In silico comparison of our data with the signature of FD craniofacial samples highlighted common traits, such as the upregulation of ADAM (A Disintegrin and Metalloprotease) proteins and other matrix-related factors, and of PDE7B (Phosphodiesterase 7B), which can be considered as a buffering process, activated to compensate for excess cAMP. We also observed high levels of CEBPs (CCAAT-Enhancer Binding Proteins) in both data sets, factors related to browning of white fat. This is the first analysis of the reaction of human skeletal stem/progenitor cells to the introduction of the FD mutation and we believe it provides a useful background for further studies on the molecular basis of the disease and for the identification of novel potential therapeutic targets.


Assuntos
Células da Medula Óssea/fisiologia , Diferenciação Celular/genética , Cromograninas/genética , Displasia Fibrosa Óssea/patologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Células-Tronco/fisiologia , Proteínas ADAM/metabolismo , Adipogenia/genética , Tecido Adiposo Branco/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Cromograninas/metabolismo , Simulação por Computador , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/metabolismo , Conjuntos de Dados como Assunto , Displasia Fibrosa Óssea/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Mutação com Ganho de Função , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Osteogênese/genética , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Estromais/fisiologia , Regulação para Cima
13.
Am J Physiol Regul Integr Comp Physiol ; 318(2): R227-R233, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31774307

RESUMO

Doxorubicin (DOX) is a highly effective antineoplastic agent used in cancer treatment. Unfortunately, clinical use of DOX is limited due to the development of dose-dependent toxicity to cardiac and respiratory (i.e., diaphragm) muscles. After administration, DOX preferentially localizes to the inner mitochondrial membrane, where it promotes cellular toxicity via enhanced mitochondrial reactive oxygen species (ROS) production. Although recent evidence suggests that amelioration of mitochondrial ROS emission preserves cardiorespiratory muscle function following DOX treatment, the mechanisms responsible for this protection remain unknown. Therefore, we tested the hypothesis that DOX-induced mitochondrial ROS production is required to stimulate pathological signaling by the autophagy/lysosomal system (ALS), the ubiquitin-proteasome pathway (UPP), and the unfolded protein response (UPR). Cause and effect were determined by administration of the mitochondria-targeted peptide SS-31 to DOX-treated animals. Interestingly, while SS-31 abrogated aberrant ROS emission in cardiorespiratory muscles of DOX-treated animals, our results revealed muscle-specific regulation of effector pathways. In the heart, SS-31 prevented DOX-induced proteolytic signaling through the ALS and UPP. In contrast, ALS signaling was inhibited by SS-31 in the diaphragm, but the UPP was not affected. UPR signaling was activated in both muscles at eukaryotic translation initiation factor 2α (eIF2α) S51 in the heart and diaphragm of DOX-treated animals and was attenuated with SS-31 treatment in both tissues. However, downstream signaling of eIF2α (activating transcription factor 4 and CCAAT/enhancer-binding protein homologous protein) was diminished in the heart but upregulated in the diaphragm with DOX. Collectively, these results show that DOX-induced ROS production plays distinct roles in the regulation of cardiac and diaphragm muscle proteolysis.


Assuntos
Antibióticos Antineoplásicos/toxicidade , Diafragma/efeitos dos fármacos , Doxorrubicina/toxicidade , Cardiopatias/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cardiotoxicidade , Diafragma/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Cardiopatias/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas/efeitos dos fármacos
14.
Ann Rheum Dis ; 79(2): 242-253, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31780527

RESUMO

OBJECTIVES: Haematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to both myeloid and lymphoid cell lineages. We reasoned that the aberrancies of immune cells in systemic lupus erythematosus (SLE) could be traced back to HSPCs. METHODS: A global gene expression map of bone marrow (BM)-derived HSPCs was completed by RNA sequencing followed by pathway and enrichment analysis. The cell cycle status and apoptosis status of HSPCs were assessed by flow cytometry, while DNA damage was assessed via immunofluorescence. RESULTS: Transcriptomic analysis of Lin-Sca-1+c-Kit+ haematopoietic progenitors from diseased lupus mice demonstrated a strong myeloid signature with expanded frequencies of common myeloid progenitors (CMPs)-but not of common lymphoid progenitors-reminiscent of a 'trained immunity' signature. CMP profiling revealed an intense transcriptome reprogramming with suppression of granulocytic regulators indicative of a differentiation arrest with downregulation trend of major regulators such as Cebpe, Cebpd and Csf3r, and disturbed myelopoiesis. Despite the differentiation arrest, frequencies of BM neutrophils were markedly increased in diseased mice, suggesting an alternative granulopoiesis pathway. In patients with SLE with severe disease, haematopoietic progenitor cells (CD34+) demonstrated enhanced proliferation, cell differentiation and transcriptional activation of cytokines and chemokines that drive differentiation towards myelopoiesis, thus mirroring the murine data. CONCLUSIONS: Aberrancies of immune cells in SLE can be traced back to the BM HSPCs. Priming of HSPCs and aberrant regulation of myelopoiesis may contribute to inflammation and risk of flare. TRIAL REGISTRATION NUMBER: 4948/19-07-2016.


Assuntos
Reprogramação Celular/imunologia , Células-Tronco Hematopoéticas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Células Mieloides/imunologia , Transcriptoma/imunologia , Animais , Apoptose/imunologia , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular/imunologia , Mapeamento Cromossômico , Dano ao DNA , Citometria de Fluxo , Imunofluorescência , Fator Estimulador de Colônias de Granulócitos/metabolismo , Granulócitos/imunologia , Linfócitos/imunologia , Camundongos
15.
Insect Mol Biol ; 29(2): 256-270, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31840914

RESUMO

Cyclic adenosine monophosphate (cAMP) response element binding protein (CREB)-binding protein (CBP or CREBBP) plays important roles in regulating gene transcription and animal development. However, the process by which CBP is up-regulated to impact insect development is unknown. In this study, the regulatory mechanism of Bombyx mori CBP (BmCBP) expression induced by 20-hydroxyecdysone (20E) was investigated. In the Bo. mori cell line, DZNU-Bm-12, 20E enhanced BmCBP transcription and histone H3K27 acetylation. BmCBP RNA interference (RNAi) resulted in decreased histone H3K27 acetylation. Additionally, the luciferase activity analysis revealed that the transcription factor, Bo. mori CCAAT/enhancer-binding protein gamma (BmC/EBPg), activated BmCBP transcription, which was suppressed by BmC/EBPg RNAi and promoted by BmC/EBPg overexpression. Electrophoretic mobility shift assay and chromatin immunoprecipitation results demonstrated that BmC/EBPg could bind to the C/EBP cis-regulatory elements in two positions of the BmCBP promoter. Moreover, BmC/EBPg transcription was enhanced by the 20E receptor (BmEcR), which bound to the BmC/EBPg promoter. BmEcR RNAi significantly inhibited the transcriptional levels of BmC/EBPg and BmCBP in the presence of 20E. Furthermore, the BmEcR-BmC/EBPg pathway regulated the acetylation levels of histone H3K27. Altogether, these results indicate that BmEcR enhances the expression of BmC/EBPg, which binds to the BmCBP promoter, activates BmCBP expression and leads to histone H3K27 acetylation.


Assuntos
Bombyx/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Acetilação , Animais , Bombyx/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Histonas/metabolismo , Proteínas de Insetos/metabolismo
16.
Nutrients ; 11(11)2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31726767

RESUMO

In this study, we investigated the effects of black ginseng (BG) and ginsenoside Rb1, which induced browning effects in 3T3-L1 and primary white adipocytes (PWATs) isolated from C57BL/6 mice. BG and Rb1 suppressed the expressions of CCAAT/enhancer-binding protein alpha (C/EBPα) and sterol regulatory element-binding transcription factor-1c (SREBP-1c), whereas the expression level of peroxisome proliferator-activated receptor gamma (PPARγ) was increased. Furthermore, BG and Rb1 enhanced the protein expressions of the brown-adipocyte-specific markers PR domain containing 16 (PRDM16), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), and uncoupling protein 1 (UCP1). These results were further supported by immunofluorescence images of mitochondrial biogenesis. In addition, BG and Rb1 induced expressions of brown-adipocyte-specific marker proteins by AMP-activated protein kinase (AMPK) activation. BG and Rb1 exert antiobesity effects by inducing browning in 3T3-L1 cells and PWATs through AMPK-mediated pathway activation. We suggest that BG and Rb1 act as potential functional antiobesity food agents.


Assuntos
Adipócitos Marrons/efeitos dos fármacos , Adipócitos Brancos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Ginsenosídeos/farmacologia , Panax , Extratos Vegetais/farmacologia , Proteína Desacopladora 1/metabolismo , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Biogênese de Organelas , PPAR gama/metabolismo , Panax/química , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Extratos Vegetais/isolamento & purificação , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima
17.
Nat Commun ; 10(1): 4705, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31624244

RESUMO

DNA methylation, repressive histone marks, and PIWI-interacting RNA (piRNA) are essential for the control of retrotransposon silencing in the mammalian germline. However, it remains unknown how these repressive epigenetic pathways crosstalk to ensure retrotransposon silencing in the male germline. Here, we show that UHRF1 is responsible for retrotransposon silencing and cooperates with repressive epigenetic pathways in male germ cells. Conditional loss of UHRF1 in postnatal germ cells causes DNA hypomethylation, upregulation of retrotransposons, the activation of a DNA damage response, and switches in the global chromatin status, leading to complete male sterility. Furthermore, we show that UHRF1 interacts with PRMT5, an arginine methyltransferase, to regulate the repressive histone arginine modifications (H4R3me2s and H3R2me2s), and cooperates with the PIWI pathway during spermatogenesis. Collectively, UHRF1 regulates retrotransposon silencing in male germ cells and provides a molecular link between DNA methylation, histone modification, and the PIWI pathway in the germline.


Assuntos
Proteínas Argonauta/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Metilação de DNA , Proteína-Arginina N-Metiltransferases/genética , Retroelementos/genética , Espermatozoides/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas Argonauta/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Feminino , Inativação Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , Espermatogênese/genética , Ubiquitina-Proteína Ligases/metabolismo
18.
Elife ; 82019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31637998

RESUMO

Monocyte counts are increased during human tuberculosis (TB) but it has not been determined whether Mycobacterium tuberculosis (Mtb) directly regulates myeloid commitment. We demonstrated that exposure to Mtb directs primary human CD34+ cells to differentiate into monocytes/macrophages. In vitro myeloid conversion did not require type I or type II IFN signaling. In contrast, Mtb enhanced IL-6 responses by CD34+ cell cultures and IL-6R neutralization inhibited myeloid differentiation and decreased mycobacterial growth in vitro. Integrated systems biology analysis of transcriptomic, proteomic and genomic data of large data sets of healthy controls and TB patients established the existence of a myeloid IL-6/IL6R/CEBP gene module associated with disease severity. Furthermore, genetic and functional analysis revealed the IL6/IL6R/CEBP gene module has undergone recent evolutionary selection, including Neanderthal introgression and human pathogen adaptation, connected to systemic monocyte counts. These results suggest Mtb co-opts an evolutionary recent IFN-IL6-CEBP feed-forward loop, increasing myeloid differentiation linked to severe TB in humans.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Interferons/metabolismo , Interleucina-6/metabolismo , Monócitos/metabolismo , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Antígenos CD34 , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Hidrolases , Interferons/genética , Interleucina-6/genética , Macrófagos/microbiologia , Monócitos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Células Mieloides/fisiologia , Proteômica , Receptores de Interleucina-6 , Índice de Gravidade de Doença , Transcriptoma , Tuberculose/metabolismo
19.
Int J Mol Sci ; 20(18)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487963

RESUMO

The TORC2 gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2'-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of TORC2 in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine TORC2, we cloned a 1990 bp of the 5' untranslated region (5'UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5'UTR flanking region. The core promoter region of the TORC2 gene was identified at location -314 to -69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors (CCAAT/enhancer-binding protein C/BEP, X-box binding protein 1 XBP1, Insulinoma-associated 1 INSM1, and Zinc finger protein 263 ZNF263) were identified as the transcriptional regulators of TORC2 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that C/EBP, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region. We can conclude that TORC2 gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.


Assuntos
Adipócitos/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Adipócitos/citologia , Adipogenia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Bovinos , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Transcriptoma
20.
Oncol Rep ; 42(5): 2117-2129, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31545467

RESUMO

It has been reported that kruppel­like factor 17 (KLF17) acts as a tumour suppressor in several tissues and cancer cells, however, the molecular roles, the underlying mechanisms and clinical significance of KLF17 in colorectal cancer (CRC) have not been completely elucidated. In the present study, KLF17 protein expression was detected in 140 primary CRCs and paired adjacent non­tumour tissues using immunohistochemistry with tissue microarrays. The KLF17 mRNA expression was determined in 4 CRC cell lines and 20 pairs of the aforementioned tissues using reverse transcription quantitative polymerase chain reaction. The correlation between KLF17 expression and clinicopathologic characteristics was determined. Next, the functions of KLF17 in CRC were examined by cell proliferation, colony formation, adhesion, invasion and mouse xenograft assays. Methylation­specific PCR and bisulfite sequencing PCR were also carried out to investigate the promoter methylation status of KLF17 in CRC cells and tissues and explore the effects of lentiviral­mediated RNAi of UHRF1 on the methylation and expression of KLF17. The results revealed that KLF17 expression was abnormally decreased in CRC and associated with lymph node metastasis and unfavorable overall survival. Moreover, ectopic KLF17 expression suppressed CRC cell growth and invasion in vitro and in vivo. In addition, the downregulation of KLF17 was associated with the hypermethylation of the CpG nucleotides on the KLF17 promoter. The knockdown of the epigenetic regulator UHRF1 reduced the methylation level of the KLF17 promoter and inhibited CRC cell adhesion, invasion and epithelial­mesenchymal transition by upregulating KLF17. The present findings indicated that KLF17 may act as a tumour suppressor gene in CRC and a potential independent prognostic biomarker in CRC patients. UHRF1 can suppress KLF17 expression through the hypermethylation of its promoter in CRC. These results offer insights into the KLF17 expression regulation in CRC and suggest an inhibitory effect of KLF17 on tumourigenesis.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Neoplasias Colorretais/patologia , Regulação para Baixo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Prognóstico , Regiões Promotoras Genéticas , Análise de Sobrevida , Análise Serial de Tecidos
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