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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204880

RESUMO

Dysregulated mammalian target of rapamycin (mTOR) activity is associated with various neurodevelopmental disorders ranging from idiopathic autism spectrum disorders (ASD) to syndromes caused by single gene defects. This suggests that maintaining mTOR activity levels in a physiological range is essential for brain development and functioning. Upon activation, mTOR regulates a variety of cellular processes such as cell growth, autophagy, and metabolism. On a molecular level, however, the consequences of mTOR activation in the brain are not well understood. Low levels of cholesterol are associated with a wide variety of neurodevelopmental disorders. We here describe numerous genes of the sterol/cholesterol biosynthesis pathway to be transcriptionally regulated by mTOR complex 1 (mTORC1) signaling in vitro in primary neurons and in vivo in the developing cerebral cortex of the mouse. We find that these genes are shared targets of the transcription factors SREBP, SP1, and NF-Y. Prenatal as well as postnatal mTORC1 inhibition downregulated expression of these genes which directly translated into reduced cholesterol levels, pointing towards a substantial metabolic function of the mTORC1 signaling cascade. Altogether, our results indicate that mTORC1 is an essential transcriptional regulator of the expression of sterol/cholesterol biosynthesis genes in the developing brain. Altered expression of these genes may be an important factor contributing to the pathogenesis of neurodevelopmental disorders associated with dysregulated mTOR signaling.


Assuntos
Colesterol/genética , Neurônios/metabolismo , Proteínas Quinases/genética , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Serina-Treonina Quinases TOR/genética , Animais , Autofagia/genética , Fator de Ligação a CCAAT/genética , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Colesterol/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Neurogênese/genética , Cultura Primária de Células , Transdução de Sinais/genética , Transcrição Genética/genética
2.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200173

RESUMO

Neuropeptides serve as neurohormones and local paracrine regulators that control neural networks regulating behavior, endocrine system and sensorimotor functions. Their expression is characterized by exceptionally restricted profiles. Circuit-specific and adaptive expression of neuropeptide genes may be defined by transcriptional and epigenetic mechanisms controlled by cell type and subtype sequence-specific transcription factors, insulators and silencers. The opioid peptide dynorphins play a critical role in neurological and psychiatric disorders, pain processing and stress, while their mutations cause profound neurodegeneration in the human brain. In this review, we focus on the prodynorphin gene as a model for the in-depth epigenetic and transcriptional analysis of expression of the neuropeptide genes. Prodynorphin studies may provide a framework for analysis of mechanisms relevant for regulation of neuropeptide genes in normal and pathological human brain.


Assuntos
Encéfalo/metabolismo , Encefalinas/genética , Epigênese Genética/genética , Precursores de Proteínas/genética , Transcrição Genética/genética , Analgésicos Opioides/metabolismo , Animais , Epigenômica/métodos , Regulação da Expressão Gênica/genética , Humanos , Neuropeptídeos/genética
3.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065495

RESUMO

(1) Background: Blue light is important for the formation of maize stomata, but the signal network remains unclear. (2) Methods: We replaced red light with blue light in an experiment and provided a complementary regulatory network for the stomatal development of maize by using transcriptome and metabolomics analysis. (3) Results: Exposure to blue light led to 1296 differentially expressed genes and 419 differential metabolites. Transcriptome comparisons and correlation signaling network analysis detected 55 genes, and identified 6 genes that work in the regulation of the HY5 module and MAPK cascade, that interact with PTI1, COI1, MPK2, and MPK3, in response to the substitution of blue light in environmental adaptation and signaling transduction pathways. Metabolomics analysis showed that two genes involved in carotenoid biosynthesis and starch and sucrose metabolism participate in stomatal development. Their signaling sites located on the PHI1 and MPK2 sites of the MAPK cascade respond to blue light signaling. (4) Conclusions: Blue light remarkably changed the transcriptional signal transduction and metabolism of metabolites, and eight obtained genes worked in the HY5 module and MAPK cascade.


Assuntos
Redes Reguladoras de Genes/genética , Transcriptoma/genética , Zea mays/genética , Regulação da Expressão Gênica de Plantas/genética , Luz , Metabolômica/métodos , Transdução de Sinais/genética , Transcrição Genética/genética
4.
Nat Commun ; 12(1): 3760, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145295

RESUMO

Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells. While the notion that spontaneous DNA damage at telomeres is required to initiate ALT, the molecular triggers of this physiological telomere instability are largely unknown. We previously proposed that the telomeric long noncoding RNA TERRA may represent one such trigger; however, given the lack of tools to suppress TERRA transcription in cells, our hypothesis remained speculative. We have developed Transcription Activator-Like Effectors able to rapidly inhibit TERRA transcription from multiple chromosome ends in an ALT cell line. TERRA transcription inhibition decreases marks of DNA replication stress and DNA damage at telomeres and impairs ALT activity and telomere length maintenance. We conclude that TERRA transcription actively destabilizes telomere integrity in ALT cells, thereby triggering BIR and promoting telomere elongation. Our data point to TERRA transcription manipulation as a potentially useful target for therapy.


Assuntos
RNA Longo não Codificante/genética , Homeostase do Telômero/genética , Telômero/genética , Transcrição Genética/genética , Linhagem Celular Tumoral , Quebra Cromossômica , Dano ao DNA/genética , Replicação do DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/genética
5.
PLoS Pathog ; 17(6): e1009596, 2021 06.
Artigo em Inglês | MEDLINE | ID: covidwho-1249581

RESUMO

The rapid evolution of RNA viruses has been long considered to result from a combination of high copying error frequencies during RNA replication, short generation times and the consequent extensive fixation of neutral or adaptive changes over short periods. While both the identities and sites of mutations are typically modelled as being random, recent investigations of sequence diversity of SARS coronavirus 2 (SARS-CoV-2) have identified a preponderance of C->U transitions, proposed to be driven by an APOBEC-like RNA editing process. The current study investigated whether this phenomenon could be observed in datasets of other RNA viruses. Using a 5% divergence filter to infer directionality, 18 from 36 datasets of aligned coding region sequences from a diverse range of mammalian RNA viruses (including Picornaviridae, Flaviviridae, Matonaviridae, Caliciviridae and Coronaviridae) showed a >2-fold base composition normalised excess of C->U transitions compared to U->C (range 2.1x-7.5x), with a consistently observed favoured 5' U upstream context. The presence of genome scale RNA secondary structure (GORS) was the only other genomic or structural parameter significantly associated with C->U/U->C transition asymmetries by multivariable analysis (ANOVA), potentially reflecting RNA structure dependence of sites targeted for C->U mutations. Using the association index metric, C->U changes were specifically over-represented at phylogenetically uninformative sites, potentially paralleling extensive homoplasy of this transition reported in SARS-CoV-2. Although mechanisms remain to be functionally characterised, excess C->U substitutions accounted for 11-14% of standing sequence variability of structured viruses and may therefore represent a potent driver of their sequence diversification and longer-term evolution.


Assuntos
Mamíferos/virologia , Mutação , Vírus de RNA/genética , SARS-CoV-2/genética , Desaminases APOBEC/metabolismo , Animais , Sequência de Bases , COVID-19/virologia , Citidina/genética , Dano ao DNA/fisiologia , Evolução Molecular , Regulação Viral da Expressão Gênica , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Humanos , Conformação de Ácido Nucleico , Filogenia , Edição de RNA/fisiologia , Vírus de RNA/classificação , RNA Viral/química , RNA Viral/genética , SARS-CoV-2/química , SARS-CoV-2/classificação , Análise de Sequência de RNA , Transcrição Genética/genética , Uridina/genética
6.
Nat Commun ; 12(1): 3628, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131135

RESUMO

We present TensorSignatures, an algorithm to learn mutational signatures jointly across different variant categories and their genomic localisation and properties. The analysis of 2778 primary and 3824 metastatic cancer genomes of the PCAWG consortium and the HMF cohort shows that all signatures operate dynamically in response to genomic states. The analysis pins differential spectra of UV mutagenesis found in active and inactive chromatin to global genome nucleotide excision repair. TensorSignatures accurately characterises transcription-associated mutagenesis in 7 different cancer types. The algorithm also extracts distinct signatures of replication- and double strand break repair-driven mutagenesis by APOBEC3A and 3B with differential numbers and length of mutation clusters. Finally, TensorSignatures reproduces a signature of somatic hypermutation generating highly clustered variants at transcription start sites of active genes in lymphoid leukaemia, distinct from a general and less clustered signature of Polη-driven translesion synthesis found in a broad range of cancer types. In summary, TensorSignatures elucidates complex mutational footprints by characterising their underlying processes with respect to a multitude of genomic variables.


Assuntos
Genômica , Mutação , Neoplasias/genética , Algoritmos , Sequência de Bases , Citidina Desaminase/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Análise Mutacional de DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Epigenômica , Humanos , Antígenos de Histocompatibilidade Menor/genética , Modelos Genéticos , Mutagênese , Proteínas/genética , Transcrição Genética/genética
7.
Cell ; 184(13): 3474-3485.e11, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: covidwho-1240208

RESUMO

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/genética , Exorribonucleases/genética , Metiltransferases/genética , SARS-CoV-2/genética , Sequência de Aminoácidos , COVID-19/virologia , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Alinhamento de Sequência , Transcrição Genética/genética , Replicação Viral/genética
8.
Nat Commun ; 12(1): 3806, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155213

RESUMO

Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.


Assuntos
Elementos Facilitadores Genéticos/genética , Mutação com Ganho de Função/genética , Regiões Promotoras Genéticas/genética , Regulação da Expressão Gênica , Humanos , Família Multigênica , Mutação Puntual , Transcrição Genética/genética , alfa-Globinas/genética , Talassemia alfa/genética
9.
Nat Commun ; 12(1): 3859, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162879

RESUMO

Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range. However, available NIR optogenetic systems consist of several protein components of large size and multidomain structure. Here, we engineer single-component NIR systems consisting of evolved photosensory core module of Idiomarina sp. bacterial phytochrome, named iLight, which are smaller and packable in adeno-associated virus. We characterize iLight in vitro and in gene transcription repression in bacterial and gene transcription activation in mammalian cells. Bacterial iLight system shows 115-fold repression of protein production. Comparing to multi-component NIR systems, mammalian iLight system exhibits higher activation of 65-fold in cells and faster 6-fold activation in deep tissues of mice. Neurons transduced with viral-encoded iLight system exhibit 50-fold induction of fluorescent reporter. NIR light-induced neuronal expression of green-light-activatable CheRiff channelrhodopsin causes 20-fold increase of photocurrent and demonstrates efficient spectral multiplexing.


Assuntos
Gammaproteobacteria/genética , Regulação da Expressão Gênica , Neurônios/metabolismo , Optogenética/métodos , Transcrição Genética/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Feminino , Gammaproteobacteria/metabolismo , Células HeLa , Humanos , Raios Infravermelhos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Neurônios/citologia , Espectroscopia de Luz Próxima ao Infravermelho
10.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064134

RESUMO

Bone is a dynamic tissue constantly responding to environmental changes such as nutritional and mechanical stress. Bone homeostasis in adult life is maintained through bone remodeling, a controlled and balanced process between bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoblasts secrete matrix, with some being buried within the newly formed bone, and differentiate to osteocytes. During embryogenesis, bones are formed through intramembraneous or endochondral ossification. The former involves a direct differentiation of mesenchymal progenitor to osteoblasts, and the latter is through a cartilage template that is subsequently converted to bone. Advances in lineage tracing, cell sorting, and single-cell transcriptome studies have enabled new discoveries of gene regulation, and new populations of skeletal stem cells in multiple niches, including the cartilage growth plate, chondro-osseous junction, bone, and bone marrow, in embryonic development and postnatal life. Osteoblast differentiation is regulated by a master transcription factor RUNX2 and other factors such as OSX/SP7 and ATF4. Developmental and environmental cues affect the transcriptional activities of osteoblasts from lineage commitment to differentiation at multiple levels, fine-tuned with the involvement of co-factors, microRNAs, epigenetics, systemic factors, circadian rhythm, and the microenvironments. In this review, we will discuss these topics in relation to transcriptional controls in osteogenesis.


Assuntos
Osteogênese/genética , Fatores de Transcrição/genética , Transcrição Genética/genética , Animais , Osso e Ossos/fisiologia , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Humanos
11.
PLoS Pathog ; 17(6): e1009596, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34061905

RESUMO

The rapid evolution of RNA viruses has been long considered to result from a combination of high copying error frequencies during RNA replication, short generation times and the consequent extensive fixation of neutral or adaptive changes over short periods. While both the identities and sites of mutations are typically modelled as being random, recent investigations of sequence diversity of SARS coronavirus 2 (SARS-CoV-2) have identified a preponderance of C->U transitions, proposed to be driven by an APOBEC-like RNA editing process. The current study investigated whether this phenomenon could be observed in datasets of other RNA viruses. Using a 5% divergence filter to infer directionality, 18 from 36 datasets of aligned coding region sequences from a diverse range of mammalian RNA viruses (including Picornaviridae, Flaviviridae, Matonaviridae, Caliciviridae and Coronaviridae) showed a >2-fold base composition normalised excess of C->U transitions compared to U->C (range 2.1x-7.5x), with a consistently observed favoured 5' U upstream context. The presence of genome scale RNA secondary structure (GORS) was the only other genomic or structural parameter significantly associated with C->U/U->C transition asymmetries by multivariable analysis (ANOVA), potentially reflecting RNA structure dependence of sites targeted for C->U mutations. Using the association index metric, C->U changes were specifically over-represented at phylogenetically uninformative sites, potentially paralleling extensive homoplasy of this transition reported in SARS-CoV-2. Although mechanisms remain to be functionally characterised, excess C->U substitutions accounted for 11-14% of standing sequence variability of structured viruses and may therefore represent a potent driver of their sequence diversification and longer-term evolution.


Assuntos
Mamíferos/virologia , Mutação , Vírus de RNA/genética , SARS-CoV-2/genética , Desaminases APOBEC/metabolismo , Animais , Sequência de Bases , COVID-19/virologia , Citidina/genética , Dano ao DNA/fisiologia , Evolução Molecular , Regulação Viral da Expressão Gênica , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Humanos , Conformação de Ácido Nucleico , Filogenia , Edição de RNA/fisiologia , Vírus de RNA/classificação , RNA Viral/química , RNA Viral/genética , SARS-CoV-2/química , SARS-CoV-2/classificação , Análise de Sequência de RNA , Transcrição Genética/genética , Uridina/genética
12.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063857

RESUMO

Survivors of sepsis often suffer from prolonged post-critical illness syndrome secondary to the immune system's reprogramming. It is unclear if this process is static and pervasive due to methodological difficulties studying long-term outcomes of sepsis. The purpose of this study is to evaluate transcriptional profiles longitudinally in Drosophila melanogaster in the aftermath of sepsis to provide preliminary data for targets playing a role in post-sepsis immunostasis. Adult Drosophila melanogaster were infected with E. coli, and survivors were euthanized at 7, 14, and 21 days. Control flies were subjected to sham stress. Gene profiling was done with RNA-seq, and potential miRNA factors were computed. Profiling identified 55 unique genes at seven days, 61 unique genes at 14 days, and 78 genes at 21 days in sepsis survivors vs. sham control. Each post-sepsis timepoint had a distinctive transcriptional pattern with a signature related to oxidative stress at seven days, neuronal signal transduction at 14 days, and metabolism at 21 days. Several potential miRNA patterns were computed as potentially affecting several of the genes expressed in sepsis survivors. Our study demonstrated that post-sepsis changes in the transcriptome profile are dynamic and extend well into the Drosophila melanogaster natural life span.


Assuntos
Sepse/genética , Transcrição Genética/genética , Animais , Estado Terminal , Drosophila melanogaster/genética , Escherichia coli/genética , Perfilação da Expressão Gênica/métodos , Longevidade/genética , MicroRNAs/genética , Estresse Oxidativo/genética , RNA-Seq/métodos , Transdução de Sinais/genética , Sobreviventes , Transcriptoma/genética
13.
Cell ; 184(13): 3474-3485.e11, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34143953

RESUMO

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/genética , Exorribonucleases/genética , Metiltransferases/genética , SARS-CoV-2/genética , Sequência de Aminoácidos , COVID-19/virologia , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Alinhamento de Sequência , Transcrição Genética/genética , Replicação Viral/genética
14.
Cell Rep ; 35(6): 109108, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: covidwho-1202346

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses subgenomic RNA (sgRNA) to produce viral proteins for replication and immune evasion. We apply long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA upregulates earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of open reading frame 1ab (ORF1ab) containing nsp1 joins to ORF10, and the 3' untranslated region (UTR) upregulates at 48 h post-infection in human cell lines. We identify double-junction sgRNA containing both TRS-dependent and -independent junctions. We find multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA and that sgRNA modifications are stable across transcript clusters, host cells, and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle.


Assuntos
COVID-19/genética , SARS-CoV-2/genética , Transcrição Genética/genética , Animais , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Epigênese Genética , Genoma Viral/genética , Humanos , Evasão da Resposta Imune , Fases de Leitura Aberta , RNA Viral/genética , Transcriptoma , Células Vero , Proteínas Virais/genética
15.
Cancer Med ; 10(10): 3413-3426, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33932125

RESUMO

Metastasis to regional lymph nodes or distal organs predicts the progression of the disease and poor prognosis in esophageal squamous cell carcinoma (ESCC). Previous studies demonstrated that BTB and CNC homology 1 (BACH1) participates in various types of tumor metastasis. However, the function of BACH1 in ESCC was rarely reported. The present study demonstrated that BACH1 protein was overexpressed in ESCC tissues compared with paired esophageal epithelial tissues according to immunohistochemical staining (IHC). Higher levels of BACH1 mRNA were associated with decreased overall survival (OS) and shorter disease-free survival (DFS) of ESCC patients based on an analysis of The Cancer Genome Atlas (TCGA) datasets. BACH1 significantly enhanced the migration and invasion of ESCC in vitro. Mechanistically, BACH1 promoted the epithelial-mesenchymal transition (EMT) by directly activating the transcription of CDH2, SNAI2, and VIM, as determined by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR). BACH1 overexpression significantly enhanced CDH2 promoter activity according to the results of a luciferase assay. The results of subsequent experiments indicated that BACH1 enhanced the growth of tumor xenografts. The density of CD31+ blood vessels and the expression of vascular endothelial growth factor C (VEGFC) in tumor xenografts were significantly associated with BACH1 levels according to the results of IHC and immunofluorescence (IF) analyses performed in vivo. Moreover, ChIP-qPCR analysis demonstrated that the transcriptional activity of VEGFC was also upregulated by BACH1. Thus, BACH1 contributes to ESCC metastasis and tumorigenesis by partially facilitating the EMT and angiogenesis, and BACH1 may be a promising therapeutic target or molecular marker in ESCC.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Neovascularização Patológica/genética , Animais , Biomarcadores Tumorais/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Intervalo Livre de Doença , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neovascularização Patológica/patologia , Regiões Promotoras Genéticas/genética , Transcrição Genética/genética , Regulação para Cima/genética , Fator C de Crescimento do Endotélio Vascular/genética
16.
Cancer Med ; 10(10): 3346-3357, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33932127

RESUMO

BACKGROUND: Chronic alcohol consumption is more frequently associated with advanced, aggressive hepatocellular carcinoma (HCC) tumors. Alcohol adversely impacts ER/Golgi membrane trafficking and Golgi protein N-glycosylation in hepatocytes; these effects have been attributed (in part) to dysregulated adenosine diphosphate-ribosylation factor (ARF) GTPase signaling. Here, we investigated the role of the ARF GTPase guanine exchange factor PSD4 in HCC progression. METHODS: R-based bioinformatics analysis was performed on publicly available array data. Modulating gene expression was accomplished via lentiviral vectors. Gene expression was analyzed using quantitative real-time PCR and immunoblotting. PSD4 promoter methylation was assessed using quantitative methylation-specific PCR. Phospho-p65(S276)/DNMT1 binding to the PSD4 promoter was analyzed via chromatin immunoprecipitation. We constructed ethanol/DEN-induced and DEN only-induced transgenic murine models of HCC. RESULTS: We identified PSD4 as a hypermethylated, suppressed gene in alcohol-related HCC tumors; however, PSD4 was not dysregulated in all-cause HCC tumors. Certain HCC cell lines also displayed varying degrees of PSD4 downregulation. PSD4 overexpression or knockdown decreased and increased cell migration and invasiveness, respectively. Mechanistically, PSD4 transcription was repressed by TNF-α-induced phospho-p65(S276)'s recruitment of DNA methyltransferase 1 (DNMT1), resulting in PSD4 promoter methylation. PSD4 inhibited pro-EMT CDC42 activity, resulting in downregulation of E-cadherin and upregulation of N-cadherin and vimentin. Hepatocyte-specific PSD4 overexpression reduced ethanol/DEN-induced HCC tumor progression and EMT marker expression in vivo. CONCLUSIONS: PSD4 is a hypermethylated, suppressed gene in alcohol-related HCC tumors that negatively modulated pro-EMT CDC42 activity. Furthermore, we present a novel phospho-NF-κB p65(S276)/DNMT1-mediated promoter methylation mechanism by which TNF-α/NF-κB signaling represses PSD4 transcription in HCC cells.


Assuntos
Álcoois/efeitos adversos , Carcinoma Hepatocelular/genética , Epigênese Genética/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Neoplasias Hepáticas/genética , NF-kappa B/genética , Transcrição Genética/genética , Fator de Necrose Tumoral alfa/genética , Consumo de Bebidas Alcoólicas/genética , Animais , Caderinas/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/genética , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Transgênicos , Gravidez , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Fator de Transcrição RelA/genética
17.
Cell Rep ; 35(6): 109108, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33961822

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses subgenomic RNA (sgRNA) to produce viral proteins for replication and immune evasion. We apply long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA upregulates earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of open reading frame 1ab (ORF1ab) containing nsp1 joins to ORF10, and the 3' untranslated region (UTR) upregulates at 48 h post-infection in human cell lines. We identify double-junction sgRNA containing both TRS-dependent and -independent junctions. We find multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA and that sgRNA modifications are stable across transcript clusters, host cells, and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle.


Assuntos
COVID-19/genética , SARS-CoV-2/genética , Transcrição Genética/genética , Animais , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Epigênese Genética , Genoma Viral/genética , Humanos , Evasão da Resposta Imune , Fases de Leitura Aberta , RNA Viral/genética , Transcriptoma , Células Vero , Proteínas Virais/genética
18.
Gene ; 791: 145717, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-33991649

RESUMO

ZNFO is a Krüppel-associated box (KRAB) containing zinc finger transcription factor, which is exclusively expressed in bovine oocytes. Previous studies have demonstrated that ZNFO possesses an intrinsic transcriptional repressive activity and is essential for early embryonic development in cattle. However, the mechanisms regulating ZNFO transcription remain elusive. In the present study, the core promoter that controls the ZNFO basal transcription was identified. A 1.7 kb 5' regulatory region of the ZNFO gene was cloned and its promoter activity was confirmed by a luciferase reporter assay. A series of 5' deletion in the ZNFO promoter followed by luciferase reporter assays indicated that the core promoter region has to include the sequence located within 57 bp to 31 bp upstream of the transcription start site. Sequence analysis revealed that a putative USF1/USF2 binding site (GGTCACGTGACC) containing an E-box motif (CACGTG) is located within the essential region. Depletion of USF1/USF2 by RNAi and E-box mutation analysis demonstrated that the USF1/USF2 binding site is required for the ZNFO basal transcription. Furthermore, EMSA and super-shift assays indicated that the observed effects are dependent on the specific interactions between USF proteins and the ZNFO core promoter. From these results, it is concluded that USF1 and USF2 are essential for the basal transcription of the ZNFO gene.


Assuntos
Oócitos/metabolismo , Fatores de Transcrição/genética , Fatores Estimuladores Upstream/genética , Animais , Sequência de Bases/genética , Sítios de Ligação , Bovinos/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Elementos E-Box/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Herança Materna/genética , Oócitos/fisiologia , Oogênese/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Genética/genética , Fatores Estimuladores Upstream/metabolismo , Dedos de Zinco/genética
19.
Mol Cell ; 81(8): 1631-1639, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33826920

RESUMO

Spatial transcriptional profiling provides gene expression information within the important anatomical context of tissue architecture. This approach is well suited to characterizing solid tumors, which develop within a complex landscape of malignant cells, immune cells, and stroma. In a single assay, spatial transcriptional profiling can interrogate the role of spatial relationships among these cell populations as well as reveal spatial patterns of relevant oncogenic genetic events. The broad utility of this approach is reflected in the array of strategies that have been developed for its implementation as well as in the recent commercial development of several profiling platforms. The flexibility to apply these technologies to both hypothesis-driven and discovery-driven studies allows widespread applicability in research settings. This review discusses available technologies for spatial transcriptional profiling and several applications for their use in cancer research.


Assuntos
Perfilação da Expressão Gênica/métodos , Neoplasias/genética , Transcrição Genética/genética , Animais , Expressão Gênica/genética , Humanos
20.
Mol Cell Biol ; 41(7): e0004721, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-33875574

RESUMO

In budding tunicates, aging accompanies a decrease in the gene expression of mitochondrial transcription factor A (Tfam), and the in vivo transfection of Tfam mRNA stimulates the mitochondrial respiratory activity of aged animals. The gene expression of both the transcriptional repressor Yin-Yang-1 (YY1) and corepressor Sirtuin6 (Sirt6) increased during aging, and the cotransfection of synthetic mRNA of YY1 and Sirt6 synergistically downregulated Tfam gene expression. Pulldown assays of proteins indicated that YY1-associated factor 2 (YAF2) was associated with both YY1 and SIRT6. Protein cross-linking confirmed that YAF2 bound YY1 and SIRT6 with a molar ratio of 1:1. YY1 was bound to CCAT- or ACAT-containing oligonucleotides in the 5' flanking region of the Tfam gene. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) showed that SIRT6 specifically induced the histone H3 lysine 9 (H3K9) deacetylation of the Tfam upstream region. YY1 and YAF2 accelerated SIRT6-induced H3K9 deacetylation. YY1 and Sirt6 mRNA transfection attenuated mitochondrial respiratory gene expression and blocked MitoTracker fluorescence. In contrast, the SIRT6 inhibitor and Tfam mRNA antagonized the inhibitory effects of YY1 and Sirt6, indicating that Tfam acts on mitochondria downstream of YY1 and Sirt6. We concluded that in the budding tunicate Polyandrocarpa misakiensis, YY1 recruits SIRT6 via YAF2 to the TFAM gene, resulting in aging-related mitochondrial downregulation.


Assuntos
Envelhecimento/fisiologia , Mitocôndrias/metabolismo , Urocordados/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Proteínas Mitocondriais/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Urocordados/genética , Fator de Transcrição YY1/genética
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