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1.
Nat Commun ; 11(1): 5061, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033262

RESUMO

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3's catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.


Assuntos
Reprogramação Celular , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Catálise , Proliferação de Células , Senescência Celular , Desmetilação , Elementos Facilitadores Genéticos/genética , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Ativação Transcricional/genética
2.
Gene ; 758: 144954, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32683079

RESUMO

Teosinte branched1/cycloidea/proliferating cell factor1 (TCP) is a plant-specific protein family member involved in plant growth and development. However, the functions of most members of the cotton TCP family are unknown. In this study, the GbTCP5 gene encodes a sea-island cotton class II TCP CIN subclass transcription factor. The GbTCP5 transcription factor is located in the nucleus, has transcriptional activation activity, and can bind to TCP II cis-acting elements. GbTCP5 was widely expressed in tissues with the highest transcript level in the calyx. GbTCP5 is expressed at different developmental stages of the fiber and has significantly high transcriptional level expression in the fibers at 20, 30 and 35 days post anthesis (DPA). Heterologous overexpression of the GbTCP5 gene increased root hair length, root hair and stem trichome density, and stem lignin content in transgenic Arabidopsis compared to the wild type (WT). GbTCP5 binds the promoters of the GL3, EGL3, CPC, MYB46, LBD30, CesA4, VND7, CCOMT1, and CAD5 genes to upregulate their expression. Moreover, the homologous genes of these genes are expressed in the fibers of different developmental stages of the sea-island cotton fiber. These results indicate that GbTCP5 regulates root hair development and secondary wall formation in Arabidopsis and may be a candidate gene for improving cotton fiber quality.


Assuntos
Arabidopsis/genética , Gossypium/genética , Lignina/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Fatores de Transcrição/genética , Tricomas/crescimento & desenvolvimento , Arabidopsis/crescimento & desenvolvimento , Fibra de Algodão/análise , Proteínas de Ligação a DNA/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética
3.
Nat Commun ; 11(1): 3420, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647127

RESUMO

Remyelination of the peripheral and central nervous systems (PNS and CNS, respectively) is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac-eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination.


Assuntos
Fator 1 de Elongação de Peptídeos/metabolismo , Remielinização/genética , Ativação Transcricional/genética , Acetilação , Envelhecimento/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Lisina Acetiltransferase 5/metabolismo , Camundongos , Modelos Biológicos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Fatores de Transcrição SOXE/metabolismo , Fator de Transcrição STAT3/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Teofilina/farmacologia , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos
4.
PLoS Biol ; 18(7): e3000747, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644995

RESUMO

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Staphylococcus aureus/metabolismo , Biblioteca Gênica , Engenharia Genética , Loci Gênicos , Genoma Humano , Células HEK293 , Humanos , Nucleotídeos/genética , Fenótipo , Reprodutibilidade dos Testes , Ativação Transcricional/genética
5.
Proc Natl Acad Sci U S A ; 117(31): 18701-18710, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690679

RESUMO

Yin Yang 1 (YY1) is a DNA-binding transcription factor that either activates or represses gene expression. YY1 has previously been implicated in the transcriptional silencing of many retroviruses by binding to DNA sequences in the U3 region of the viral long terminal repeat (LTR). We here show that YY1 overexpression leads to profound activation, rather than repression, of human T lymphotropic virus type 1 (HTLV-1) expression, while YY1 down-regulation reduces HTLV-1 expression. The YY1 responsive element mapped not to YY1 DNA-binding sites in the HTLV-1 LTR but to the R region. The HTLV-1 R sequence alone is sufficient to provide YY1 responsiveness to a nonresponsive promoter, but only in the sense orientation and only when included as part of the mRNA. YY1 binds to the R region of HTLV-1 RNA in vitro and in vivo, leading to increased transcription initiation and elongation. The findings indicate that YY1 is a potent transactivator of HTLV-1 gene expression acting via binding viral RNA, rather than DNA.


Assuntos
Regulação Viral da Expressão Gênica/genética , Vírus Linfotrópico T Tipo 1 Humano , RNA/metabolismo , Sequências Repetidas Terminais/genética , Fator de Transcrição YY1 , Células HEK293 , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Ligação Proteica/genética , RNA/genética , Ativação Transcricional/genética , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
6.
PLoS One ; 15(7): e0236403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32716961

RESUMO

Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells.


Assuntos
Autofagia/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Ativação Transcricional/genética , Aminoácidos/deficiência , Autofagia/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células HCT116 , Células HEK293 , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Sirolimo/farmacologia , Ativação Transcricional/efeitos dos fármacos , Proteína com Valosina/metabolismo
7.
Nucleic Acids Res ; 48(15): 8393-8407, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32619221

RESUMO

The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR's anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription. Using mice with a point mutation in GR's zinc finger, that still tether via protein-protein interactions while being unable to recognize DNA, we demonstrate that DNA binding is essential for both transcriptional activation and repression. Performing ChIP-Seq, RNA-Seq and proteomics under inflammatory conditions, we show that DNA recognition is required for the assembly of a functional co-regulator complex to mediate glucocorticoid responses. Our findings may contribute to the development of safer immunomodulators with fewer side effects.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Inflamação/genética , Receptores de Glucocorticoides/genética , Animais , DNA/metabolismo , Regulação da Expressão Gênica/genética , Glucocorticoides/genética , Glucocorticoides/metabolismo , Humanos , Inflamação/patologia , Camundongos , Domínios e Motivos de Interação entre Proteínas/genética , RNA-Seq , Ativação Transcricional/genética
8.
Nucleic Acids Res ; 48(15): 8332-8348, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32633757

RESUMO

Negative cofactor 2 (NC2), including two subunits NC2α and NC2ß, is a conserved positive/negative regulator of class II gene transcription in eukaryotes. It is known that NC2 functions by regulating the assembly of the transcription preinitiation complex. However, the exact role of NC2 in transcriptional regulation is still unclear. Here, we reveal that, in Neurospora crassa, NC2 activates catalase-3 (cat-3) gene transcription in the form of heterodimer mediated by histone fold (HF) domains of two subunits. Deletion of HF domain in either of two subunits disrupts the NC2α-NC2ß interaction and the binding of intact NC2 heterodimer to cat-3 locus. Loss of NC2 dramatically increases histone variant H2A.Z deposition at cat-3 locus. Further studies show that NC2 recruits chromatin remodeling complex INO80C to remove H2A.Z from the nucleosomes around cat-3 locus, resulting in transcriptional activation of cat-3. Besides HF domains of two subunits, interestingly, C-terminal repression domain of NC2ß is required not only for NC2 binding to cat-3 locus, but also for the recruitment of INO80C to cat-3 locus and removal of H2A.Z from the nucleosomes. Collectively, our findings reveal a novel mechanism of NC2 in transcription activation through recruiting INO80C to remove H2A.Z from special H2A.Z-containing nucleosomes.


Assuntos
Catalase/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Transcrição Genética , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Genes MHC da Classe II/genética , Histonas/genética , Neurospora crassa/genética , Nucleossomos/genética , Nucleossomos/ultraestrutura , Fosfoproteínas/ultraestrutura , Ligação Proteica/genética , Fatores de Transcrição/ultraestrutura , Ativação Transcricional/genética
9.
Nucleic Acids Res ; 48(15): 8408-8430, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32663283

RESUMO

The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.


Assuntos
Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética , Ativação Transcricional/genética , Adenosina Trifosfatases/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/genética
10.
Gene ; 760: 144990, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32721476

RESUMO

The MYB transcription factors are involved in the regulation of plant secondary metabolism, cell development and morphogenesis, and stress response. Here, a full-length, 816-bp NtMYB4a cDNA, which encodes a protein comprising 271 amino acids, was isolated from tobacco leaves. Phylogenetic analysis revealed that NtMYB4a is most similar to Nicotiana. attenuata MYB4, followed by Eriobotrya japonica MYB4, and NtMYB4a clustered with transcriptional activators rather than repressors. Subcellular localization assays showed that NtMYB4 localized in the nucleus, membrane, and cytoplasm. Expression analyses revealed differential expression of NtMYB4a among different tissues and organs and between different developmental stages, with most expression occurring in the stems and leaves during the full-bloom stage. Moreover, NtMYB4a expression was induced by cold, NaCl, PEG, abscisic acid, methyl jasmonate, and dark stressors, and the expression patterns and maximum expression levels varied with the type of stress. Overexpression of NtMYB4a upregulated NtPAL, Nt4CL, NtCHS, NtCHI, NtF3H, NtDFR, NtANS, and NtUFGT, which resulted in increased anthocyanin content in the tobacco corolla and darker colors. However, CRISPR/Cas9-mediated knockout of NtMYB4a downregulated NtPAL, NtC4H, Nt4CL, NtCHS, NtCHI, NtF3H, NtANS, and NtUFGT, which resulted in reduced anthocyanin content, and lighter corolla colors. These results indicated that NtMYB4a positively regulates anthocyanin biosynthesis and is involved in abiotic stress responses in tobacco plants.


Assuntos
Tabaco/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Sequência de Aminoácidos , Antocianinas/biossíntese , Regulação da Expressão Gênica de Plantas/genética , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Estresse Fisiológico/genética , Tabaco/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética
11.
Respir Res ; 21(1): 182, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664949

RESUMO

BACKGROUND: Severe acute respiratory syndrome (SARS)-CoV-2-induced coronavirus disease-2019 (COVID-19) is a pandemic disease that affects > 2.8 million people worldwide, with numbers increasing dramatically daily. However, there is no specific treatment for COVID-19 and much remains unknown about this disease. Angiotensin-converting enzyme (ACE)2 is a cellular receptor of SARS-CoV-2. It is cleaved by type II transmembrane serine protease (TMPRSS)2 and disintegrin and metallopeptidase domain (ADAM)17 to assist viral entry into host cells. Clinically, SARS-CoV-2 infection may result in acute lung injury and lung fibrosis, but the underlying mechanisms of COVID-19 induced lung fibrosis are not fully understood. METHODS: The networks of ACE2 and its interacting molecules were identified using bioinformatic methods. Their gene and protein expressions were measured in human epithelial cells after 24 h SARS-CoV-2 infection, or in existing datasets of lung fibrosis patients. RESULTS: We confirmed the binding of SARS-CoV-2 and ACE2 by bioinformatic analysis. TMPRSS2, ADAM17, tissue inhibitor of metalloproteinase (TIMP)3, angiotensinogen (AGT), transformation growth factor beta (TGFB1), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) A and fibronectin (FN) were interacted with ACE2, and the mRNA and protein of these molecules were expressed in lung epithelial cells. SARS-CoV-2 infection increased ACE2, TGFB1, CTGF and FN1 mRNA that were drivers of lung fibrosis. These changes were also found in lung tissues from lung fibrosis patients. CONCLUSIONS: Therefore, SARS-CoV-2 binds with ACE2 and activates fibrosis-related genes and processes to induce lung fibrosis.


Assuntos
Infecções por Coronavirus/genética , Regulação da Expressão Gênica , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Fibrose Pulmonar/genética , Síndrome do Desconforto Respiratório do Adulto/genética , Vírus da SARS/genética , China , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/fisiopatologia , Progressão da Doença , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Pandemias/estatística & dados numéricos , Pneumonia Viral/epidemiologia , Pneumonia Viral/fisiopatologia , Prevalência , Fibrose Pulmonar/epidemiologia , Fibrose Pulmonar/etiologia , Receptores Virais/metabolismo , Síndrome do Desconforto Respiratório do Adulto/diagnóstico , Síndrome do Desconforto Respiratório do Adulto/epidemiologia , Medição de Risco , Análise de Sobrevida , Transcrição Genética , Ativação Transcricional/genética
12.
Respir Res ; 21(1): 182, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: covidwho-647112

RESUMO

BACKGROUND: Severe acute respiratory syndrome (SARS)-CoV-2-induced coronavirus disease-2019 (COVID-19) is a pandemic disease that affects > 2.8 million people worldwide, with numbers increasing dramatically daily. However, there is no specific treatment for COVID-19 and much remains unknown about this disease. Angiotensin-converting enzyme (ACE)2 is a cellular receptor of SARS-CoV-2. It is cleaved by type II transmembrane serine protease (TMPRSS)2 and disintegrin and metallopeptidase domain (ADAM)17 to assist viral entry into host cells. Clinically, SARS-CoV-2 infection may result in acute lung injury and lung fibrosis, but the underlying mechanisms of COVID-19 induced lung fibrosis are not fully understood. METHODS: The networks of ACE2 and its interacting molecules were identified using bioinformatic methods. Their gene and protein expressions were measured in human epithelial cells after 24 h SARS-CoV-2 infection, or in existing datasets of lung fibrosis patients. RESULTS: We confirmed the binding of SARS-CoV-2 and ACE2 by bioinformatic analysis. TMPRSS2, ADAM17, tissue inhibitor of metalloproteinase (TIMP)3, angiotensinogen (AGT), transformation growth factor beta (TGFB1), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) A and fibronectin (FN) were interacted with ACE2, and the mRNA and protein of these molecules were expressed in lung epithelial cells. SARS-CoV-2 infection increased ACE2, TGFB1, CTGF and FN1 mRNA that were drivers of lung fibrosis. These changes were also found in lung tissues from lung fibrosis patients. CONCLUSIONS: Therefore, SARS-CoV-2 binds with ACE2 and activates fibrosis-related genes and processes to induce lung fibrosis.


Assuntos
Infecções por Coronavirus/genética , Regulação da Expressão Gênica , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Fibrose Pulmonar/genética , Síndrome do Desconforto Respiratório do Adulto/genética , Vírus da SARS/genética , China , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/fisiopatologia , Progressão da Doença , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Pandemias/estatística & dados numéricos , Pneumonia Viral/epidemiologia , Pneumonia Viral/fisiopatologia , Prevalência , Fibrose Pulmonar/epidemiologia , Fibrose Pulmonar/etiologia , Receptores Virais/metabolismo , Síndrome do Desconforto Respiratório do Adulto/diagnóstico , Síndrome do Desconforto Respiratório do Adulto/epidemiologia , Medição de Risco , Análise de Sobrevida , Transcrição Genética , Ativação Transcricional/genética
13.
Biol Res ; 53(1): 25, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503642

RESUMO

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS: We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS: Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ß-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ß-catenin and protein level of ß-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ß-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION: bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Expressão Gênica , Células HEK293 , Humanos , Hipóxia/genética , Fator 1 Induzível por Hipóxia/genética , Reação em Cadeia da Polimerase em Tempo Real , Ativação Transcricional/genética
14.
Cell Prolif ; 53(7): e12823, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32515533

RESUMO

OBJECTIVES: Over the past years, growing attention has been paid to deciphering the pivotal role of long non-coding RNAs (lncRNAs) in regulating the occurrence and development of human malignancies, cervical cancer (CC) included. Nonetheless, the regulatory role of lncRNA BBOX1 antisense RNA 1 (BBOX1-AS1) has not been explored as yet. MATERIAL AND METHODS: The expression of BBOX1-AS1 was detected by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8), colony formation, TUNEL, Western blot, transwell and immunofluorescence assays testified the critical role of BBOX1-AS1 in CC. The relationship between RNAs (BBOX1-AS1, miR-361-3p, HOXC6 and HuR) was analysed by luciferase reporter, RNA Immunoprecipitation (RIP) and RNA pull-down assays. RESULTS: BBOX1 antisense RNA 1 antisense RNA 1 was revealed to be highly expressed in CC. Decreased expression of BBOX1-AS1 had suppressive effects on CC cell growth and migration. Molecular mechanism assays verified that BBOX1-AS1 had negative interaction with miR-361-3p in CC. Additionally, homeobox C6 (HOXC6) was validated to be a downstream target of miR-361-3p in CC. Furthermore, ELAV-like RNA-binding protein 1, also known as HuR, was uncovered to be capable of regulating the mRNA stability of HOXC6 in CC. More importantly, rescue assays delineated that knockdown of HuR after overexpressing miR-361-3p could reverse BBOX1-AS1 upregulation-mediated effect on CC progression. Similarly, the function induced by BBOX1-AS1 upregulation on CC progression could be countervailed by HOXC6 depletion. CONCLUSIONS: BBOX1 antisense RNA 1 facilitates CC progression by upregulating HOXC6 expression via miR-361-3p and HuR.


Assuntos
Proteína Semelhante a ELAV 1/genética , Proteínas de Homeodomínio/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Regulação para Cima/genética , Neoplasias do Colo do Útero/genética , gama-Butirobetaína Dioxigenase/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Ativação Transcricional/genética , Neoplasias do Colo do Útero/patologia
15.
Cell Prolif ; 53(7): e12853, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32537867

RESUMO

BACKGROUND: Hypoxia-inducible factors (HIFs) are thought to play important roles in the carcinogenesis and progression of VHL-deficient clear cell renal cell carcinoma (ccRCC). METHODS: The roles of HIF-1/2α in VHL-deficient clear cell renal cell carcinoma were evaluated by bioinformatics analysis, immunohistochemistry staining and Kaplan-Meier survival analysis. The downstream genes that counteract the cancer-promoting effect of HIF were analysed by unbiased proteomics and verified by in vitro and in vivo assays. RESULTS: There was no correlation between the high protein level of HIF-1/2α and the poor prognosis of ccRCC patients in our large set of clinical data. Furthermore, NDRG1 was found to be up-regulated by both HIF-1α and -2α at the cellular level and in ccRCC tissues. Intriguingly, the high NDRG1 expression was correlated with lower Furman grade, TNM stage and longer survival for ccRCC patients compared with the low NDRG1 expression. In addition, NDRG1 suppressed the expression of series oncogenes as well as the proliferation, metastasis and invasion of VHL-deficient ccRCC cells in vitro and vivo. CONCLUSIONS: Our study demonstrated that HIF downstream gene of NDRG1 may counteract the cancer-promoting effect of HIF. These results provided evidence that NDRG1 may be a potential prognostic biomarker as well as a therapeutic target in ccRCC.


Assuntos
Carcinoma de Células Renais/genética , Proteínas de Ciclo Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Renais/genética , Regulação para Cima/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Gravidez , Ativação Transcricional/genética , Adulto Jovem
16.
PLoS One ; 15(6): e0233784, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492024

RESUMO

Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.


Assuntos
Clonagem de Organismos/veterinária , Citomegalovirus/genética , Técnicas de Transferência Nuclear/veterinária , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética , Animais , Animais Geneticamente Modificados , Azacitidina/farmacologia , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Metilação de DNA/efeitos dos fármacos , Cães , Transferência Embrionária/veterinária , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Gravidez , Transfecção , Transgenes
17.
Nat Commun ; 11(1): 2243, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382029

RESUMO

Cells rely on a diverse repertoire of genes for maintaining homeostasis, but the transcriptional networks underlying their expression remain poorly understood. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator. It is essential in Drosophila, and haploinsufficiency of the human KANSL1 subunit results in the Koolen-de Vries syndrome. Here, we perform a genome-wide RNAi screen and identify the BET protein BRD4 as an evolutionary conserved co-factor of the NSL complex. Using Drosophila and mouse embryonic stem cells, we characterise a recruitment hierarchy, where NSL-deposited histone acetylation enables BRD4 recruitment for transcription of constitutively active genes. Transcriptome analyses in Koolen-de Vries patient-derived fibroblasts reveals perturbations with a cellular homeostasis signature that are evoked by the NSL complex/BRD4 axis. We propose that BRD4 represents a conserved bridge between the NSL complex and transcription activation, and provide a new perspective in the understanding of their functions in healthy and diseased states.


Assuntos
Histonas/metabolismo , Ativação Transcricional/fisiologia , Acetilação , Animais , Células Cultivadas , Cromatina/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epigenômica , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Gravidez , Regiões Promotoras Genéticas/genética , Interferência de RNA/fisiologia , Ativação Transcricional/genética
18.
Nat Commun ; 11(1): 2286, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385332

RESUMO

Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report that widespread promoter-proximal RNA polymerase II pausing in resting macrophages is marked by co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.


Assuntos
Anti-Inflamatórios/metabolismo , Regulação da Expressão Gênica , Inflamação/genética , Macrófagos/patologia , Fatores de Transcrição/metabolismo , Animais , Cromatina/metabolismo , Interleucina-10/metabolismo , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Camundongos , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Genética , Ativação Transcricional/genética
19.
Mol Cell ; 78(5): 890-902.e6, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32416068

RESUMO

Acidic transcription activation domains (ADs) are encoded by a wide range of seemingly unrelated amino acid sequences, making it difficult to recognize features that promote their dynamic behavior, "fuzzy" interactions, and target specificity. We screened a large set of random 30-mer peptides for AD function in yeast and trained a deep neural network (ADpred) on the AD-positive and -negative sequences. ADpred identifies known acidic ADs within transcription factors and accurately predicts the consequences of mutations. Our work reveals that strong acidic ADs contain multiple clusters of hydrophobic residues near acidic side chains, explaining why ADs often have a biased amino acid composition. ADs likely use a binding mechanism similar to avidity where a minimum number of weak dynamic interactions are required between activator and target to generate biologically relevant affinity and in vivo function. This mechanism explains the basis for fuzzy binding observed between acidic ADs and targets.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Fatores de Transcrição/genética , Ativação Transcricional/genética , Sequência de Aminoácidos/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Ligação a DNA/metabolismo , Aprendizado Profundo , Ligação Proteica , Domínios Proteicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia
20.
PLoS Genet ; 16(3): e1008645, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226006

RESUMO

The gene products that drive early development are critical for setting up developmental trajectories in all animals. The earliest stages of development are fueled by maternally provided mRNAs until the zygote can take over transcription of its own genome. In early development, both maternally deposited and zygotically transcribed gene products have been well characterized in model systems. Previously, we demonstrated that across the genus Drosophila, maternal and zygotic mRNAs are largely conserved but also showed a surprising amount of change across species, with more differences evolving at the zygotic stage than the maternal stage. In this study, we use comparative methods to elucidate the regulatory mechanisms underlying maternal deposition and zygotic transcription across species. Through motif analysis, we discovered considerable conservation of regulatory mechanisms associated with maternal transcription, as compared to zygotic transcription. We also found that the regulatory mechanisms active in the maternal and zygotic genomes are quite different. For maternally deposited genes, we uncovered many signals that are consistent with transcriptional regulation at the level of chromatin state through factors enriched in the ovary, rather than precisely controlled gene-specific factors. For genes expressed only by the zygotic genome, we found evidence for previously identified regulators such as Zelda and GAGA-factor, with multiple analyses pointing toward gene-specific regulation. The observed mechanisms of regulation are consistent with what is known about regulation in these two genomes: during oogenesis, the maternal genome is optimized to quickly produce a large volume of transcripts to provide to the oocyte; after zygotic genome activation, mechanisms are employed to activate transcription of specific genes in a spatiotemporally precise manner. Thus the genetic architecture of the maternal and zygotic genomes, and the specific requirements for the transcripts present at each stage of embryogenesis, determine the regulatory mechanisms responsible for transcripts present at these stages.


Assuntos
Desenvolvimento Embrionário/genética , RNA Mensageiro Estocado/genética , Fatores de Transcrição/genética , Animais , Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Genômica/métodos , Proteínas Nucleares/genética , Oócitos/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Zigoto/metabolismo
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