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1.
Anticancer Res ; 41(9): 4295-4304, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475049

RESUMO

BACKGROUND/AIM: Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults. The aim of this study was to elucidate the molecular pathogenesis of sporadic RCC in Taiwan. MATERIALS AND METHODS: Fifteen patients with RCC were screened for mutations in the von Hippel-Lindau (VHL) gene by PCR and Sanger sequencing. The methylation status of promoters of 24 tumor suppressor genes by methylation sensitive multiplex ligation-dependent probe amplification analysis was also determined. RESULTS: Inactivation of the VHL gene was observed in 5 cases: three missense somatic mutations, one promoter methylation, and one small deletion. In RCCs, methylation was most frequently observed in APC (100%), CDKN2B (92.9%), CASP8, MLH1_167, and KLLN (85.7.4%), but not in FHIT, MLH1_463, DAPK1, or HIC1 (0%). CONCLUSION: In addition to VHL inactivation, promoter methylation of APC may be a universal pathognomonic event in the tumorigenesis of RCC and a candidate diagnostic and therapeutic biomarker.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Carcinoma de Células Renais/diagnóstico , Metilação de DNA , Neoplasias Renais/diagnóstico , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Detecção Precoce de Câncer , Epigênese Genética , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Mutação de Sentido Incorreto , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Deleção de Sequência
2.
BMC Plant Biol ; 21(1): 408, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493199

RESUMO

BACKGROUND: Mung bean (Vigna radiata) is a warm-season legume crop and belongs to the papilionoid subfamily of the Fabaceae family. China is the leading producer of mung bean in the world. Mung bean has significant economic and health benefits and is a promising species with broad adaptation ability and high tolerance to environmental stresses. OSCA (hyperosmolality-gated calcium-permeable channel) gene family members play an important role in the modulation of hypertonic stress, such as drought and salinity. However, genome-wide analysis of the OSCA gene family has not been conducted in mung bean. RESULTS: We identified a total of 13 OSCA genes in the mung bean genome and named them according to their homology with AtOSCAs. All the OSCAs were phylogenetically split into four clades. Phylogenetic relationship and synteny analyses showed that the VrOSCAs in mung bean and soybean shared a relatively conserved evolutionary history. In addition, three duplicated VrOSCA gene pairs were identified, and the duplicated VrOSCAs gene pairs mainly underwent purifying selection pressure during evolution. Protein domain, motif and transmembrane analyses indicated that most of the VrOSCAs shared similar structures with their homologs. The expression pattern showed that except for VrOSCA2.1, the other 12 VrOSCAs were upregulated under treatment with ABA, PEG and NaCl, among which VrOSCA1.4 showed the largest increased expression levels. The duplicated genes VrOSCA2.1/VrOSCA2.2 showed divergent expression, which might have resulted in functionalization during subsequent evolution. The expression profiles under ABA, PEG and NaCl stress revealed a functional divergence of VrOSCA genes, which agreed with the analysis of cis-acting regulatory elements in the promoter regions of VrOSCA genes. CONCLUSIONS: Collectively, the study provided a systematic analysis of the VrOSCA gene family in mung bean. Our results establish an important foundation for functional and evolutionary analysis of VrOSCAs and identify genes for further investigation of their ability to confer abiotic stress tolerance in mung bean.


Assuntos
Osmorregulação/genética , Proteínas de Plantas/genética , Vigna/fisiologia , Ácido Abscísico/farmacologia , Arabidopsis/genética , Duplicação Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genoma de Planta , Estudo de Associação Genômica Ampla , Família Multigênica , Oryza/genética , Pressão Osmótica , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Cloreto de Sódio/farmacologia , Soja/genética , Estresse Fisiológico/genética , Sintenia , Vigna/efeitos dos fármacos , Vigna/genética
3.
BMC Plant Biol ; 21(1): 411, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34496770

RESUMO

BACKGROUND: The phytohormone ethylene controls many processes in plant development and acts as a key signaling molecule in response to biotic and abiotic stresses: it is rapidly induced by flooding, wounding, drought, and pathogen attack as well as during abscission and fruit ripening. In kiwifruit (Actinidia spp.), fruit ripening is characterized by two distinct phases: an early phase of system-1 ethylene biosynthesis characterized by absence of autocatalytic ethylene, followed by a late burst of autocatalytic (system-2) ethylene accompanied by aroma production and further ripening. Progress has been made in understanding the transcriptional regulation of kiwifruit fruit ripening but the regulation of system-1 ethylene biosynthesis remains largely unknown. The aim of this work is to better understand the transcriptional regulation of both systems of ethylene biosynthesis in contrasting kiwifruit organs: fruit and leaves. RESULTS: A detailed molecular study in kiwifruit (A. chinensis) revealed that ethylene biosynthesis was regulated differently between leaf and fruit after mechanical wounding. In fruit, wound ethylene biosynthesis was accompanied by transcriptional increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO) and members of the NAC class of transcription factors (TFs). However, in kiwifruit leaves, wound-specific transcriptional increases were largely absent, despite a more rapid induction of ethylene production compared to fruit, suggesting that post-transcriptional control mechanisms in kiwifruit leaves are more important. One ACS member, AcACS1, appears to fulfil a dominant double role; controlling both fruit wound (system-1) and autocatalytic ripening (system-2) ethylene biosynthesis. In kiwifruit, transcriptional regulation of both system-1 and -2 ethylene in fruit appears to be controlled by temporal up-regulation of four NAC (NAM, ATAF1/2, CUC2) TFs (AcNAC1-4) that induce AcACS1 expression by directly binding to the AcACS1 promoter as shown using gel-shift (EMSA) and by activation of the AcACS1 promoter in planta as shown by gene activation assays combined with promoter deletion analysis. CONCLUSIONS: Our results indicate that in kiwifruit the NAC TFs AcNAC2-4 regulate both system-1 and -2 ethylene biosynthesis in fruit during wounding and ripening through control of AcACS1 expression levels but not in leaves where post-transcriptional/translational regulatory mechanisms may prevail.


Assuntos
Actinidia/genética , Etilenos/biossíntese , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Actinidia/metabolismo , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Liases/genética , Liases/metabolismo , Lycopersicon esculentum/genética , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
4.
Molecules ; 26(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34443682

RESUMO

Atherosclerosis is the main cause of cardiovascular diseases which in turn, lead to the highest number of mortalities globally. This pathophysiological condition is developed due to a constant elevated level of plasma cholesterols. Statin is currently the widely used treatment in reducing the level of cholesterols, however, it may cause adverse side effects. Therefore, there is an urgent need to search for new alternative treatment. PCSK9 is an enzyme responsible in directing LDL-receptor (LDL-R)/LDL-cholesterols (LDL-C) complex to lysosomal degradation, preventing the receptor from recycling back to the surface of liver cells. Therefore, PCSK9 offers a potential target to search for small molecule inhibitors which inhibit the function of this enzyme. In this study, a marine invertebrate Acanthaster planci, was used to investigate its potential in inhibiting PCSK9 and lowering the levels of cholesterols. Cytotoxicity activity of A. planci on human liver HepG2 cells was carried out using the MTS assay. It was found that methanolic extract and fractions did not exhibit cytotoxicity effect on HepG2 cell line with IC50 values of more than 30 µg/mL. A compound deoxythymidine also did not exert any cytotoxicity activity with IC50 value of more than 4 µg/mL. Transient transfection and luciferase assay were conducted to determine the effects of A. planci on the transcriptional activity of PCSK9 promoter. Methanolic extract and Fraction 2 (EF2) produced the lowest reduction in PCSK9 promoter activity to 70 and 20% of control at 12.5 and 6.25 µg/mL, respectively. In addition, deoxythymidine also decreased PCSK9 promoter activity to the lowest level of 60% control at 3.13 µM. An in vivo study using Sprague Dawley rats demonstrated that 50 and 100 mg/kg of A. planci methanolic extract reduced the total cholesterols and LDL-C levels to almost similar levels of untreated controls. The level of serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) showed that the administration of the extract did not produce any toxicity effect and cause any damage to rat liver. The results strongly indicate that A. planci produced a significant inhibitory activity on PCSK9 gene expression in HepG2 cells which may be responsible for inducing the uptake of cholesterols by liver, thus, reducing the circulating levels of total cholesterols and LDL-C. Interestingly, A. planci also did show any adverse hepato-cytotoxicity and toxic effects on liver. Thus, this study strongly suggests that A. planci has a vast potential to be further developed as a new class of therapeutic agent in lowering the blood cholesterols and reducing the progression of atherosclerosis.


Assuntos
Colesterol/sangue , Pró-Proteína Convertase 9/antagonistas & inibidores , Estrelas-do-Mar/química , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Morte Celular , Proliferação de Células , LDL-Colesterol/sangue , Células Hep G2 , Humanos , Luciferases/metabolismo , Masculino , Metanol , Regiões Promotoras Genéticas/genética , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Ratos Sprague-Dawley , Timidina/farmacologia , Triglicerídeos/sangue
5.
Nutrients ; 13(8)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34444938

RESUMO

l-Arginine is an important nutrient in the infant diet that significantly regulates the maturation of the immune system in neonates, including the maturation of CD4+ T cells. The biological activities of CD4+ T cells differ substantially between neonates and adults, and these differences may be governed by epigenetic processes. Investigating these differences and the causative processes may help understand neonatal and developmental immunity. In this study, we compared the functional DNA methylation profiles in CD4+ T cells of neonates and adults, focusing on the role of l-arginine supplementation. Umbilical cord blood and adult CD4+ T cells were cultured with/without l-arginine treatment. By comparing DNA methylation in samples without l-arginine treatment, we found that CD4+ T cells of neonatal cord blood generally showed higher DNA methylation than those of adults (average CpG methylation percentage 0.6305 for neonate and 0.6254 for adult, t-test p-value < 0.0001), suggesting gene silencing in neonates. By examining DNA methylation patterns of CpG dinucleotides induced by l-arginine treatment, we found that more CpG dinucleotides were hypomethylated and more genes appeared to be activated in neonatal T-cells as compared with adult. Genes activated by l-arginine stimulation of cord blood samples were more enriched regarding immune-related pathways. CpG dinucleotides at IL-13 promoter regions were hypomethylated after l-arginine stimulation. Hypomethylated CpG dinucleotides corresponded to higher IL-13 gene expression and cytokine production. Thus, DNA methylation partially accounts for the mechanism underlying differential immune function in neonates. Modulatory effects of l-arginine on DNA methylation are gene-specific. Nutritional intervention is a potential strategy to modulate immune function of neonates.


Assuntos
Arginina/administração & dosagem , Linfócitos T CD4-Positivos/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Adulto , Ilhas de CpG , Suplementos Nutricionais , Epigênese Genética , Sangue Fetal/metabolismo , Expressão Gênica , Humanos , Imunidade/genética , Recém-Nascido , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-13/genética , Interleucina-13/metabolismo , Regiões Promotoras Genéticas
6.
FASEB J ; 35(9): e21833, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34365659

RESUMO

Macrophages are the principal component of the innate immune system. They play very crucial and multifaceted roles in the pathogenesis of inflammatory vascular diseases. There is an increasing recognition that transcriptionally dynamic macrophages are the key players in the pathogenesis of inflammatory vascular diseases. In this context, the accumulation and aberrant activation of macrophages in the subendothelial layers govern atherosclerotic plaque development. Macrophage-mediated inflammation is an explicitly robust biological response that involves broad alterations in inflammatory gene expression. Thus, cell-intrinsic negative regulatory mechanisms must exist which can restrain inflammatory response in a spatiotemporal manner. In this study, we identified CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) as one such cell-intrinsic negative regulator of inflammation. Our in vivo studies show that myeloid-CITED2-deficient mice on the Apoe-/- background have larger atherosclerotic lesions on both control and high-fat/high-cholesterol diets. Our integrated transcriptomics and gene set enrichment analyses studies show that CITED2 deficiency elevates STAT1 and interferon regulatory factor 1 (IRF1) regulated pro-inflammatory gene expression in macrophages. At the molecular level, our studies identify that CITED2 deficiency elevates IFNγ-induced STAT1 transcriptional activity and STAT1 enrichment on IRF1 promoter in macrophages. More importantly, siRNA-mediated knockdown of IRF1 completely reversed elevated pro-inflammatory target gene expression in CITED2-deficient macrophages. Collectively, our study findings demonstrate that CITED2 restrains the STAT1-IRF1 signaling axis in macrophages and limits the development of atherosclerotic plaques.


Assuntos
Aterosclerose/genética , Fator Regulador 1 de Interferon/genética , Proteínas Repressoras/genética , Fator de Transcrição STAT1/genética , Transdução de Sinais/genética , Transativadores/genética , Animais , Feminino , Inflamação/genética , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Células RAW 264.7 , Transcrição Genética/genética
7.
FASEB J ; 35(9): e21827, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383980

RESUMO

Neuron-derived orphan receptor 1, NR4A3 (Nor1)/NR4A3 is an orphan nuclear receptor involved in the transcriptional control of developmental and neurological functions. Oxidative stress-induced conditions are primarily associated with neurological defects in humans, yet the impact on Nor1-mediated transcription of neuronal genes remains with unknown mechanism. Here, we demonstrate that Nor1 is a non-conventional target of SUMO2/3 conjugation at Lys-137 contained in an atypic ψKxSP motif referred to as the pSuM. Nor1 pSuM SUMOylation differs from the canonical process with the obligate phosphorylation of Ser-139 by Ras signaling to create the required negatively charged interface for SUMOylation. Additional phosphorylation at sites flanking the pSuM is also mediated by the coordinated action of protein kinase casein kinase 2 to function as a small ubiquitin-like modifier enhancer, regulating Nor1-mediated transcription and proteasomal degradation. Nor1 responsive genes involved in cell proliferation and metabolism, such as activating transcription factor 3, cyclin D1, CASP8 and FADD-like apoptosis regulator, and enolase 3 were upregulated in response to pSuM disruption in mouse HT-22 hippocampal neuronal cells and human neuroblastoma SH-SY5Y cells. We also identified critical antioxidant genes, such as catalase, superoxide dismutase 1, and microsomal glutathione S-transferase 2, as responsive targets of Nor1 under pSuM regulation. Nor1 SUMOylation impaired gene transcription through less effective Nor1 chromatin binding and reduced enrichment of histone H3K27ac marks to gene promoters. These effects resulted in decreased neuronal cell growth, increased apoptosis, and reduced survival to oxidative stress damage, underlying the role of pSuM-modified Nor1 in redox homeostasis. Our findings uncover a hierarchical post-translational mechanism that dictates Nor1 non-canonical SUMOylation, disrupting Nor1 transcriptional competence, and neuroprotective redox sensitivity.


Assuntos
Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Sumoilação/genética , Animais , Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinase do Ponto de Checagem 2/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Hipocampo/metabolismo , Homeostase/genética , Humanos , Camundongos , Neuroblastoma/genética , Neurônios/metabolismo , Oxirredução , Estresse Oxidativo/genética , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Regulação para Cima/genética
9.
Nat Genet ; 53(8): 1177-1186, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34341563

RESUMO

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates ß-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to ß-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.


Assuntos
Fator de Ligação a CCAAT/genética , Hemoglobina Fetal/genética , Fator de Transcrição GATA1/genética , gama-Globinas/genética , Animais , Sítios de Ligação , Células COS , Sistemas CRISPR-Cas , Linhagem Celular , Chlorocebus aethiops , Células Eritroides , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
10.
FASEB J ; 35(9): e21667, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34405442

RESUMO

Long noncoding RNAs (lncRNAs) are central regulators of the inflammatory response and play an important role in inflammatory diseases. PINT has been reported to be involved in embryonic development and tumorigenesis. However, the potential functions of PINT in the innate immune system are largely unknown. Here, we revealed the transcriptional regulation of inflammatory genes by PINT, whose expression is primarily dependent on the NF-κB signaling pathway in human and mouse macrophage and intestinal epithelial cell lines. Functionally, PINT selectively regulates the expression of TNF-α in basal and LPS-stimulated cells. Mechanistically, PINT acts as a modular scaffold of p65 and EZH2 to coordinate their localization and specify their binding to the target genes. Further, a high expression level of PINT was detected in intestinal mucosal tissues from patients with ulcerative colitis (UC). Together, these findings demonstrate that PINT acts as an activator of inflammatory responses, highlighting the importance of this lncRNA as a potential therapeutic target in infectious diseases and inflammatory diseases.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , Fator de Transcrição RelA/metabolismo , Transcrição Genética , Fator de Necrose Tumoral alfa/genética , Animais , Linhagem Celular , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Citocinas/genética , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transcrição Genética/genética
11.
J Biomed Nanotechnol ; 17(7): 1284-1292, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34446132

RESUMO

This study aimed to introduce nano-gold PCR for detection of TERT methylation, and explore the correlation between TERT methylation and prognosis of hepatocellular carcinoma (HCC). From March 2016 to March 2018, 154 HBV carriers treated in our hospital were enrolled in the study and divided into HCC (68 cases), cirrhosis (45 cases) and chronic hepatitis (CH) groups (41 cases) based on clinical disease. HCC patients were further divided into methylation (30 cases) and non-methylation (38 cases) subgroup based on methylation status of the TERT. TERT methylation of HCC specimens were 44.12% and 35.24% by nano-PCR and conventional PCR, respectively. The TERT methylation and TERT expression in HCC specimens were higher than for cirrhosis and CH specimens. A significant positive correlation was observed between TERT methylation and TERT expression. AFP, Edmondson classification, tumor size, hilar lymph node and intrahepatic metastasis, and TNM staging in the methylation group were higher than in non-methylation group. Further, overall survival and progression-free survival were significantly shorter. Nano-gold PCR is more sensitive in detecting TERT methylation. As CHB progresses, TERT methylation increases. Greater methylation of the gene is associated with worse prognosis in HCC patients.


Assuntos
Carcinoma Hepatocelular , Hepatite B Crônica , Hepatite B , Neoplasias Hepáticas , Telomerase , Carcinoma Hepatocelular/genética , Metilação de DNA , Ouro , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Humanos , Neoplasias Hepáticas/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Telomerase/genética
12.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361039

RESUMO

Plant G proteins are versatile components of transmembrane signaling transduction pathways. The deficient mutant of heterotrimeric G protein leads to defects in plant growth and development, suggesting that it regulates the GA pathway in Arabidopsis. However, the molecular mechanism of G protein regulation of the GA pathway is not understood in plants. In this study, two G protein ß subunit (AGB1) mutants, agb1-2 and N692967, were dwarfed after exogenous application of GA3. AGB1 interacts with the DNA-binding domain MYB62, a GA pathway suppressor. Transgenic plants were obtained through overexpression of MYB62 in two backgrounds including the wild-type (MYB62/WT Col-0) and agb1 mutants (MYB62/agb1) in Arabidopsis. Genetic analysis showed that under GA3 treatment, the height of the transgenic plants MYB62/WT and MYB62/agb1 was lower than that of WT. The height of MYB62/agb1 plants was closer to MYB62/WT plants and higher than that of mutants agb1-2 and N692967, suggesting that MYB62 is downstream of AGB1 in the GA pathway. qRT-PCR and competitive DNA binding assays indicated that MYB62 can bind MYB elements in the promoter of GA2ox7, a GA degradation gene, to activate GA2ox7 transcription. AGB1 affected binding of MYB62 on the promoter of GA2ox7, thereby negatively regulating th eactivity of MYB62.


Assuntos
Proteínas de Arabidopsis/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Giberelinas/metabolismo , Arabidopsis , Proteínas de Arabidopsis/genética , Sítios de Ligação , Subunidades beta da Proteína de Ligação ao GTP/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
13.
Biol Res ; 54(1): 25, 2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362460

RESUMO

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARα) is associated with diabetic retinopathy (DR), and the underlying mechanism is still unclear. Aim of this work was to investigate the mechanism of PPARα in DR. METHODS: Human retinal capillary pericytes (HRCPs) were treated with high glucose (HG) to induce DR cell model. DR mouse model was established by streptozotocin injection, and then received 5-Aza-2-deoxycytidine (DAC; DNA methyltransferase inhibitor) treatment. Hematoxylin-eosin staining was performed to assess retinal tissue damage. PPARα methylation was examined by Methylation-Specific PCR. Flow cytometry and DCFH-DA fluorescent probe was used to estimate apoptosis and reactive oxygen species (ROS). The interaction between DNA methyltransferase-1 (DNMT1) and PPARα promoter was examined by Chromatin Immunoprecipitation. Quantitative real-time PCR and western blot were performed to assess gene and protein expression. RESULTS: HG treatment enhanced the methylation levels of PPARα, and repressed PPARα expression in HRCPs. The levels of apoptotic cells and ROS were significantly increased in HRCPs in the presence of HG. Moreover, DNMT1 was highly expressed in HG-treated HRCPs, and DNMT1 interacted with PPARα promoter. PPARα overexpression suppressed apoptosis and ROS levels of HRCPs, which was rescued by DNMT1 up-regulation. In DR mice, DAC treatment inhibited PPARα methylation and reduced damage of retinal tissues. CONCLUSION: DNMT1-mediated PPARα methylation promotes apoptosis and ROS levels of HRCPs and aggravates damage of retinal tissues in DR mice. Thus, this study may highlight novel insights into DR pathogenesis.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Retinopatia Diabética , PPAR alfa/genética , Retina/patologia , Animais , Apoptose , Células Cultivadas , Metilação de DNA , Diabetes Mellitus , Modelos Animais de Doenças , Humanos , Metilação , Camundongos , Regiões Promotoras Genéticas , Retina/citologia
14.
Blood Adv ; 5(15): 3002-3015, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34351390

RESUMO

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.


Assuntos
Proteínas de Ligação a DNA , Eritropoese , Fatores de Transcrição , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Eritroides/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica , Humanos , Células K562 , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361106

RESUMO

Enhancers regulate multiple genes via higher-order chromatin structures, and they further affect cancer progression. Epigenetic changes in cancer cells activate several cancer-specific enhancers that are silenced in normal cells. These cancer-specific enhancers are potential therapeutic targets of cancer. However, the functions and regulation networks of colorectal-cancer-specific enhancers are still unknown. In this study, we profile colorectal-cancer-specific enhancers and reveal their regulation network through the analysis of HiChIP data that were derived from a colorectal cancer cell line and Hi-C and RNA-seq data that were derived from tissue samples by in silico analysis and in vitro experiments. Enhancer-promoter loops in colorectal cancer cells containing colorectal-cancer-specific enhancers are involved in more than 50% of the topological associated domains (TADs) changed in colorectal cancer cells compared to normal colon cells. In addition, colorectal-cancer-specific enhancers interact with 152 genes that are significantly and highly expressed in colorectal cancer cells. These colorectal-cancer-specific enhancer target genes include ITGB4, RECQL4, MSLN, and GDF15. We propose that the regulation network of colorectal-cancer-specific enhancers plays an important role in the progression of colorectal cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Elementos Facilitadores Genéticos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias Colorretais/genética , Progressão da Doença , Humanos , Células Tumorais Cultivadas
16.
Am J Hum Genet ; 108(9): 1611-1630, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34343493

RESUMO

Genome-wide association studies (GWASs) have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). Because ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWASs identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. Because AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 introduced via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression and altering melanocyte growth phenotypes upon exposure.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 7 , Loci Gênicos , Melanócitos/metabolismo , Melanoma/genética , Receptores de Hidrocarboneto Arílico/genética , Neoplasias Cutâneas/genética , Alelos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Cromatina/química , Cromatina/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Melanócitos/efeitos da radiação , Melanoma/metabolismo , Melanoma/patologia , Dibenzodioxinas Policloradas/toxicidade , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Banho de Sol , Raios Ultravioleta/efeitos adversos
17.
J Microbiol ; 59(9): 871-878, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34449059

RESUMO

Anti-virulence therapeutic strategies are promising alternatives against drug-resistant pathogens. Outer membrane protein A (OmpA) plays a versatile role in the pathogenesis and antimicrobial resistance of Acinetobacter baumannii. Therefore, OmpA is an innovative target for anti-virulence therapy against A. baumannii. This study aimed to develop a high-throughput screening (HTS) system to discover small molecules inhibiting the ompA promoter activity of A. baumannii and screen chemical compounds using the bacterial growth-based HTS system. The ompA promoter and open reading frame of nptI fusion plasmids that controlled the expression of nptI encoding resistance to kanamycin by the ompA promoter were constructed and then transformed into A. baumannii ATCC 17978. This reporter strain was applied to screen small molecules inhibiting the ompA promoter activity in a chemical library. Of the 7,520 chemical compounds, 15 exhibited ≥ 70% growth inhibition of the report strain cultured in media containing kanamycin. Three compounds inhibited the expression of ompA and OmpA in the outer membrane of A. baumannii ATCC 17978, which subsequently reduced biofilm formation. In conclusion, our reporter strain is useful for large-scale screening of small molecules inhibiting the ompA expression in A. baumannii. Hit compounds identified by the HTS system are promising scaffolds to develop novel therapeutics against A. baumannii.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/patogenicidade , Acinetobacter baumannii/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Avaliação Pré-Clínica de Medicamentos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Virulência/efeitos dos fármacos
18.
Methods Mol Biol ; 2351: 25-39, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382182

RESUMO

Post-transcriptional processing strongly affects the stability and the relative quantification of RNA molecules, so that steady-state levels of mature RNA, such as mRNAs, rarely reflect accurately the rate of in situ transcription in nuclei by RNA polymerases (RNAPs). The "Global Run-on Sequencing (GRO-Seq)" method, developed in 2008, combines the nuclear run-on assay with next-generation deep sequencing to detect nascent RNA levels to annotate the positions, the relative levels and the orientation of transcriptionally engaged RNA polymerase II (RNAPII) molecules genome-wide. Thus, GRO-Seq is a powerful method to infer mechanistic insights into the multiple levels of transcriptional regulation such as promoter-proximal pausing of RNAP, bidirectional transcription, and enhancer activity. Here, we describe a protocol for mammalian cells that can reliably detect low abundant nascent RNA from both coding and noncoding genomic regions. This protocol can easily be adapted for most mammalian cells to define the transcriptionally active regions of the genome and to measure dynamic transcriptional responses with high sensitivity upon external stimuli.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Polimerase II/metabolismo , Análise de Sequência de RNA/métodos , Transcrição Genética , Elementos Facilitadores Genéticos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Controle de Qualidade , RNA/genética , RNA/isolamento & purificação , RNA não Traduzido/genética
19.
Methods Mol Biol ; 2351: 3-22, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382181

RESUMO

Knowledge in gene transcription and chromatin regulation has been intensely studied for decades, but thanks to next-generation sequencing (NGS) techniques there has been a major leap forward in the last few years. Historically, identification of specific enhancer elements has led to the identification of master transcription factors (TFs) in the 1990s. Genetic and biochemical experiments have identified the key regulators controlling RNA polymerase II (RNAPII) transcription and structurally analyses have elucidated detailed mechanisms. NGS and the development of chromatin immunoprecipitation (ChIP) have accelerated the gain of knowledge in the recent years. By now, we have a dazzling wealth of techniques that are currently used to put gene expression into a genome-wide context. This book is an attempt to assemble useful protocols for many researchers within and nearby research areas. In general, these innovative techniques focus on enhancer and promoter studies. The techniques should also be of interest for related fields such as DNA repair and replication.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Genética , Animais , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Metilação de DNA , Epigênese Genética , Histonas/metabolismo , Humanos , Ligação Proteica , RNA Polimerase II/metabolismo
20.
Methods Mol Biol ; 2351: 67-90, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382184

RESUMO

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation of the Mammalian Genome (FANTOM) consortia. These datasets constitute open resource for TSS annotations and gene expression analysis. Here, we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE "LQ-ssCAGE", which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50 ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.


Assuntos
Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Capuzes de RNA , Sítio de Iniciação de Transcrição , Bases de Dados Genéticas , Regulação da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Reprodutibilidade dos Testes , Fluxo de Trabalho
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