RESUMO
Liver fibrosis is caused by chronic liver damage and results in the aberrant accumulation of extracellular matrix during disease progression. Despite the identification of the HAT enzyme p300 as a major factor for liver fibrosis, the development of therapeutic agents targeting the regulation of p300 has not been reported. We validated a novel p300 inhibitor (A6) on the improvement of liver fibrosis using two mouse models, mice on a choline-deficient high-fat diet and thioacetamide-treated mice. We demonstrated that pathological hall-marks of liver fibrosis were significantly diminished by A6 treatment through Masson's trichrome and Sirius red staining on liver tissue and found that A6 treatment reduced the expression of matricellular protein genes. We further showed that A6 treatment improved liver fibrosis by reducing the stability of p300 protein via disruption of p300 binding to AKT. Our findings suggest that targeting p300 through the specific inhibitor A6 has potential as a major therapeutic avenue for treating liver fibrosis. [BMB Reports 2023; 56(2): 114-119].
Assuntos
Histonas , Cirrose Hepática , Camundongos , Animais , Histonas/metabolismo , Fígado/metabolismo , Modelos Animais de Doenças , Dieta HiperlipídicaRESUMO
Forkhead box protein M1 (FOXM1) is a key transcription factor (TF) that regulates a common set of genes related to the cell cycle in various cell types. However, the mechanism by which FOXM1 controls the common gene set in different cellular contexts is unclear. In this study, a comprehensive meta-analysis of genome-wide FOXM1 binding sites in ECC-1, GM12878, K562, MCF-7, and SK-N-SH cell lines was conducted to predict FOXM1-driven gene regulation. Consistent with previous studies, different TF binding motifs were identified at FOXM1 binding sites, while the NFY binding motif was found at 81% of common FOXM1 binding sites in promoters of cell cycle-related genes. The results indicated that FOXM1 might control the gene set through interaction with the NFY proteins, while cell type-specific genes were predicted to be regulated by enhancers with FOXM1 and cell type-specific TFs. We also found that the high expression level of FOXM1 was significantly associated with poor prognosis in nine types of cancer. Overall, these results suggest that FOXM1 is predicted to function as a master regulator of the cell cycle through the interaction of NFY-family proteins, and therefore the inhibition of FOXM1 could be an attractive strategy for cancer therapy.
Assuntos
Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Redes Reguladoras de Genes , Neoplasias/genética , Sítios de Ligação , Ciclo Celular , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação , Regulação Neoplásica da Expressão Gênica , Humanos , Células K562 , Células MCF-7 , Neoplasias/metabolismo , Prognóstico , Regulação para Cima , Sequenciamento Completo do GenomaRESUMO
Next-generation sequencing (NGS) techniques have been used to generate various molecular maps including genomes, epigenomes, and transcriptomes. Transcriptomes from a given cell population can be profiled via RNA-seq. However, there is no simple way to assess the characteristics of RNA-seq data systematically. In this study, we provide a simple method that can intuitively evaluate RNA-seq data using two different principal component analysis (PCA) plots. The gene expression PCA plot provides insights into the association between samples, while the transcript integrity number (TIN) score plot provides a quality map of given RNA-seq data. With this approach, we found that RNA-seq datasets deposited in public repositories often contain a few low-quality RNA-seq data that can lead to misinterpretations. The effect of sampling errors for differentially expressed gene (DEG) analysis was evaluated with ten RNA-seq data from invasive ductal carcinoma tissues and three RNA-seq data from adjacent normal tissues taken from a Korean breast cancer patient. The evaluation demonstrated that sampling errors, which select samples that do not represent a given population, can lead to different interpretations when conducting the DEG analysis. Therefore, the proposed approach can be used to avoid sampling errors prior to RNA-seq data analysis.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Análise de Componente Principal , RNA/genética , Transcriptoma/genética , Expressão Gênica , Humanos , Análise de Sequência de RNARESUMO
Although GPR64 has an important role for male fertility, its physiological roles in the female reproductive system are still unknown. In the present study, immunohistochemical analysis reveals a spatiotemporal expression of GPR64 in the uterus during early pregnancy. Observation of remarkable induction of GPR64 expression in uterine decidual cells points to its potential physiological significance on decidualization. The decidualization of uterine stromal cells is a key event in implantation. Progesterone (P4) signaling is crucial for the decidualization of the endometrial stromal cells for successful pregnancy. Therefore, we examined ovarian steroid hormone regulation of GPR64 expression in the murine uterus. P4 induced GPR64 expression in the epithelial and stromal cells of the uterus in ovariectomized wild-type mice, but not in PRKO mice. ChIP analysis confirmed that PGR proteins were recruited on progesterone response element of Gpr64 gene in the uteri of wild-type mice treated with P4. Furthermore, the expression of GPR64 was increased in human endometrial stromal cells (hESCs) during in vitro decidualization. Interestingly, small interfering RNA (siRNA)-mediated knockdown of GPR64 in hESCs remarkably reduced decidualization. These results suggest that Gpr64 has a crucial role in the decidualization of endometrial stromal cells.
Assuntos
Decídua/citologia , Endométrio/citologia , Progesterona/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Decídua/efeitos dos fármacos , Decídua/metabolismo , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Feminino , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos , Gravidez , Receptores Acoplados a Proteínas G/genética , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Regulação para CimaRESUMO
Cell lines are often used as in vitro tools to mimic certain types of in vivo system; several cell lines, including MCF-7 and T47D, have been widely used in breast cancer studies without investigating the cell lines' characteristics. In this study, we compared the genome-wide binding profiles of ERα, PR, and P300, and the gene expression changes between MCF-7 and T47D cell lines that represent the luminal A subtype of breast cancer. Surprisingly, several thousand genes were differentially expressed under estrogenic condition. In addition, ERα, PR, and P300 binding to regulatory elements showed distinct genomic landscapes between MCF-7 and T47D cell lines in the same hormonal states. In particular, the T47D cell line was markedly susceptible to progesterone, whereas the MCF-7 cell line did not respond to progesterone in the presence of estrogen. Consistently, changes in the expression level of the PR-target gene, STAT5A, were only observed in the T47D cell line, not the MCF-7 cell line, when treated with progesterone. Overall, the results highlight the importance of selecting appropriate cell lines for breast cancer studies and suggest that T47D cell lines can be an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer.
Assuntos
Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Progesterona/farmacologia , Transcriptoma , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Células MCF-7 , Modelos Biológicos , Especificidade de Órgãos , Ligação Proteica , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The imprinting and transcription of the 500 kb genomic region surrounding the mouse Peg3 is predicted to be regulated by the Peg3-differentially methylated region (DMR). In the current study, this prediction was tested using a mutant mouse line lacking this potential imprinting control region (ICR). At the organismal level, paternal and maternal transmission of this knockout (KO) allele caused either reduced or increased growth rates in the mouse, respectively. In terms of the imprinting control, the paternal transmission of the KO allele resulted in bi-allelic expression of the normally maternally expressed Zim2, whereas the maternal transmission switched the transcriptionally dominant allele for Zfp264 (paternal to maternal). However, the allele-specific DNA methylation patterns of the DMRs of Peg3, Zim2 and Zim3 were not affected in the mice that inherited the KO allele either paternally or maternally. In terms of the transcriptional control, the paternal transmission caused a dramatic down-regulation in Peg3 expression, but overall up-regulation in the other nearby imprinted genes. Taken together, deletion of the Peg3-DMR caused global changes in the imprinting and transcription of the Peg3 domain, confirming that the Peg3-DMR is an ICR for this imprinted domain.
Assuntos
Metilação de DNA , Impressão Genômica , Fatores de Transcrição Kruppel-Like/genética , Alelos , Animais , Animais Recém-Nascidos , Sítios de Ligação/genética , Western Blotting , Encéfalo/metabolismo , Cruzamentos Genéticos , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dedos de Zinco/genéticaRESUMO
Zfp42/Rex1 (reduced expression gene 1) is a well-known stem-cell marker that has been duplicated from YY1 in the eutherian lineage. In the current study, we characterized the in vivo roles of Rex1 using a mutant mouse line disrupting its transcription. In contrast to the ubiquitous expression of YY1, Rex1 is expressed only during spermatogenesis and early embryogenesis and also in a very limited area of the placenta. Yet, the gene dosage of Rex1 is very critical for the survival of the late-stage embryos and neonates. This delayed phenotypic consequence suggests potential roles for Rex1 in establishing and maintaining unknown epigenetic modifications. Consistently, Rex1-null blastocysts display hypermethylation in the differentially methylated regions (DMRs) of Peg3 and Gnas imprinted domains, which are known to contain YY1 binding sites. Further analyses confirmed in vivo binding of Rex1 only to the unmethylated allele of these two regions. Thus, Rex1 may function as a protector for these DMRs against DNA methylation. Overall, the functional connection of Rex1 to genomic imprinting represents another case where newly made genes have co-evolved with lineage-specific phenomena.
Assuntos
Alelos , Blastocisto/metabolismo , Metilação de DNA/fisiologia , Impressão Genômica/fisiologia , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Mutantes , Fatores de Transcrição/genéticaRESUMO
To develop a cell-based assay to screen for human dopamine D(1) receptor agonists or antagonists from medicinal plant extracts, a stable Chinese hamster ovary (CHO) cell line (CHO-D1R) expressing the human dopamine D(1) receptor was established using an expression vector containing a scaffold attachment region (SAR) element. CHO-D1R cells showed specific binding to [(3)H]-SCH23390 with high affinity (K(d)=1.47+/-0.17 nM) and dose-dependent responses for the dopamine-mediated stimulation of cAMP concentrations (EC(50)=20.6+/-1.44 nM). The screening of medicinal plant extracts using cell-based cAMP assays revealed that an extract of Gleditsia sinensis Lam., which is known to be rich in saponin, had strong antagonist activity for the D(1) receptor. From the activity-guided fractionation and chemical structural analysis of the G. sinensis extract, a compound called gleditsioside F was isolated and was identified to have antagonist activity for the D(1) receptor. Gleditsioside F showed very effective D(1) antagonist activity by inhibiting ligand binding to the D(1) receptor as well as by inhibiting dopamine-mediated increases in cAMP concentration.
Assuntos
Bioensaio/métodos , Antagonistas de Dopamina/isolamento & purificação , Antagonistas de Dopamina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Receptores de Dopamina D1/antagonistas & inibidores , Animais , Produtos Biológicos/análise , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Células CHO , Cricetinae , Cricetulus , Antagonistas de Dopamina/química , Gleditsia/química , Humanos , Ligantes , Receptores de Dopamina D1/metabolismo , Saponinas/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Hepatocyte growth factor (HGF) is a potent multi-functional protein that affects morphogenesis, cell migration, organ regeneration, and tumor invasion in various tissues, and has thus been considered to have potential as a therapeutic target in various diseases. In our current study, we established Chinese hamster ovary (CHO) cells overexpressing recombinant human HGF (rhHGF) protein and in a 5 day batch culture process using a 7.5l bioreactor (5l working volume) and serum-free medium these cells could produce over 13 mg/l of rhHGF protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in about a 35% recovery rate. This purified rhHGF was found to be a mixture of inactive pro-HGF and an active heterodimeric form of this protein with a higher molecular weight than its counterpart expressed from insect cells. This finding suggests that the glycosylation of rhHGF protein in CHO cells differs from that in insect cells. Inactive pro-HGF was found to rapidly convert to the active heterodimeric form of HGF in the presence of FBS (Fetal Bovine Serum), suggesting that this process would occur also when injected into human body. We further demonstrate in cell proliferation and scattering activity assays that our purified rhHGF protein preparation is functionally active with a half-maximal effective concentration of 36.3 ng/ml.
Assuntos
Fator de Crescimento de Hepatócito/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Cricetinae , CricetulusRESUMO
BACKGROUND: The transcription factor Yin Yang 1 (YY1) is a ubiquitously expressed, multifunctional protein that controls a large number of genes and biological processes in vertebrates. As a general transcription factor, the proper levels of YY1 protein need to be maintained for the normal function of cells and organisms. However, the mechanism for the YY1 homeostasis is currently unknown. RESULTS: The current study reports that the YY1 gene locus of all vertebrates contains a cluster of its own DNA-binding sites within the 1st intron. The intact structure of these DNA-binding sites is absolutely necessary for transcriptional activity of the YY1 promoter. In an inducible cell line system that over-expresses an exogenous YY1 gene, the overall increased levels of YY1 protein caused a reduction in transcription levels of the endogenous YY1 gene. Reversion to the normal levels of YY1 protein restored the transcriptional levels of the endogenous YY1 to normal levels. This homeostatic response was also mediated through its cluster of YY1 binding sites. CONCLUSION: Taken together, the transcriptional level of YY1 is self-regulated through its internal DNA-binding sites. This study identifies YY1 as the first known autoregulating transcription factor in mammalian genomes.
Assuntos
Ativação Transcricional , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Evolução Molecular , Homeostase , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Ligação ProteicaRESUMO
In this study, we identified an antisense transcript to ZIM2 (zinc finger imprinted gene 2) in the human, called ZIM2as. Sequence analysis of the 110 kb region spanned by this transcript revealed a cluster of tandemly repeated sequence in the human, orangutan, and chimpanzee as well as a loss of approximately 70 kb from the corresponding region in the rhesus. The homologous region in most mammals contains a cluster of olfactory receptor (OLFR) genes, but this gene cluster has been lost from the primate lineage. Expression analyses confirmed that ZIM2as is expressed in the human brain and testis. Two CpG islands near the promoter region of ZIM2as showed different methylation patterns in these three species. The CpG island distal to ZIM2as showed an allele-specific DNA methylation pattern in the human testis, while the CpG island proximal to the ZIM2as promoter showed a mosaic methylation pattern in the chimpanzee. The methylation status of several nearby zinc finger genes was unchanged among the primates tested. Overall, this study reports the presence of a previously unreported primate-specific antisense transcript in the PEG3 imprinted domain, suggesting that the formation of this transcript may coincide with the loss of the OLFR cluster.
Assuntos
Fatores de Transcrição Kruppel-Like/genética , Primatas/genética , RNA Antissenso/isolamento & purificação , Animais , Clonagem Molecular , Deleção de Genes , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Macaca mulatta/genética , Masculino , Pan troglodytes/genética , Filogenia , Pongo pygmaeus/genética , RNA Antissenso/genética , Receptores Odorantes/genéticaRESUMO
BACKGROUND: Two evolutionarily Conserved Sequence Elements, CSE1 and CSE2 (YY1 binding sites), are found within the 3.8-kb CpG island surrounding the bidirectional promoter of two imprinted genes, Peg3 (Paternally expressed gene 3) and Usp29 (Ubiquitin-specific protease 29). This CpG island is a likely ICR (Imprinting Control Region) that controls transcription of the 500-kb genomic region of the Peg3 imprinted domain. RESULTS: The current study investigated the functional roles of CSE1 and CSE2 in the transcriptional control of the two genes, Peg3 and Usp29, using cell line-based promoter assays. The mutation of 6 YY1 binding sites (CSE2) reduced the transcriptional activity of the bidirectional promoter in the Peg3 direction in an orientation-dependent manner, suggesting an activator role for CSE2 (YY1 binding sites). However, the activity in the Usp29 direction was not detectable regardless of the presence/absence of YY1 binding sites. In contrast, mutation of CSE1 increased the transcriptional activity of the promoter in both the Peg3 and Usp29 directions, suggesting a potential repressor role for CSE1. The observed repression by CSE1 was also orientation-dependent. Serial mutational analyses further narrowed down two separate 6-bp-long regions within the 42-bp-long CSE1 which are individually responsible for the repression of Peg3 and Usp29. CONCLUSION: CSE2 (YY1 binding sites) functions as an activator for Peg3 transcription, while CSE1 acts as a repressor for the transcription of both Peg3 and Usp29.
Assuntos
Sequência Conservada , Endopeptidases/metabolismo , Evolução Molecular , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Células 3T3 , Animais , Sequência de Bases/genética , Sítios de Ligação , Sequência Conservada/genética , Elementos Facilitadores Genéticos , Ordem dos Genes , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Ativação Transcricional/genética , Proteases Específicas de Ubiquitina , Fator de Transcrição YY1/metabolismoRESUMO
Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. VEGF165 is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human VEGF165 (rhVEGF165) protein. The production rate of the established CHO cells was over 80 mg/l of rhVEGF165 protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The rhVEGF165 protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified rhVEGF165 protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the rhVEGF165 protein in CHO cells differed from that in insect cells. The purified rhVEGF165 protein in this study was functionally active with a half-maximal effective concentration of 3.8 ng/ ml and specific activity of 2.5 x 105 U/mg.
Assuntos
Biotecnologia/métodos , Células CHO/metabolismo , Proteínas Recombinantes/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Reatores Biológicos , Células Cultivadas , Cricetinae , Cricetulus , Meios de Cultura , Células Endoteliais , Endotélio Vascular/citologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
We describe the development of an efficient expression system suitable for the stable expression of recombinant genes in Chinese hamster ovary (CHO) cells using the human interferon beta SAR element. The insertion of two copies of the human interferon beta SAR element at the 5' and 3' flanking regions of the beta-galactosidase reporter gene increased the frequency of beta-galactosidase positive colonies by up to 75% and enhanced beta-galactosidase expression by 15- to 20-fold after G418 selection or 30- to 40-fold at the initial stage of the MTX selection procedure. Deletion analysis showed that the whole DNA regions of the human interferon beta SAR element are required for beta-galactosidase expression enhancement. The developed expression system was also highly effective at enhancing the stable expression of two therapeutically important proteins, namely, erythropoietin (EPO) and hepatocyte growth factor (HGF). We isolated stable colonies with expression levels of 47 microg/10(6) cells/day for EPO and 13 microg/10(6) cells/day for HGF, suggesting that the developed expression system based on the human beta SAR element is suitable for expressing high levels of recombinant proteins in CHO cells.