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1.
Article in Chinese | WPRIM | ID: wpr-1039065

ABSTRACT

Cells not only contain membrane-bound organelles (MBOs), but also membraneless organelles (MLOs) formed by condensation of many biomacromolecules. Examples include RNA-protein granules such as nucleoli and PML nuclear bodies (PML-NBs) in the nucleus, as well as stress granules and P-bodies in the cytoplasm. Phase separation is the basic organizing principle of the form of the condensates or membraneless organelles (MLOs) of biomacromolecules including proteins and nucleic acids. In particular, liquid-liquid phase separation (LLPS) compartmentalises and concentrates biological macromolecules into liquid condensates. It has been found that phase separation of biomacromolecules requires some typical intrinsic characteristics, such as intrinsically disordered regions, modular domains and multivalent interactions. The phase separation of biomacromolecules plays a key role in many important cell activities. In recent years, the phase separation of biomacromolecules phase has become a focus of research in gene transcriptional regulation. Transcriptional regulatory elements such as RNA polymerases, transcription factors (TFs), and super enhancers (SEs) all play important roles through phase separation. Our group has previously reported for the first time that long-term inactivation or absence of assembly factors leads to the formation of condensates of RNA polymerase II (RNAPII) subunits in the cytoplasm, and this process is reversible, suggesting a novel regulatory model of eukaryotic transcription machinery. The phase separation of biomacromolecules provides a biophysical understanding for the rapid transmission of transcriptional signals by a large number of TFs. Moreover, phase separation during transcriptional regulation is closely related to the occurrence of cancer. For example, the activation of oncogenes is usually associated with the formation of phase separation condensates at the SEs. In this review, the intrinsic characteristics of the formation of biomacromolecules phase separation and the important role of phase separation in transcriptional regulation are reviewed, which will provide reference for understanding basic cell activities and gene regulation in cancer.

2.
Article in Chinese | WPRIM | ID: wpr-1004896

ABSTRACT

GATA-binding protein 1 (GATA1), an important hematopoietic transcription factor, specifically regulates the proliferation and differentiation of erythroid and megakaryoid cells at the transcription level, which maintains the normal development and maturation of these two lineages. The functional structure of GATA1 is composed of one N-terminal transactivation domain (N-TAD) and two zinc fingers (NF and CF). GATA1 is highly conserved in different species. Alteration of GATA1 expression or function will lead to transcriptional disorder of erythrocyte and megakaryocyte related genes, resulting in various clinical phenotypes. This article reviews the molecular structure of GATA1, its transcriptional regulation in erythrocyte and megakaryocyte, and the hereditary hematopoietic regulatory disorders of these two lineages caused by GATA1 mutations.

3.
Chinese Journal of Biotechnology ; (12): 1670-1683, 2023.
Article in Chinese | WPRIM | ID: wpr-981162

ABSTRACT

Overexpression of Krüppel like factor 2 (Klf2) or Klf7 inhibits adipocyte formation. However, it remains unclear whether Klf2 regulates klf7 expression in adipose tissue. In this study, oil red O staining and Western blotting were employed to study the effect of Klf2 overexpression on the differentiation of chicken preadipocytes. The results showed that Klf2 overexpression inhibited the differentiation of chicken preadipocytes induced by oleate and the expression of pparγ, while promoted klf7 expression in chicken preadipocytes. Spearman correlation analysis was used to study the correlation between the expression data of klf2 and klf7 in the adipose tissue of both human and chicken. The results showed that there was a significantly positive correlation between the expression of klf2 and klf7 in adipose tissues (r > 0.1). Luciferase reporter assay showed that overexpression of Klf2 significantly promoted the activity of chicken klf7 promoter (-241/-91, -521/-91, -1 845/-91, -2 286/-91, -1 215/-91; P < 0.05). In addition, the activity of klf7 promoter (-241/-91) reporter in chicken preadipocytes was significantly positively correlated with the amount of klf2 overexpression plasmid transfected (Tau=0.917 66, P=1.074×10-7). Moreover, Klf2 overexpression significantly promoted the mRNA expression of klf7 in chicken preadipocytes (P < 0.05). In conclusion, upregulation of klf7 expression might be one of the pathways that Klf2 inhibits chicken adipocyte differentiation, and the sequence from -241 bp to -91 bp upstream chicken klf7 translation start site might mediate the regulation of Klf2 on klf7 transcription.


Subject(s)
Animals , Humans , Chickens/genetics , Kruppel-Like Transcription Factors/metabolism , Transcription Factors/metabolism , Adipocytes/metabolism , Adipose Tissue/metabolism
4.
Article in Chinese | WPRIM | ID: wpr-1029470

ABSTRACT

Objective:To investigate the mechanism of cyclic AMP receptor protein (CRP) in regulating the siderophore enterobactin-related gene entC of carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods:A mutant strain with crp gene deletion strain (Δ crp) and a complementary strain (c-Δ crp) were constructed using CRKP-27 as the wild-type strain. The influence of CRP on the secretion of siderophore by CRKP was analyzed by chrome azurol sulfonate (CAS) quantitative assay. RT-qPCR and lacZ reporter gene fusion assay were used to detect the regulatory effect of CRP on entC gene expression and its promoter. Electric mobility shift assay (EMSA) was performed to detect the binding of CRP to the entC promoter region and the binding sequence was analyzed by DNase Ⅰ footprinting assay. Results:The Δ crp and c-Δ crp strains were successfully constructed. Compared with the wild-type and c-Δ crp strains, the Δ crp strain could secrete more siderophore under both normal and iron-deficient conditions, but the difference was statistically significant only under normal condition ( P<0.05). The relative expression of entC gene at mRNA level was significantly lower in the Δ crp strain than that in the wild-type and c-Δ crp strains under both normal and iron-deficient conditions (both P<0.05). The promoter of entC gene in the Δ crp strain was less active than that in the wild-type and c-Δ crp strains under both normal and iron-deficient conditions (both P<0.05). EMSA showed that with the increase of CRP protein, the distance of entC probe from the positive pole was shortened and blocked. DNase Ⅰ footprinting assay further identified the specific binding site of the entC promoter region to CRP as 5′-AAGGTGATAAATGCGTCTCATTTTCAA-3′. Conclusions:The CRP protein in CRKP could specifically bind to the entC promoter region and directly promote its expression at transcriptional level.

5.
Chinese Journal of Biologicals ; (12): 1143-1148, 2023.
Article in Chinese | WPRIM | ID: wpr-996608

ABSTRACT

@#Membrane fusion is essential for all life activities of eukaryotes and the fusion process requires the interaction of different vesicular transporters,which are highly conserved in eukaryotes,to coordinate specifically and facilitate the fusion of different biofilms. Vesicle associated membrane protein 8(VAMP8)mainly locates in vesicular as well as lysosomal membranes,and plays a vital role in the fusion of many different biofilms. This paper focused on the molecular structure,transcriptional regulation,post-translational modification and biological function of VAMP8 as well as the research progress on its correlation with human diseases,so as to provide new ideas for the treatment of related diseases and the development of effective therapeutics targeting VAMP8.

6.
Article in Chinese | WPRIM | ID: wpr-1015646

ABSTRACT

Tripartite motif containing protein 7 (TRIM7), as a member of the E3 ubiquitin ligase TRIM family, plays an important regulatory role in immune regulation, metabolism and other physiological processes. The aberrant expression of TRIM7 is closely related to the development and progression of hepatocellular carcinoma (HCC) and it shows a complex regulatory role. However, the regulatory mechanism for the expression of TRIM7 in HCC remains unknown. In this study, multiple online databases were used to analyze the expression of TRIM7 in HCC and data indicated that TRIM7 expression was upregulated in HCC and correlated to poor prognosis. Subsequently, the transcription factor binding sites in the TRIM7 promoter region were analyzed using UCSC and JASPAR databases, and the results showed that TRIM7 promoter contains four SP1 binding sites. In this work, we demonstrated that SP1 could directly bind to its binding sites in TRIM7 promoter and positively regulate the transcriptional activity driven by the TRIM7 promoter using dual luciferase reporter experiments and the ChIP-PCR method. Moreover, our results also showed SP1 overexpression upregulated the expression of TRIM7 at both mRNA and protein levels (P<0. 01),and SP1 inhibitor, mithramycin A, could reverse the activated effect of SP1 on TRIM7 expression (P<0. 01). In conclusion, this study preliminarily reveals the regulatory mechanism of TRIM7 upregulation in HCC, which provides an important theoretical basis for further study of the gene function, early diagnosis and targeted therapy.

7.
Article in English | WPRIM | ID: wpr-929232

ABSTRACT

Natural products (NPs), especially those from traditional herbal medicines, can evidently modulate human gene expression at multiple levels, leading to a wide diversity of bioactivities. Although numerous bio-functions of NPs for human body have been found, there is little understanding about how NPs achieve it, as less attention was drawn to the definite mechnism by which NPs regulate gene expression. Furthermore, based on the rapidly advancing knowledge of mechanisms for gene regulation in recent years, newly-understood mechanisms, such as post-transcriptional regulation, are found to be involved in NP-elicited bio-effects, providing a new perspective on understanding the role of NPs in gene expression. Therefore, in the current review, we summarize the function of NPs in gene expression from the perspectives of transcriptional, post-transcriptional, and post-translational regulation, which will reinforce the understanding of NP-induced effects in gene expression and facilitate the exploration of more NPs with potential therapeutic effects.


Subject(s)
Humans , Biological Products/pharmacology , Gene Expression , Gene Expression Regulation
8.
Chinese Journal of Biotechnology ; (12): 4719-4730, 2022.
Article in Chinese | WPRIM | ID: wpr-970343

ABSTRACT

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.


Subject(s)
Vibrio parahaemolyticus/metabolism , Escherichia coli/metabolism , Bacterial Proteins/metabolism , Transcription Factors/genetics , Gene Expression Regulation, Bacterial
9.
Zhongguo Zhong Yao Za Zhi ; (24): 412-418, 2022.
Article in Chinese | WPRIM | ID: wpr-927983

ABSTRACT

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.


Subject(s)
Amino Acid Sequence , Cloning, Molecular , Geranyltranstransferase/genetics , Pogostemon , Transcription Factors/genetics
10.
Article in Chinese | WPRIM | ID: wpr-1015752

ABSTRACT

Macrophage polarization is a process of phenotypic regulation based on the surroundingstimulus environment, which is generally polarized into two phenotypes, namely classical activated M1macrophages and alternative activated M2 macrophages. In short, M1 macrophages are characterized aspro-inflammatory and anti-tumor; M2 macrophages are anti-inflammatory and pro-tumor. Macrophagepolarization is considered to be a key regulator of human physiology and pathology processes, and itseffectiveness depends on the coordinated expression of key factors, whose expression is finely regulated bymicroRNAs (miRNAs) at the post-transcriptional level. MicroRNAs are small non-coding RNAs that havethe ability to regulate gene expression and cellular network processes. More and more evidence shows thatmiRNAs play an important role in regulating macrophage polarization. Therefore, in this review, miRNAsregulating macrophage polarization to M1 / M2 type and have bidirectional regulation function are listed, and how they regulate macrophage polarization through transcription factors and their potential in thetreatment of inflammation and tumor.

11.
Article in Chinese | WPRIM | ID: wpr-1015759

ABSTRACT

As constitutive cofactors of bacterial RNA polymerase (RNAP), sigma70 and sigma54 areinvolved in the regulation of transcription initiation for different genes in prokaryotic cells, respectively. sigma70 is responsible for the spontaneous transcription initiation of housekeeping genes, while sigma54blocks DNA from entering RNAP by way of steric blockade and inhibits the initiation of gene transcriptionafter forming a complex with RNAP. When the cell environment changes, specific stress signals willinduce conformational changes of sigma54 through the bacterial enhancer binding protein (bEBP), release the inhibition of sigma54 on RNAP, and initiate sigma54-dependent gene transcription. Recentstructural biology studies have revealed the structures of several complexes for sigma54-dependenttranscription initiation, including holoenzyme, closed complex, two intermediate complexes and opencomplex. By analyzing the structure of these transcription initiation complexes, we introduce the structuralchanges of the complex during transcription initiation in this article. The functions of sigma54 and bEBPin the process of transcription initiation are described and analyzed. This article is helpful to understandthe changes of transcription initiation on molecular level, and provides a reference for deep understandingof the molecular mechanism of sigma54 and bEBP in promoting transcription initiation.

12.
Article in Chinese | WPRIM | ID: wpr-1015780

ABSTRACT

AR (androgen receptor) and CCAT2 are two prostate cancer (PCa)-related genes whereas their relationship is not yet reported. AR is the classical major functional gene in PCa progression. CCAT2, a non-coding gene, was identified based on big-data GWAS (Genome-Wide Association Studies) in the year of 2013. Androgen deprivation therapy (ADT) is usually used to treat PCa in the early stage. After persistent androgen deprivation, PCa would generally lead to castration resistant prostate cancer (CRPC), whereas the mechanism is yet unclear. Here we explore the function of AR and CCAT2 in PCa progression, especially their relation in androgen sensitive and insensitive cell model LNCap and DU145. We found a loop between AR and CCAT2 transcription by over-expression and knock-down strategies. In DU145 cells, G-CCAT2 activated AR mRNA level 2. 6 times, while T-CCAT2 inhibited it to 0. 2 times (P<0. 05). In LNCaP cells, G-CCAT2 could activate AR mRNA levels 1. 5 times, and TCCAT2 had no significant effect (P<0. 05). Under overexpression of AR in DU145 cells, the expression of CCAT2 increased 2. 9 times (P < 0. 05). The abundance of CCAT2 decreased to 0. 48 (P < 0. 05) in LNCaP cells by AR knock-down. Reporter gene analysis showed that CCAT2 could function on the AR promoter. We then performed CCK8 assays and AR protein level detection as supplement for the new gene CCAT2 studies. Finally we primarily studied some target genes that are related to AR and CCAT2 . The results showed that the G-CCAT2 transcript could activate AR expression in LNCap cells while UCCAT2 had no significant effect. In DU145 cells, G-CCAT2 exhibited a more relative stronger activation effect on AR, and U-CCAT2 could inhibit AR transcription. AR activates the transcriptional activity of CCAT2 in both cell lines, suggesting a feedback regulation between them. Our data showed that there would be a feedback loop between CCAT2 and AR, which may indicate a new method for PCa treatment.

13.
Biol. Res ; 55: 8-8, 2022. ilus
Article in English | LILACS | ID: biblio-1383912

ABSTRACT

BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.


Subject(s)
Animals , Mice , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Bacterial Outer Membrane Proteins/genetics , Porins/genetics , Porins/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology
14.
Article in Chinese | WPRIM | ID: wpr-931226

ABSTRACT

Suppression of cellular O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) can repress prolifera-tion and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insulin gene transcription,we designed a cell-based fluorescent reporter capable of sensing cellular O-GlcNAcylation in HEK293T cells.The fluorescent reporter mainly consists of a reporter (green fluorescent protein (GFP)),an internal reference (red fluorescent protein),and an operator (neuronal differentiation 1),which serves as a "sweet switch" to control GFP expression in response to cellular O-GlcNAcylation changes.The fluorescent reporter can efficiently sense reduced levels of cellular O-GlcNAcylation in several cell lines.Using the fluorescent reporter,we screened 120 natural products and obtained one compound,sesamin,which could markedly inhibit protein O-GlcNAcylation in HeLa and human colorectal carcinoma-116 cells and repress their migration in vitro.Altogether,the present study demonstrated the development of a novel strategy for anti-tumor drug screening,as well as for con-ducting gene transcription studies.

15.
Chinese Journal of Biotechnology ; (12): 1205-1228, 2021.
Article in Chinese | WPRIM | ID: wpr-878625

ABSTRACT

Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level, including insertion, knockout, replacement and point mutation of specific DNA fragments. The ultimate principle of genome editing technology relying on engineered nucleases is to generate double-stranded DNA breaks at specific locations in genome and then repair them through non-homologous end joining or homologous recombination. With the intensive study of these nucleases, genome editing technology develops rapidly. The most used nucleases include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats associated Cas proteins. Based on introducing the development and principles of above mentioned genome editing technologies, we review the research progress of CRISPR/Cas9 system in the application fields of identification of gene function, establishment of disease model, gene therapy, immunotherapy and its prospect.


Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing , Technology , Transcription Activator-Like Effector Nucleases/metabolism
16.
Article in Chinese | WPRIM | ID: wpr-933996

ABSTRACT

Objective:To study the transcriptional regulation of pilABCD by the master quorum sensing (QS) regulator OpaR in Vibrio parahaemolyticus. Methods:Total RNAs were extracted from the wild type (WT) and opaR mutant (Δ opaR) strain. Quantitative real-time PCR (qPCR) was employed to calculate the transcriptional variation of pilA (the first gene of pilABCD operon) between WT and Δ opaR. The regulatory DNA region of pilABCD was cloned into the corresponding restriction endonuclease sites of pHRP309 harboring a promoterless lacZ reporter gene. The recombinant pHRP309 plasmid was then transferred into WT and Δ opaR, respectively, to detect the β-galactosidase activity in cellular extracts using a β-Galactosidase Enzyme Assay System (Promega). The primer extension assay was applied to map the transcription start site of pilABCD using the total RNAs extracted from the WT strain as the template. The regulatory DNA region of pilABCD was amplified by PCR, and the over-expressed His-OpaR was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Thereafter, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-OpaR to the target DNA in vitro, and the DNase I footprinting assay was further employed to detect the DNA-binding sites of His-OpaR within the target DNA. Results:The results of qPCR and LacZ fusion assays showed that OpaR activated the transcription of pilABCD, leading to a gradual increase in the expression level of pilA with the extension of culture time. The primer extension assay detected only one transcription start site located at 155 bp upstream of pilA. The results of EMSA and DNase Ⅰ footprinting assays showed that His-OpaR protected two DNA regions located from -246 to -197 bp and -181 to -131 bp upstream of pilA. Conclusions:Vibrio parahaemolyticus OpaR activated the transcription of pilABCD in a direct manner.

17.
J Biosci ; 2020 Jan; : 1-7
Article | IMSEAR | ID: sea-214354

ABSTRACT

The nucleosome presents a formidable barrier to DNA-templated transcription by the RNA polymerase IImachinery. Overcoming this transcriptional barrier in a locus-specific manner requires sequence-specificrecognition of nucleosomal DNA by ‘pioneer’ transcription factors (TFs). Cell fate decisions, in turn, dependon the coordinated action of pioneer TFs at cell lineage-specific gene regulatory elements. Although it isalready appreciated that pioneer factors play a critical role in cell differentiation, our understanding of thestructural and biochemical mechanisms by which they act is still rapidly expanding. Recent research hasrevealed novel insight into modes of nucleosome-TF binding and uncovered kinetic principles by whichnucleosomal DNA compaction affects both TF binding and residence time. Here, we review progress and arguethat these structural and kinetic studies suggest new models of gene regulation by pioneer TFs.

18.
Electron. j. biotechnol ; Electron. j. biotechnol;43: 55-61, Jan. 2020. tab, ilus, graf
Article in English | LILACS | ID: biblio-1087522

ABSTRACT

Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.


Subject(s)
Humans , Matrix Metalloproteinase 12/genetics , CRISPR-Cas Systems , Luciferases/genetics , Transcription, Genetic , Cell Communication , Cell Line , Promoter Regions, Genetic/genetics , Cell Culture Techniques , Extracellular Matrix , Gene Knock-In Techniques , Clustered Regularly Interspaced Short Palindromic Repeats
19.
Chinese Journal of Biotechnology ; (12): 879-890, 2020.
Article in Chinese | WPRIM | ID: wpr-826888

ABSTRACT

Human parvovirus B19 (B19 virus) is one of the two parvoviruses that cause human diseases. As an important pathogen to humans, it causes infectious erythema in children, acute aplastic anemia, fetal edema and death. In this review, we focus on the recent advances in the molecular virology of B19V, such as viral genotypes, viral receptor, genomic features and viral replication, viral transcription and post-transcription regulation, viral nonstructural and structural protein features and functions, viral diagnosis and antiviral agents, to provide reference for further study of B19 pathogenesis mechanisms, treatment and diagnostic strategies.


Subject(s)
Humans , Antiviral Agents , DNA, Viral , Genetics , Erythema Infectiosum , Diagnosis , Virology , Genotype , Parvovirus B19, Human , Genetics , Virology , Virus Replication
20.
Article in Chinese | WPRIM | ID: wpr-848145

ABSTRACT

BACKGROUND: Since non-coding RNAs maintain bone homeostasis through various pathways, applications of non-coding RNAs as bioactive molecules in bone tissue engineering for bone defect repair has become an increasing area of interest. OBJECTIVE: To introduce non-coding RNAs as bioactive molecules in bone tissue engineering. METHODS: A computer-based online search of Web of Science, PubMed, SpringerLink databases was performed by the first author between December 2018 and March 2019 using the search terms “bone tissue engineering, ncRNA (miRNA, siRNA or lncRNA), scaffold, drug delivery system” to retrieve papers published during 2004-2019. A total of 1754 papers were preliminarily retrieved, and 95 of them were eligible for final analysis. RESULTS AND CONCLUSION: Because non-coding RNAs play a key role in osteogenic differentiation, they can be used as important bioactive factors for bone tissue engineering. At present, bone tissue engineering repair methods based on non-coding RNA bioactive factors have become a research hotspot in bone defect repair. There are two major application strategies: (1) The non-coding RNA transcription within the seed cells is purposefully altered and combines with the bone tissue-engineered scaffold to promote bone defect repair. (2) a specifically designed bone engineered scaffold can controllably and purposefully alter the expression of non-coding RNA in the seed cells, which promotes bone defect repair. In addition, the function of more and more non-coding RNAs has been identified in the process of bone regeneration. This shows good application prospects of non-coding RNAs.

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