Overexpression of chicken Klf2 promotes klf7 transcription and inhibits adipocyte differentiation / 生物工程学报

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.

Hematopoietic regulation of transcription factor GATA1 and mutations causing hereditary erythroid and megakaryocytic disorders / 中国输血杂志

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.

Research progress on vesicle-associated membrane protein 8 / 中国生物制品学杂志

@#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.

ToxR represses the synthesis of c-di-GMP in Vibrio parahaemolyticus / 生物工程学报

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.

Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress

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.

Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter / 中国中药杂志

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.

Advances in the role of natural products in human gene expression / 中国天然药物

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.

Transcriptional regulation of pilABCD by OpaR in Vibrio parahaemolyticus / 中华微生物学和免疫学杂志

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.

A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs / 药物分析学报

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.

CRISPR/Cas9 technology in disease research and therapy: a review / 生物工程学报

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.

Picking a nucleosome lock: Sequence- and structure-specific recognition of the nucleosome

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.

The luciferase reporter system of the MMP12 endogenous promoter for investigating transcriptional regulation of the human MMP12 gene

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.

Application and research of non-coding RNA in bone tissue engineering with cells and scaffold / 中国组织工程研究

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.

Advances in molecular biology research on human parvovirus B19 / 生物工程学报

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.

PXR: a center of transcriptional regulation in cancer

Pregnane X receptor (PXR, NR1I2) is a prototypical member of the nuclear receptor superfamily. PXR can be activated by both endobiotics and xenobiotics. As a key xenobiotic receptor, the cellular function of PXR is mostly exerted by its binding to the regulatory gene sequences in a ligand-dependent manner. Classical downstream target genes of PXR participate in xenobiotic responses, such as detoxification, metabolism and inflammation. Emerging evidence also implicates PXR signaling in the processes of apoptosis, cell cycle arrest, proliferation, angiogenesis and oxidative stress, which are closely related to cancer. Here, we discussed, in addition to the characterization of PXR , the biological function and regulatory mechanism of PXR signaling in cancer, and its potential for the targeted prevention and therapeutics.

Research progress on effect of AP2/ERF transcription factors in regulating secondary metabolite biosynthesis / 中国中药杂志

AP2/ERF transcription factor is a kind of transcription factors widely existing in plants, and contains at least a conserved AP2/ERF domain composed of about 60-70 amino acids. AP2/ERF transcription factors are widely involved in a variety of physiological processes in plants, including plant development, fruit ripening, flower development and other plant development processes, as well as such stress response processes as damage, pathogen defense, high-salt condition and drought. In recent years, secondary metabolic engineering that takes transcription factors as genetic manipulation targets has developed rapidly in improving the content of active ingredients and the quality of medicinal plants. This paper reviews the recent progress in the regulation of secondary metabolites biosynthesis with AP2/ERF transcription factors, and provides theoretical basis for the exploration of efficient regulatory targets, the regulation of secondary metabolites in medicinal plants, the targeted improvement of the content of active ingredients in traditional Chinese medicine, and the sustainable supply of high-quality traditional Chinese medicines.

MicroRNA-383-5p inhibits the progression of gastric carcinoma via targeting HDAC9 expression

MicroRNAs (miRNAs), as post-transcriptional regulators, have been reported to be involved in the initiation and progression of various types of cancer, including gastric cancer (GC). The present study aimed to investigate the role of miR-383-5p in gastric carcinogenesis. Cell viability was analyzed using CCK-8 kit. Annexin V-fluorescein isothiocyanate/propidium iodide double staining was used to evaluate cell apoptosis. The expression levels of miR-383-5p and histone deacetylase 9 (HDAC9) mRNA in GC tissues and cell lines were analyzed using RT-qPCR. The protein expression of HDAC9 was detected by western blotting. We found that HDAC9 was up-regulated and miR-383-5p was down-regulated in GC tissues and cell lines. High HDAC9 expression or low miR-383-5p expression was closely related to poor prognosis and metastasis in GC patients. HDAC9 knockout or miR-383-5p mimics led to growth inhibition and increased apoptosis in AGS and SGC-7901 cells. More importantly, we validated that miR-383-5p as a post-transcriptional regulator inhibited HDAC9 expression and was inversely correlated with HDAC9 expression in GC tissues. miR-383-5p had the opposite effects to HDAC9 in gastric carcinogenesis. miR-383-5p played an important role in gastric carcinogenesis, and it is one of the important mechanisms to regulate oncogenic HDAC9 in GC, which might be helpful in the development of novel therapeutic strategies for the treatment of GC.

Research progress in oxidative stress induced by antibiotics in bacteria / 中华微生物学和免疫学杂志

Antibiotics can not only combine with specific targets, but also induce a cascade of re-active oxygen species ( ROS) in bacteria. Antioxidant defense system plays an important role in scavenging ROS and repairing injury against oxidative stress. However, the mechanism of ROS accumulation in bacteria and the action of ROS are very complicated. Therefore, this paper reviewed the mechanisms of bacterial re-sponding to oxidative stress in the existence of antibiotics. In addition, we analyzed the main problems lying in the current study and the issues needing further consideration.

Nkx-2.5 Regulates MDR1 Expression via Its Upstream Promoter in Breast Cancer Cells

BACKGROUND: Increased expression of MDR1 gene is one of the major mechanisms responsible for multidrug resistance in cancer cells. Two alternative promoters, upstream and downstream, are responsible for transcription of MDR1 gene in the human. However, the molecular mechanism regarding the transactivation of MDR1 upstream promoter (USP) has not been determined. METHODS: Dual-luciferase reporter gene assays were used to assess the effect of Nkx-2.5 on MDR1 USP activity using reporter plasmids for human MDR1 USP and its mutants. MDR1 mRNA level was examined by quantitative real-time PCR. The direct binding of Nkx-2.5 to the USP of MDR1 was evaluated by promoter enzyme immunoassays and chromatin immunoprecipitation assays.

Roles of TRIM28 in tumorigenesis and embryogenesis / 上海交通大学学报（医学版）

TRIM28, known as a kind of macro-molecules, consists of multiple domains and belongs to one of human tripartite motif-containing proteins families. This superfamily contains more than 60 proteins in total and is characterized by four conservative domains, also referred to the RBCC domain, which is a RING (really interesting new gene) finger, two B-boxes (type 1 and type 2) and a leucine zipper coiled-coil region. TRIM28 plays its pivotal roles in transcriptional co-activation or co-repression by interacting directly with transcription factors, within which is a Kruppel-associated box domain (KRAB domain). Moreover, it is critical during the development of embryos, the differentiation of cells and the regulation of cancers.