Mechanism for synergistic effect of IRF4 and MITF on tyrosinase promoter / 中南大学学报(医学版)

Objective:To investigate the mechanism for the synergistic effect of interferon regulatory factor 4 (IRF4) and microphthalmia-associated transcription factor (MITF) on tyrosinase (TYR)promoter.Methods:The synergistic transcriptional effect,subcellular localization,and protein-protein interaction for IRF4 and MITF were observed by luciferase assay,immunofluorescence,GST-pull down,and co-immunoprecipitation,respectively.Results:IRF4 and MITF proteins were co-expressed in the cell nucleus.IRF4 augmented the transcriptional function of MITF (but not the mutant MITF) to activate the expression of the TYR promoter,but with no effect on other MITF-specific target promoters.IRF4 alone did not affect TYR promoter significantly.No direct interaction between the two proteins was noted.Conclusion:IRF4 and MITF exert a specifically synergistic effect on activation of TYR promoter through IRF4-mediated upregulation of transcriptional function of MITF.This synergistic effect is mainly regulated by MITF;DNA might be involved in the interaction between the two proteins.

Light-inducible CRISPR/Cas9 system for control of gene expression:research advances / 国际药学研究杂志

Cas9 is a RNA-guided double stranded DNA nuclease that participates in the CRISPR/Cas9 system. Wide-type Cas9 directly silences the expression of target gene by gene splicing. The engineered dCas9 protein with the mutation at D10A and H840A lacks the Cas9' s endonuclease function but keeps its DNA binding activity. dCas9 can activate special genes by fusing with transcription activator. Meanwhile,it can inhibit the gene transcription by directly binding to the target gene and stop gene transcrip?tion. Combination of light sensitive structures and CRISPR can produce light-inducible CRISPR/Cas9 system for control of gene expres?sion. This system is able to activate or inhibit gene expression via the use of controlling blue light(470 nm). In this review,we mainly discuss the development of the light inducible CRISPR/Cas9 system as well as its application in the control of gene expression.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

The complexity of ToxT-dependent transcription in Vibrio cholerae.

Vibrio cholerae is the causative agent of the disease cholera, characterized by profuse watery diarrhoea. Two of the main virulence factors associated with the disease are cholera toxin (CT) and toxin-coregulated pilus (TCP). Expression of CT and TCP is regulated via a complex cascade of factors that respond to environmental signals, but ultimately ToxT is the direct transcriptional activator of the genes encoding CT and TCP. Recent studies have begun to unveil the mechanisms behind ToxT-dependent transcription. We review current knowledge of transcriptional activation by ToxT and the environmental stimuli that allow ToxT to regulate virulence gene expression, resulting in cholera pathogenesis.

The relation of forkhead box gene and growth / 国际儿科学杂志

Forkhead box(Fox) proteins characterized by a conserved winged-helix DNA binding domain constitute a large family of transcription factors.Fox proteins regulate the gene transcription not only by recruiting various coactivators like most transcription factors,but also by binding to the condensed chromosomes directly,which can remodel chromatin structure and generate a combinatorial effect with other transcription factors to cause significant changes in expression.The diverse biological processes that Fox proteins influence,such as embryonic development,aging and immunoregulation,have also been covered in this article.

Isolation of a Rice WRKY Gene OsWRKY52,Whose Expression Is Induced by Magnaporthe grisea / 生物化学与生物物理进展

WRKY proteins, a big family of transcription factors, are involved in regulation diverse developmental and physiological processes in plants. Here, a novel WRKY gene, OsWRKY52, was isolated from a rice cDNA library. This gene included an open reading frame of 1 719 bp in length, and the deduced polypeptide contained 572 amino acids,sharing 54% identity with a WRKY1 protein from Avena sativa. Expression of OsWRKY52 gene was induced rapidly by Magnaporthe grisea in the incompatible interaction with rice plant. OsWRKY52 protein, expressed prokaryotically bound specifically to W box cis elements derived from the promoter of a rice PR1a. Transcriptional activation assay was performed by a yeast one- hybrid method. Regions of transactivation were identified to be the N-terminal serine- and threonine-rich islands and the C-terminal acidic domain of OsWRKY52. These results suggest that OsWRKY52, as a transcription activator, may be involved in defense responses against Magnaporthe grisea in rice plants.

Clone and expression of nuclear factor ?B p65 TAD / 第三军医大学学报

Objective To acquire NF-?B p65 TAD(transcription activation domain) and construct its eukaryotic expression vector and express it in endothelial cells.Methods Human umbilical vein endothelial cells(HUVECs) were cultured and total RNA was extracted.The p65 TAD gene was amplified by RT-PCR.After sequencing,the p65 TAD gene was inserted into the eukaryotic expression vector pEGFP-N1 with the green fluorescence protein,named pEGFP-N1-p65 TAD.pEGFP-N1-p65 TAD was transfected into HUVECs and its expression was observed under fluorescence microscope and analyzed by Western blotting.Results p65 TAD(288-548) was cloned successfully.The constructed plasmid including p65 TAD gene was identical to the designed.p65 TAD gene was expressed successfully in HUVECs.Conclusion The construction of eukaryotic expression vector including p65 TAD gene and its expression in HUVECs are very successful.