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Robust transcriptional activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems

Molecular Plant 2018年第2期11卷 245-256页

作者： Levi G. Lowder Jianping Zhou Yingxiao Zhang Aimee Malzahn Zhaohui Zhong Tzung-Fu Hsieh Daniel F. Voytas Yong Zhang Yiping Qi Department of Biology East Carolina University Greenville NC 27858 USA Department of Biotechnology School of Life Science and Technology Center for informational Biology University of Electronic Science and Technology of ChinaChengdu 610054 China Department of Plant Science and Landscape Architecture University of Maryland College Park MD 20742 USA Department of Plant and Microbial Biology and Plants for Human Health Institute North Carolina State University North Carolina Research Campus KannapolisNC 28O81 USA Department of Genetics Ceil Biology and Development Center for Genome Engineering University of Minnesota Minneapolis MN 55455 USA Institute for Bioscience and Biotechnology Research University of Maryland Rockville MD 20850 USA

User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR- associated protein 9 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCasg-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 （designated CRISPR-Act2.0） yielded stronger transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector activation （mTALE-Act） system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.

关键词： CRISPR gRNA2.0 MS2-VP64 TALE-VP64 multiplex transcriptional activation

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Wheat kinase TaSnRK2.4 forms a functional module with phosphatase TaPP2C01 and transcription factor TaABF2 to regulate drought response

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The Crop Journal 2024年第2期12卷 384-400页

作者： Yanyang Zhang Xiaoyang Hou Tianjiao Li Ziyi Wang Jiaqi Zhang Chunlin Zhang Xianchang Liu Xinxin Shi Wanrong Duan Kai Xiao State Key Laboratory of North China Crop Improvement and Regulation Baoding 071001HebeiChina College of Agronomy Hebei Agricultural UniversityBaoding 071001HebeiChina

SNF1-related protein kinase 2(SnRK2)family members are essential components of the plant abscisic acid(ABA)signaling pathway initiated by osmotic stress and triggering a drought stress *** study characterized the molecular properties of TaSnRK2.4 and its function in mediating adaptation to drought in Triticum *** of TaSnRK2.4 were upregulated upon drought and ABA signaling and associated with drought-and ABA-responsive cis-elements ABRE and DRE,and MYB and MYC binding sites in the promoter as indicated by reporter GUS protein staining and activity driven by truncations of the *** two-hybrid,BiFC,and Co-IP assays indicated that TaSnRK2.4 protein interacts with TaPP2C01 and an ABF transcription factor(TF)*** results suggested that TaSnRK2.4 forms a functional TaPP2C01-TaSnRK2.4-TaABF2 module with its upstream and downstream *** analysis revealed that TaSnRK2.4 and TaABF2 positively regulate drought tolerance whereas TaPP2C01 acts negatively by modulating stomatal movement,osmotic adjustment,reactive oxygen species(ROS)homeostasis,and root *** analysis,yeast one-hybrid,and transcriptional activation assays indicated that several osmotic stress-responsive genes,including TaSLAC1-4,TaP5CS3,TaSOD5,TaCAT1,and TaPIN4,are regulated by *** analysis verified their functions in positively regulating stomatal movement(TaSLAC1-4),proline accumulation(TaP5CS3),SOD activity(TaSOD5),CAT activity(TaCAT1),and root morphology(TaPIN4).There were high correlations between plant biomass and yield with module transcripts in a wheat variety panel cultivated under drought conditions in the *** findings provide insights into understanding plant drought response underlying the SnRK2 signaling pathway in common wheat.

关键词： Triticum aestivum SnRK2.4 kinase Gene expression Protein interaction Transgene analysis transcriptional activation

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AP-1 and SP1 trans-activate the expression of hepatic CYP1A1 and CYP2A6 in the bioactivation of AFB1 in chicken

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Science China(Life Sciences) 2024年第7期67卷 1468-1478页

作者： Jiang Deng Jia-Cheng Yang Yue Feng Ze-Jing Xu Kamil Kuča Meng Liu Lv-Hui Sun State Key Laboratory of Agricultural Microbiology Hubei Hongshan LaboratoryFrontiers Science Center for Animal Breeding and Sustainable ProductionCollege of Animal Sciences and TechnologyHuazhong Agricultural UniversityWuhan 430070China Department of Chemistry Faculty of ScienceUniversity of Hradec KraloveHradec Kralove 50003Czech Republic

Dietary exposure to aflatoxin B1(AFB1)is harmful to the health and performance of domestic *** hepatic cytochrome P450s(CYPs),CYP1A1 and CYP2A6,are the primary enzymes responsible for the bioactivation of AFB1to the highly toxic exo-AFB1-8,9-epoxide(AFBO)in ***,the transcriptional regulation mechanism of these CYP genes in the liver of chicks in AFB1metabolism remains ***-luciferase reporter assay,bioinformatics and site-directed mutation results indicated that specificity protein 1(SP1)and activator protein-1(AP-1)motifs were located in the core region-1,063/-948,-606/-541 of the CYP1A1 promoter as well as-636/-595,-503/-462,-147/-1 of the CYP2A6 ***,overexpression and decoy oligodeoxynucleotide technologies demonstrated that SP1 and AP-1 were pivotal transcriptional activators regulating the promoter activity of CYP1A1 and ***,bioactivation of AFB1to AFBO could be increased by upregulation of CYP1A1 and CYP2A6 expression,which was trans-activated owing to the upregulation of AP-1,rather than SP1,stimulated by AFB1-induced reactive oxygen ***,nano-selenium could reduce ROS,downregulate AP-1 expression and then decrease the expression of CYP1A1 and CYP2A6,thus alleviating the toxicity of *** conclusion,AP-1 and SP1 played important roles in the transactivation of CYP1A1 and CYP2A6 expression and further bioactivated AFB1to AFBO in chicken liver,which could provide novel targets for the remediation of aflatoxicosis in chicks.

关键词： aflatoxin B1 CYP1A1 CYP2A6 SP1 AP-1 transcriptional activation

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Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3

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Chinese Medical Journal 2024年第11期137卷 1351-1359页

作者： Jiacheng Li Ruopu Li Xue Bai Wenlong Zhang Yu Nie Shengshou Hu State Key Laboratory of Cardiovascular Disease Fuwai Hospital and Cardiovascular InstituteNational Center for Cardiovascular DiseasesChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100037China Department of Obstetrics and Gynecology Beijing Advanced Innovation Center for GenomicsThird HospitalSchool of Life SciencesPeking UniversityBeijing 100871China

Background:The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease *** regularly interspaced short palindromic repeats(CRISPR)activator(CRISPRa)is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously,providing a new strategy for cell fate *** main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver ***:The differentiation of mouse embryonic fibroblasts(MEFs)into functional induced hepatocyte-like cells(iHeps)was achieved by utilizing the CRISPRa synergistic activation mediator(SAM)system,which drove the combined expression of three endogenous transcription factors-Gata4,Foxa3,and Hnf1a-or alternatively,the expression of two transcription factors,Gata4 and *** vivo,we injected adeno-associated virus serotype 6(AAV6)carrying the CRISPRa SAM system into liver fibrotic Col1a1-Cre^(ER);Cas9^(fl/fl)mice,effectively activating the expression of endogenous Gata4 and Foxa3 in *** endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction(RT-qPCR)and RNA-seq,and the morphology and characteristics of the induced hepatocytes were observed through *** level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining,while the improvement of liver fibrosis is evaluated through Sirius red staining,alpha-smooth muscle actin(α-SMA)immunofluorescence staining,and blood alanine aminotransferase(ALT)***:activation of only two factors,Gata4 and Foxa3,via CRISPRa was sufficient to successfully induce the transformation of MEFs into *** iHeps could be expanded in vitro and displayed functional characteristics similar to th

关键词： Liver fibrosis CRISPRa Cell reprogramming transcriptional activation Hepatocytes

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The association of HpDof1.7 and HpDof5.4 with soluble sugar accumulation in pitaya fruit by transcriptionally activating sugar metabolic genes

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Food Quality and Safety 2022年第4期6卷 619-628页

作者： Zhenliang Mou Runxi Zeng Nanhui Chen Zongli Liu Zexiang Zeng Yonghua Qin Wei Shan Jianfei Kuang Wangjin Lu Jianye Chen Yating Zhao State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and Vegetables/Engineering Research Center of Southern Horticultural Products Preservation Ministry of EducationCollege of HorticultureSouth China Agricultural UniversityGuangzhouChina

Soluble sugar is one of the most important factors affecting fruit flavor and ***,we report the identification of two Dof(DNA-binding with one finger)transcription factors termed HpDof1.7 and HpDof5.4 and their roles in influencing sugar accumulation in ***1.7 and HpDof5.4 share a similar expression pattern with sugar metabolism-related genes HpSuSy1 and HpINV2,and sugar transporter genes HpTMT2 and HpSWEET14 during pitayas maturation,and their expression pattern was also consistent with the accumulation of glucose and fructose,which were the predominant sugars in ***1.7 and HpDof5.4 were both typical nucleus-localized proteins with trans-activation *** mobility shift assay revealed that HpDof1.7 and HpDof5.4 were bound to promoters of HpSuSy1,HpINV2,HpTMT2 and ***,transient expression assays in tobacco leaves showed that HpDof1.7 and HpDof5.4 increased the activities of HpSuSy1,HpINV2,HpTMT2 and HpSWEET14 promoters,thus facilitating sugar accumulation by transcriptionally enhancing sugar metabolic pathway *** findings provide a new perspective on the regulatory mechanisms of Dof transcription factors in sugar accumulation and pitaya fruit quality formation.

关键词： Pitaya sugar accumulation Dof transcription factors transcriptional activation

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CRISPR-assisted transcription activation by phaseseparation proteins

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Protein & Cell 2023年第12期14卷 874-887页

作者： Jiaqi Liu Yuxi Chen Baoting Nong Xiao Luo Kaixin Cui Zhan Li Pengfei Zhang Wenqiong Tan Yue Yang Wenbin Ma Puping Liang Zhou Songyang State Key Laboratory of Biocontrol MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging ResearchSchool of Life SciencesSun Yat-senUniversityGuangzhou510275China Sun Yat-sen Memorial Hospital Sun Yat-sen UniversityGuangzhou 510275China Lumiere Therapeutics Suzhou215000China

The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different *** CRISPR-activation(CRISPRa)platforms often require multiple components because of inefficient transcriptional ***,we fused different phase-separation proteins to dCas9-VPR(dCas9-VP64-P65-RTA)and observed robust increases in transcriptional activation ***,human NUP98(nucleoporin 98)and FUS(fused in sarcoma)IDR domains were best at enhancing dCas9-VPR activity,with dCas9-VPR-FUS IDR(VPRF)outperforming the other CRISPRa systems tested in this study in both activation efficiency and system ***9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of *** findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

关键词： CRISPR transcriptional activation phase-separation proteins

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Analyses of functional conservation and divergence reveal requirement of b HLH010/089/091 for pollen development at elevated temperature in Arabidopsis

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Journal of Genetics and Genomics 2020年第8期47卷 477-492页

作者： Ying Fu Mengyu Li Shiting Zhang Qi Yang Engao Zhu Chenjiang You Ji Qi Hong Ma Fang Chang State Key Laboratory of Genetic Engineering Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering and Institute of Biodiversity SciencesInstitute of Plant BiologySchool of Life SciencesFudan UniversityShanghai200438China Department of Biology The Huck Institutes of the Life SciencesThe Pennsylvania State UniversityUniversity ParkPA16802USA

The Arabidopsis b HLH010/089/091(basic helix-loop-helix)genes are functionally redundant and are required for both anther development and normal expression of DYT1-activated anther-related *** three genes are conserved in Brassicaceae,suggesting that each of them is under selection pressure;however,little is known about the possible functional differences among these b HLH genes and between the b HLH and DYT1 ***,we compared novel anther transcriptomic data sets from b HLH010/089/091 single and double mutants,with an anther transcriptomic data set from the wild type(WT)and a previously obtained anther transcriptomic data set from the bhlh010 bhlh089 bhlh091 triple *** results revealed molecular phenotypes that support the functional redundancy and divergence of b HLH010,b HLH089,and b HLH091,as well as the functional overlap and difference between them and ***-binding analyses revealed that DYT1 and b HLH089 specifically recognize the TCATGTGC box to activate the expression of target genes,including ATA20,EXL4,and *** addition,among genes whose expression was affected in the bhlh010 bhlh089 double and bhlh010 bhlh089 bhlh091 triple mutants,genes that are involved in the stress response and cell signaling were enriched,which included256 genes whose expression was preferentially induced by heat during early flower ***,the bhlh double mutants exhibited defective pollen development when the plants were grown under elevated temperature,suggesting that b HLH genes are important for anther gene expression under such *** results are consistent with the observation that the heat-induced expression of several genes is less in the bhlh mutants than that in the ***,our results provide important insights into the molecular mechanism underlying the activation of direct targets by DYT1-b HLH089 heterodimers and demonstrate the protective roles of b HLH010/089/091 in maintaining fertility upon heat stress.

关键词： DYT1 bHLH010/089/091 Male fertility Molecular phenotype transcriptional activation Heat response

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Isolation and Functional Analysis of the bZIP Transcription Factor Gene TaABP1 from a Chinese Wheat Landrace

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Journal of Integrative Agriculture 2012年第10期11卷 1580-1591页

作者： CAO Xin-you CHEN Ming XU Zhao-shi CHEN Yao-feng LI Lian-cheng YU Yue-hua LIU Yang-na MAYou-zhi National Key Facility for Crop Genetic Resources and Genetic Improvement Key Laboratory of Crop Genetics and BreedingMinistry of Agriculture/Institute of Crop Sciences Chinese Academy of Agricultural Sciences Crop Research Institute Shandong Academy of Agricultural Sciences College of Agronomy Northwest A&F University

In plants, basic leucine zipper （bZIP） transcription factors play important roles in regulatory processes, including stress response, pathogenic defense and light response as well as organ and tissue differentiation. Chinese wheat landrace Pingyaoxiaobaimai （PYXBM）, an original parent of drought tolerant wheat varieties grown in northern China, is significantly tolerant to abiotic stresses such as drought, cold and nutrient deficiencies. In order to isolate key stress-responsive genes and then improve stress tolerances of conventional varieties, a bZIP transcription factor gene was isolated from a cDNA library of drought-treated PYXBM using the in situ plaque hybridization method, and was designated as Triticum aestivum L. abscisic acid （ABA）-responsive element binding protein 1 （TaABP1）. It encodes 372 amino acids, and contains three conserved domains （C1-C3） in the N terminal and a bZIP domain in the C terminal which is a typical protein structure for the group member of bZIP family. transcriptional activation analysis showed that TaABP1 activated the expression of downstream reporter genes in yeast without ABA application. TaABP1 protein fused with green fluorescent protein （GFP） demonstrated that the localization of TaABP 1 protein is in the nucleus. Expression pattern assays indicated that TaABP1 was strongly induced by ABA, high salt, low temperature and drought, and its expression was stronger in stems and leaves than in the roots of wheat. Furthermore, overexpression of TaABP1 in tobacco showed significant improvement of drought tolerance. Data suggested that TaABP1 may be a good candidate gene for improving stress tolerance of wheat by genetic transformation and elucidation of the role of this gene will be useful for understanding the mechanism underlying drought tolerance of Chinese wheat landrace PYXBM.

关键词： wheat expression pattern stress tolerance sub-cellular localization transcriptional activation

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Molecular mechanism of mureidomycin biosynthesis activated by introduction of an exogenous regulatory gene ssa A into Streptomyces roseosporus

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Science China(Life Sciences) 2021年第11期64卷 1949-1963页

作者： Ning Liu Hanye Guan Guoqing Niu Lingjuan Jiang Yue Li Jihui Zhang Jine Li Huarong Tan State Key Laboratory of Microbial Resources Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China College of Life Sciences University of Chinese Academy of SciencesBeijing 100049China Biotechnology Research Center Southwest UniversityChongqing 400715China Department of Medical Research Center Peking Union Medical College HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100730China

Mureidomycins(MRDs), a group of unique uridyl-peptide antibiotics, exhibit antibacterial activity against the highly refractory pathogen Pseudomonas aeruginosa. Our previous study showed that the cryptic MRD biosynthetic gene cluster(BGC) mrd in Streptomyces roseosporus NRRL 15998 could not be activated by its endogenous regulator 02995 but activated by an exogenous activator Ssa A from sansanmycin’s BGC ssa of Streptomyces sp. strain SS. Here we report the molecular mechanism for this inexplicable regulation. EMSAs and footprinting experiments revealed that Ssa A could directly bind to a 14-nt palindrome sequence of 5′-CTGRCNNNNGTCAG-3′ within six promoter regions of mrd. Disruption of three representative target genes(SSGG-02981, SSGG-02987 and SSGG-02994) showed that the target genes directly controlled by Ssa Awere essential for MRD production. The regulatory function was further investigated by replacing six regions of SSGG-02995 with those of ssa ***, only the replacement of 343–450 nt fragment encoding the 115–150 amino acids(AA) of Ssa A could activate MRD biosynthesis. Further bioinformatics analysis showed that the 115–150 AA situated between two conserved domains of Ssa *** findings significantly demonstrate that constitutive expression of a homologous exogenous regulatory gene is an effective strategy to awaken cryptic biosynthetic pathways in Streptomyces.

关键词： mureidomycin transcriptional activation exogenous regulator—SsaA cryptic gene cluster Streptomyces roseosporus Pseudomonas aeruginosa antibiotics

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Fsh-Pc-Sce complex mediates active transcription of Cubitus interruptus （Ci）

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Journal of Molecular Cell Biology 2018年第5期10卷 437-447页

作者： Xiangdong Lv Hao Chen Shuo Zhang Zhao Zhang Chenyu Pan Yuanxin Xia Jialin Fan Wenqing Wu Yi Lu Lei Zhang Hailong Wu Yun Zhao State Key Laboratory of Cell Biology CAS Center for Excellence in Molecular Cell Science Innovation Center for Cell Signaling Network Shanghai Institute of Biochemistry and Cell Biology Chinese Academy of Sciences University of Chinese Academy of Sciences Shanghai 200031 China School of Life Science and Technology ShanghaiTech University Shanghai 201210 China

The Hedgehog （Hh） signaling pathway plays important roles in both embryonic development and adult tissue homeostasis. Such biological functions are mediated by the transcription factor Cubitus interruptus （Ci）. Yet the transcriptional regulation of the effector Ci itself is poorly investigated. Through an RNAi-based genetic screen, we identified that female sterile （1） homeotic （Fsh）, a transcription co-activator, directly activates Ci transcription. Biochemistry assays demonstrated physical interactions among Fsh, Sex combs extra （Sce）, and Polycomb （Pc）. Functional assays further showed that both Pc and Sce are required for Ci expression, which is not likely mediated by the derepression of Engraited （En）, a repressor of Ci, in Pc or Sce mutant cells. Finally, we provide evidence showing that Pc/Sce facilitates the binding of Fsh at Ci locus and that the physical interaction between Fsh and Pc is essential for Fsh-mediated Ci transcription. Taken together, we not only uncover that Ci is transcriptionally regulated by Fsh-Pc-Sce complex but also provide evidence for the coordination between Fsh and PcG proteins in transcriptional regulation.

关键词： Ci Fsh Hedgehog polycomb complex Sce transcriptional activation

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