Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunopreci...Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.展开更多
The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machiner...The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery.To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100,we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection(ChIP-GLAS).From this assay,we determined that a set of promoter fragments,including several factors in the transforming growth factor beta(TGF-β)signaling pathway,exhibited interaction with p100.The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-b signaling pathway in cell lines.展开更多
花生(Arachis hypogaea L.)是重要的经济油料作物,其生长发育、产量与品质受干旱影响。为深入了解花生的抗旱机理,本研究通过ChIP-seq对组蛋白去乙酰化酶AhHDA1和转录因子AhGLK1富集的DNA序列进行分析,揭示两者调控的下游靶基因网络。...花生(Arachis hypogaea L.)是重要的经济油料作物,其生长发育、产量与品质受干旱影响。为深入了解花生的抗旱机理,本研究通过ChIP-seq对组蛋白去乙酰化酶AhHDA1和转录因子AhGLK1富集的DNA序列进行分析,揭示两者调控的下游靶基因网络。通过比对分析,GLK-IP获得6571万clean beads,HDA-IP获得6390万clean beads,Input获得7006万clean beads,唯一比对率分别为74.97%、76.81%和76.75%。GLK-IP获得714个peak, HDA-IP获得543个peak。Peak在基因的外显子、内含子、上游、下游和基因间等功能元件均有分布。GO富集结果显示,AhGLK1-IP和AhHDA1-IP的peak相关基因在分子功能中的富集分别为35.1%和32.8%,在生物学过程中的富集分别为39.3%和44.2%,在细胞组分中的富集分别为25.5%和22.8%。KEGG信号通路富集结果显示,AhGLK1-IP相关基因显著富集在“代谢途径(metabolic pathways)”、“抗生素生物合成(biosynthesis of antibiotics)”、“二羧酸代谢(glyoxylate and dicarboxylate metabolism)”、“不同环境中微生物代谢(microbial metabolism in diverse environments)”、“碳代谢(carbon metabolism)”、“次生代谢生物合成(biosynthesis of secondary metabolites)”和“氨基酸生物合成(biosynthesis of amino acids)。而AhHDA1-IP相关基因在“N聚糖生物合成(N-glycan biosynthesis)”、“精氨酸和脯氨酸代谢(arginine and proline metabolism)”和“苯丙氨酸代谢(phenylalanine metabolism)”通路显著富集。AhGLK1-IP和AhHDA1-IP共同富集的peak有4个,在AhGLK1-IP和AhHDA1-IP特异富集的基序(motif)中存在共同的保守序列AGAA/T。研究结果为深入认识AhGLK1和AhHDA1基因的功能和了解花生响应干旱胁迫和旱后恢复生长中的调控机制具有参考价值。展开更多
Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery m...Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus,Blumeria graminis f.sp.hordei(Bgh).However,the molecular features of Hv WRKY2 in its DNA-binding and repressor functions,as well as its target genes,are uncharacterized.We show that the W-box binding of Hv WRKY2 requires an intact WRKY domain and an upstream sequence of~75 amino acids,and the Hv WRKY2 W-box binding activity is linked to its repressor function in disease resistance.Chromatin immunoprecipitation(ChIP)-seq analysis identified HvCEBiP,a putative chitin receptor gene,as a target gene of Hv WRKY2 in overexpressing transgenic barley plants.ChIP-qPCR and Electrophoretic Mobility Shift Assay(EMSA)verified the direct binding of Hv WRKY2 to a W-boxcontaining sequence in the HvCEBiP promoter.Hv CEBiP positively regulates resistance against Bgh in barley.Our findings suggest that Hv WRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern(PAMP)recognition receptor genes,suggesting that Hv CEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.展开更多
文摘Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.
基金This work was supported by grants from the National Basic Research Program(973 Program,2009CB918903)863 Project of the Ministry of Science and Technology of China(2007AA02Z115)+3 种基金NSFC(90919032,30970562,30670441,30811130394 and 30870562)Tianjin Municipal Science and Technology Commission(08ZCGHHZ01900 and 08JCYBJC07700)Specialized Fund for the Doctoral Program of Higher Education(20091202110001)Tianjin Educational Committee Foundation(2008ZD01).
文摘The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery.To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100,we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection(ChIP-GLAS).From this assay,we determined that a set of promoter fragments,including several factors in the transforming growth factor beta(TGF-β)signaling pathway,exhibited interaction with p100.The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-b signaling pathway in cell lines.
文摘花生(Arachis hypogaea L.)是重要的经济油料作物,其生长发育、产量与品质受干旱影响。为深入了解花生的抗旱机理,本研究通过ChIP-seq对组蛋白去乙酰化酶AhHDA1和转录因子AhGLK1富集的DNA序列进行分析,揭示两者调控的下游靶基因网络。通过比对分析,GLK-IP获得6571万clean beads,HDA-IP获得6390万clean beads,Input获得7006万clean beads,唯一比对率分别为74.97%、76.81%和76.75%。GLK-IP获得714个peak, HDA-IP获得543个peak。Peak在基因的外显子、内含子、上游、下游和基因间等功能元件均有分布。GO富集结果显示,AhGLK1-IP和AhHDA1-IP的peak相关基因在分子功能中的富集分别为35.1%和32.8%,在生物学过程中的富集分别为39.3%和44.2%,在细胞组分中的富集分别为25.5%和22.8%。KEGG信号通路富集结果显示,AhGLK1-IP相关基因显著富集在“代谢途径(metabolic pathways)”、“抗生素生物合成(biosynthesis of antibiotics)”、“二羧酸代谢(glyoxylate and dicarboxylate metabolism)”、“不同环境中微生物代谢(microbial metabolism in diverse environments)”、“碳代谢(carbon metabolism)”、“次生代谢生物合成(biosynthesis of secondary metabolites)”和“氨基酸生物合成(biosynthesis of amino acids)。而AhHDA1-IP相关基因在“N聚糖生物合成(N-glycan biosynthesis)”、“精氨酸和脯氨酸代谢(arginine and proline metabolism)”和“苯丙氨酸代谢(phenylalanine metabolism)”通路显著富集。AhGLK1-IP和AhHDA1-IP共同富集的peak有4个,在AhGLK1-IP和AhHDA1-IP特异富集的基序(motif)中存在共同的保守序列AGAA/T。研究结果为深入认识AhGLK1和AhHDA1基因的功能和了解花生响应干旱胁迫和旱后恢复生长中的调控机制具有参考价值。
基金supported by National Key Research and Development Program of China(2018YFD1000703,2018YFD1000700)Strategic Priority Research Program of the Chinese Academy of Sciences(XDB11020400)+3 种基金National Program on Research and Development of Transgenic Plants(2016ZX08009-003-001)Startup Fund for Advanced Talents of Lushan Botanical GardenChinese Academy of Science(2020ZWZX03 and 2020ZWZX05)the“Double Hundred and Double Thousand”Talent Project of Jiujiang City(jjsbsq2020026)。
文摘Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus,Blumeria graminis f.sp.hordei(Bgh).However,the molecular features of Hv WRKY2 in its DNA-binding and repressor functions,as well as its target genes,are uncharacterized.We show that the W-box binding of Hv WRKY2 requires an intact WRKY domain and an upstream sequence of~75 amino acids,and the Hv WRKY2 W-box binding activity is linked to its repressor function in disease resistance.Chromatin immunoprecipitation(ChIP)-seq analysis identified HvCEBiP,a putative chitin receptor gene,as a target gene of Hv WRKY2 in overexpressing transgenic barley plants.ChIP-qPCR and Electrophoretic Mobility Shift Assay(EMSA)verified the direct binding of Hv WRKY2 to a W-boxcontaining sequence in the HvCEBiP promoter.Hv CEBiP positively regulates resistance against Bgh in barley.Our findings suggest that Hv WRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern(PAMP)recognition receptor genes,suggesting that Hv CEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.
