Chromatin is the primary carrier of epigenetic information in higher eukaryotes. AtCYP71 contains both cyclophilin domain and WD40 repeats. Loss of AtCYP71 function causes drastic pleiotropic phenotypic defects. Here,...Chromatin is the primary carrier of epigenetic information in higher eukaryotes. AtCYP71 contains both cyclophilin domain and WD40 repeats. Loss of AtCYP71 function causes drastic pleiotropic phenotypic defects. Here, we show that AtCYP71 physically interacts with FAS1 and LHP1, respectively, to modulate their distribution on chromatin. The Ihpl cyp71 double mutant showed more severe phenotypes than the single mutants, suggesting that AtCYP71 and LHP1 synergistically control plant development. Such synergism was in part illustrated by the observation that LHP1 association with its specific target loci requires AtCYP71 function. We also demonstrate that AtCYP71 physically interacts with FAS1 and is indispensable for FAS1 targeting to the KNAT1 locus. Together, our data suggest that AtCYP71 is involved in fundamental processes of chromatin assembly and histone modification in plants.展开更多
目的探讨骨髓间充质干细胞(BMSCs)在平滑肌微环境中发生分化后平滑肌标志性基因乙酰化水平的变化,以及组蛋白乙酰化修饰在干细胞分化中的作用及机制。方法体外培养BMSCs和膀胱平滑肌细胞(BSMCs),选择同批次的第3代BMSCs,将与BSMCs共培...目的探讨骨髓间充质干细胞(BMSCs)在平滑肌微环境中发生分化后平滑肌标志性基因乙酰化水平的变化,以及组蛋白乙酰化修饰在干细胞分化中的作用及机制。方法体外培养BMSCs和膀胱平滑肌细胞(BSMCs),选择同批次的第3代BMSCs,将与BSMCs共培养3d的BMSCs作为实验组,未经共培养的BMSCs作为对照组,采用RT-PCR检测两组BMSCs中平滑肌α肌动蛋白(α-SMA)、钙调节蛋白(calponin)、平滑肌肌球蛋白重链(SM-MHC)的表达丰度。采用条件为80%、20次、0.5 s、8个循环的超声破碎实验组及对照组BMSCs的DNA,以H3K9抗体结合特定乙酰化位点,采用免疫共沉淀技术(ChIP)沉淀BMSCs组蛋白乙酰化位点基因,接头PCR扩增所获基因,Real-time PCR检测所获基因中3种目的基因(α-SMA、Calponin、SM-MHC)的表达水平。结果 RT-PCR检测结果显示,实验组BMSCs中的平滑肌标志性基因(α-SMA、Calponin、SM-MHC)的mRNA表达水平较对照组明显增高[分别为0.176±0.003 vs. 0.070±0.002,0.079±0.002vs.0.051±0.003,0.091±0.004vs.0.034±0.001],差异均有统计学意义(P<0.01)。分光光度计检测ChIP后获得的DNA,结果表明H3K9乙酰化抗体沉淀所获DNA浓度高于Ig G抗体,且实验组高于对照组(P<0.05)。RealtimePCR分析BMSCs分化前后目的基因组蛋白H3K9乙酰化水平,结果显示,BMSCs分化后,实验组的平滑肌标志性基因α-SMA、calponin、SM-MHC的mRNA转录水平明显高于对照组[分别为9.26±5.03 vs. 1.01±0.05,2.33±0.65 vs.0.99±0.05,2.63±0.37vs.1.00±0.03],差异均有统计学意义(P>0.05)。结论在平滑肌微环境中,BSMCs特定位点H3K9乙酰化程度的增加可促进BMSCs向BSMCs的分化。展开更多
Background Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Prev...Background Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17β-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER ^+) breast cancer cell line MCF-7. Methods After the treatment of MCF-7 cells with E2 at different concentrations ( 10^-11 mol/L, 10.9 mol/L, 10.7 mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0. 7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10^-7 mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half- site ( 1/2 ERE) element and Spl sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (CHIP) assay was also used to confirm the binding of estrogen receptor a (ERα) on BMP-6 promoter in the presence of E2. Results E2 dose dependently increased BMP-6 mRNA expression in human ER ^+ breast cancer cell line MCF-7. At a dose of 10^-7 mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P 〈 0. 05 ). Both the 1/2 ERE response element mutant form and the Spl site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter' s response to E2. Through ChIP assay, the binding of ERot on 1/2 ERE response element in BMP-6 promoter was further validated. Conclusion Estrogen induces BMP-6 expression in human ER^+ breast cancer cell line MCF-7 receptor ERα binding on 1/2 ERE展开更多
Determining the binding sites of the transcription factor is important for understanding of transcriptional regulation. Transcription factor c-Jun plays an important role in cell growth, differentiation and developmen...Determining the binding sites of the transcription factor is important for understanding of transcriptional regulation. Transcription factor c-Jun plays an important role in cell growth, differentiation and development, but the binding sites and the target genes are not clearly defined in the whole human genome. In this study, we performed a ChIP-Seq experiment to identify c-Jun binding site in the human genome. Forty-eight binding sites were selected to process further evaluation by dsDNA microarray assay. We identified 283 c-Jun binding sites in K562 cells. Data analysis showed that 48.8% binding sites located within 100 kb of the upstream of the annotated genes, 28.6% binding sites comprised consensus TRE/CRE motif (5′-TGAC/GTCA-3′, 5′-TGACGTCA-3′) and variant sequences. Forty-two out of the selected 48 binding sites were found to bind the c-Jun homodimer in dsDNA microarray analysis. Data analysis also showed that 1569 genes are located in the neighborhood of the 283 binding sites and 191 genes in the neighborhood of the 42 binding sites validated by dsDNA microarray. We consulted 38 c-Jun target genes in previous studies and 16 among these 38 genes were also detected in this study. The identification of c-Jun binding sites and potential target genes in the genome scale may improve our fundamental understanding in the molecular mechanisms underlying the transcription regulation related to c-Jun.展开更多
In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein i...In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain (in complex form). Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer's disease.展开更多
Background CCAAT/enhancer-binding protein β(C/EBPβ) is required for mitotic clonal expansion (MCE) during adipogenesis. It is still unclear how C/EBPp regulates MCE in the earlier differentiation programs of 3T3...Background CCAAT/enhancer-binding protein β(C/EBPβ) is required for mitotic clonal expansion (MCE) during adipogenesis. It is still unclear how C/EBPp regulates MCE in the earlier differentiation programs of 3T3-L1 preadipocytes. The purpose of this paper was to understand why C/EBPβ is required for preadipocyte proliferation, and identify new target genes of C/EBPP with chromatin immunoprecipitation (ChlP)-on-chip. Methods Postconfluent growth-arrested 3T3-L1 preadipocytes were induced to differentiation using a standard differentiation protocol. Chip was performed at 20 hours after induction with specific anti-C/EBPβ antibodies. The precipitated DNA was amplified, labeled and hybridized with a mouse promoter microarray. Compared with the control in which the ChIP experiment was performed with non-specific antibody, only the genes with a signal increasing more than 2 fold were considered as candidate genes.Results A total of 110 candidate genes were identified. BTG3 associated nuclear protein (SMAR1, Banp) and tripartite motif-containing 35 (Hls5, trim35) were two target genes among the 110 candidate genes which are involved in cell cycle regulation; the binding of C/EBP3 to the promoter of banp and trim35 was verified by ChlP-PCR. Conclusion C/EBPβ may regulate preadipocyte proliferation through activation of banp and trim35.展开更多
Objective:To indentify target genes of transcription factor CCA AT enhancer-binding protein P(CEBPB) in acute proinyelocytie leukemia cells induced by all-tram retinoie acid.Methods: A new strategy for high—throughpu...Objective:To indentify target genes of transcription factor CCA AT enhancer-binding protein P(CEBPB) in acute proinyelocytie leukemia cells induced by all-tram retinoie acid.Methods: A new strategy for high—throughput identification of direct target genes was established by combining chromatin immunoprecipitation(ChIP) with in vitro selection.Then,106 potential CKBPB binding fragments from the genome of the all-trans retinoie acid(ATRA)-treated NB4 cells were identified.Results:Of them,82 were mapped in proximity to known or previously predicted genes;7 were randomly picked up for further confirmation by ChlP-PCR and 3 genes (CALM,1TPR2 and 0RM2) were found to be specificaUy up-regulated in the ATRA-treated NB4 cells,indicating that they might lie the down-stream target genes of ATKA.Conclusions:Our results provided new insight into the mechanisms of ATRA-induced granulocytic differentiation.展开更多
The translation of the genotype into phenotype, represented for example by the expression of genes encod- ing enzymes required for the biosynthesis of phytochemicals that are important for interaction of plants with t...The translation of the genotype into phenotype, represented for example by the expression of genes encod- ing enzymes required for the biosynthesis of phytochemicals that are important for interaction of plants with the environment, is largely carried out by transcription factors (TFs) that recognize specific cis-regulatory elements in the genes that they control. TFs and their target genes are organized in gene regulatory net- works (GRNs), and thus uncovering GRN architecture presents an important biological challenge necessary to explain gene regulation. Linking TFs to the genes they control, central to understanding GRNs, can be car- ried out using gene- or TF-centered approaches. In this study, we employed a gene-centered approach uti- lizing the yeast one-hybrid assay to generate a network of protein-DNA interactions that participate in the transcriptional control of genes involved in the biosynthesis of maize phenolic compounds including gen- eral phenylpropanoids, lignins, and flavonoids. We identified 1100 protein-DNA interactions involving 54 phenolic gene promoters and 568 TFs. A set of 11 TFs recognized 10 or more promoters, suggesting a role in coordinating pathway gene expression. The integration of the gene-centered network with informa- tion derived from TF-centered approaches provides a foundation for a phenolics GRN characterized by in- terlaced feed-forward loops that link developmental regulators with biosynthetic genes.展开更多
文摘Chromatin is the primary carrier of epigenetic information in higher eukaryotes. AtCYP71 contains both cyclophilin domain and WD40 repeats. Loss of AtCYP71 function causes drastic pleiotropic phenotypic defects. Here, we show that AtCYP71 physically interacts with FAS1 and LHP1, respectively, to modulate their distribution on chromatin. The Ihpl cyp71 double mutant showed more severe phenotypes than the single mutants, suggesting that AtCYP71 and LHP1 synergistically control plant development. Such synergism was in part illustrated by the observation that LHP1 association with its specific target loci requires AtCYP71 function. We also demonstrate that AtCYP71 physically interacts with FAS1 and is indispensable for FAS1 targeting to the KNAT1 locus. Together, our data suggest that AtCYP71 is involved in fundamental processes of chromatin assembly and histone modification in plants.
文摘目的探讨骨髓间充质干细胞(BMSCs)在平滑肌微环境中发生分化后平滑肌标志性基因乙酰化水平的变化,以及组蛋白乙酰化修饰在干细胞分化中的作用及机制。方法体外培养BMSCs和膀胱平滑肌细胞(BSMCs),选择同批次的第3代BMSCs,将与BSMCs共培养3d的BMSCs作为实验组,未经共培养的BMSCs作为对照组,采用RT-PCR检测两组BMSCs中平滑肌α肌动蛋白(α-SMA)、钙调节蛋白(calponin)、平滑肌肌球蛋白重链(SM-MHC)的表达丰度。采用条件为80%、20次、0.5 s、8个循环的超声破碎实验组及对照组BMSCs的DNA,以H3K9抗体结合特定乙酰化位点,采用免疫共沉淀技术(ChIP)沉淀BMSCs组蛋白乙酰化位点基因,接头PCR扩增所获基因,Real-time PCR检测所获基因中3种目的基因(α-SMA、Calponin、SM-MHC)的表达水平。结果 RT-PCR检测结果显示,实验组BMSCs中的平滑肌标志性基因(α-SMA、Calponin、SM-MHC)的mRNA表达水平较对照组明显增高[分别为0.176±0.003 vs. 0.070±0.002,0.079±0.002vs.0.051±0.003,0.091±0.004vs.0.034±0.001],差异均有统计学意义(P<0.01)。分光光度计检测ChIP后获得的DNA,结果表明H3K9乙酰化抗体沉淀所获DNA浓度高于Ig G抗体,且实验组高于对照组(P<0.05)。RealtimePCR分析BMSCs分化前后目的基因组蛋白H3K9乙酰化水平,结果显示,BMSCs分化后,实验组的平滑肌标志性基因α-SMA、calponin、SM-MHC的mRNA转录水平明显高于对照组[分别为9.26±5.03 vs. 1.01±0.05,2.33±0.65 vs.0.99±0.05,2.63±0.37vs.1.00±0.03],差异均有统计学意义(P>0.05)。结论在平滑肌微环境中,BSMCs特定位点H3K9乙酰化程度的增加可促进BMSCs向BSMCs的分化。
文摘Background Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17β-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER ^+) breast cancer cell line MCF-7. Methods After the treatment of MCF-7 cells with E2 at different concentrations ( 10^-11 mol/L, 10.9 mol/L, 10.7 mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0. 7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10^-7 mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half- site ( 1/2 ERE) element and Spl sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (CHIP) assay was also used to confirm the binding of estrogen receptor a (ERα) on BMP-6 promoter in the presence of E2. Results E2 dose dependently increased BMP-6 mRNA expression in human ER ^+ breast cancer cell line MCF-7. At a dose of 10^-7 mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P 〈 0. 05 ). Both the 1/2 ERE response element mutant form and the Spl site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter' s response to E2. Through ChIP assay, the binding of ERot on 1/2 ERE response element in BMP-6 promoter was further validated. Conclusion Estrogen induces BMP-6 expression in human ER^+ breast cancer cell line MCF-7 receptor ERα binding on 1/2 ERE
基金supported by the National Natural Science Foundation of China(Nos.30973375 and 30600152)
文摘Determining the binding sites of the transcription factor is important for understanding of transcriptional regulation. Transcription factor c-Jun plays an important role in cell growth, differentiation and development, but the binding sites and the target genes are not clearly defined in the whole human genome. In this study, we performed a ChIP-Seq experiment to identify c-Jun binding site in the human genome. Forty-eight binding sites were selected to process further evaluation by dsDNA microarray assay. We identified 283 c-Jun binding sites in K562 cells. Data analysis showed that 48.8% binding sites located within 100 kb of the upstream of the annotated genes, 28.6% binding sites comprised consensus TRE/CRE motif (5′-TGAC/GTCA-3′, 5′-TGACGTCA-3′) and variant sequences. Forty-two out of the selected 48 binding sites were found to bind the c-Jun homodimer in dsDNA microarray analysis. Data analysis also showed that 1569 genes are located in the neighborhood of the 283 binding sites and 191 genes in the neighborhood of the 42 binding sites validated by dsDNA microarray. We consulted 38 c-Jun target genes in previous studies and 16 among these 38 genes were also detected in this study. The identification of c-Jun binding sites and potential target genes in the genome scale may improve our fundamental understanding in the molecular mechanisms underlying the transcription regulation related to c-Jun.
基金supported by the Natural Science Foundation of Guangdong Province,China,No.8151051501000004
文摘In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain (in complex form). Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer's disease.
基金This work was supported by the National Natural Science Foundation of China (No. 30700403).
文摘Background CCAAT/enhancer-binding protein β(C/EBPβ) is required for mitotic clonal expansion (MCE) during adipogenesis. It is still unclear how C/EBPp regulates MCE in the earlier differentiation programs of 3T3-L1 preadipocytes. The purpose of this paper was to understand why C/EBPβ is required for preadipocyte proliferation, and identify new target genes of C/EBPP with chromatin immunoprecipitation (ChlP)-on-chip. Methods Postconfluent growth-arrested 3T3-L1 preadipocytes were induced to differentiation using a standard differentiation protocol. Chip was performed at 20 hours after induction with specific anti-C/EBPβ antibodies. The precipitated DNA was amplified, labeled and hybridized with a mouse promoter microarray. Compared with the control in which the ChIP experiment was performed with non-specific antibody, only the genes with a signal increasing more than 2 fold were considered as candidate genes.Results A total of 110 candidate genes were identified. BTG3 associated nuclear protein (SMAR1, Banp) and tripartite motif-containing 35 (Hls5, trim35) were two target genes among the 110 candidate genes which are involved in cell cycle regulation; the binding of C/EBP3 to the promoter of banp and trim35 was verified by ChlP-PCR. Conclusion C/EBPβ may regulate preadipocyte proliferation through activation of banp and trim35.
文摘Objective:To indentify target genes of transcription factor CCA AT enhancer-binding protein P(CEBPB) in acute proinyelocytie leukemia cells induced by all-tram retinoie acid.Methods: A new strategy for high—throughput identification of direct target genes was established by combining chromatin immunoprecipitation(ChIP) with in vitro selection.Then,106 potential CKBPB binding fragments from the genome of the all-trans retinoie acid(ATRA)-treated NB4 cells were identified.Results:Of them,82 were mapped in proximity to known or previously predicted genes;7 were randomly picked up for further confirmation by ChlP-PCR and 3 genes (CALM,1TPR2 and 0RM2) were found to be specificaUy up-regulated in the ATRA-treated NB4 cells,indicating that they might lie the down-stream target genes of ATKA.Conclusions:Our results provided new insight into the mechanisms of ATRA-induced granulocytic differentiation.
文摘The translation of the genotype into phenotype, represented for example by the expression of genes encod- ing enzymes required for the biosynthesis of phytochemicals that are important for interaction of plants with the environment, is largely carried out by transcription factors (TFs) that recognize specific cis-regulatory elements in the genes that they control. TFs and their target genes are organized in gene regulatory net- works (GRNs), and thus uncovering GRN architecture presents an important biological challenge necessary to explain gene regulation. Linking TFs to the genes they control, central to understanding GRNs, can be car- ried out using gene- or TF-centered approaches. In this study, we employed a gene-centered approach uti- lizing the yeast one-hybrid assay to generate a network of protein-DNA interactions that participate in the transcriptional control of genes involved in the biosynthesis of maize phenolic compounds including gen- eral phenylpropanoids, lignins, and flavonoids. We identified 1100 protein-DNA interactions involving 54 phenolic gene promoters and 568 TFs. A set of 11 TFs recognized 10 or more promoters, suggesting a role in coordinating pathway gene expression. The integration of the gene-centered network with informa- tion derived from TF-centered approaches provides a foundation for a phenolics GRN characterized by in- terlaced feed-forward loops that link developmental regulators with biosynthetic genes.