Identification of p100 target promoters by chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS)

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.

HvWRKY2 acts as an immunity suppressor and targets HvCEBiP to regulate powdery mildew resistance in barley

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.

Two memory associated genes regulated by amyloid precursor protein intracellular domain Novel insights into the pathogenesis of learning and memory impairment in Alzheimer's disease

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.

The Cyclophilin AtCYP71 Interacts with CAF-1 and LHP1 and Functions in Multiple Chromatin Remodeling Processes

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.

c-Jun binding site identification in K562 cells

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.

Activation of bone morphogenetic protein-6 gene transcription in MCF-7 cells by estrogen

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 element in the BMP-6 promoter. through its

A PtrLBD39-mediated transcriptional network regulates tension wood formation in Populus trichocarpa

Tension wood(TW)is a specialized xylem tissue formed in angiosperm trees under gravitational stimulus or mechanical stresses(e.g.,bending).The genetic regulation that underlies this important mechanism remains poorly understood.Here,we used laser capture microdissection of stem xylem cells coupled with full transcriptome RNA-sequencing to analyze TW formation in Populus trichocarpa.After tree bending,PtrLBD39 was the most significantly induced transcription factor gene;it has a phylogenetically paired homolog,PtrLBD22.CRISPR-based knockout of PtrLBD39/22 severely inhibited TW formation,reducing cellulose and increasing lignin content.Transcriptomic analyses of CRISPR-based PtrLBD39/22 double mutants showed that these two genes regulate a set of TW-related genes.Chromatin immunoprecipitation sequencing(ChIP-seq)was used to identify direct targets of PtrLBD39.We integrated transcriptomic analyses and ChIP-seq assays to construct a transcriptional regulatory network(TRN)mediated by PtrLBD39.In this TRN,PtrLBD39 directly regulates 26 novel TW-responsive transcription factor genes.Our work suggests that PtrLBD39 and PtrLBD22 specifically control TW formation by mediating a TW-specific TRN in Populus.

Maize Gene Regulatory Network for Phenolic Metabolism

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.

Identification and validation of novel C/EBPp-regulated genes in preadipocyte proliferation

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.

Genome-wide binding analysis of the tomato transcription factor SlDof1 reveals its regulatory impacts on fruit ripening

The DNA binding with one finger(Dof)proteins are plant-specific transcription factors involved in a variety of biological processes.However,little is known about their functions in fruit ripening,a flowering-plant-specific process that is required for seed maturation and dispersal.Here,we found that the tomato Dof transcription factor SlDof1,is necessary for normal fruit ripening.Knockdown of SlDof1 expression by RNA interference delayed ripening-related processes,including lycopene synthesis and ethylene production.Transcriptome profiling indicated that SlDof1 influences the expression of hundreds of genes,and a chromatin immunoprecipitation sequencing revealed a large number of SlDof1 binding sites.A total of 312 genes were identified as direct targets of SlDof1,among which 162 were negatively regulated by SlDof1 and 150 were positively regulated.The SlDof1 target genes were involved in a variety of metabolic pathways,and follow-up analyses verified that SlDof1 directly regulates some well-known ripening-related genes including ACS2 and PG2A as well as transcriptional repressor genes such as SlIAA27.Our findings provide insights into the transcriptional regulatory networks underlying fruit ripening and highlight a gene potentially useful for genetic engineering to control ripening.