NF-?B Target Genes Spectrum And Study Of Gene Regulatory Networks

Nuclear transcription factor NF-?B is a kind of transcription factors,which can regulate the expression of numerous genes and express widely in many cell types.NF-?B is an important regulatory factor of immune response and inflammatory response,meanwhile NF-?B regulates many important biological processes,and the pathogenesis of various diseases is associated with abnormal activation.of NF-?B.Therefore,NF-?B plays an important role in scientific and clinical value,which is also currently an important target for transcription therapeutic drugs.The developed inhibition molecules aimed at the excessive activation of NF-?B have the phamaceutical value of reduction and elimination of the excessive activation of NF-?B,but it also may damage the normal function of NF-?B and produces toxic side effects.To prevent this condition,the research of complete and exact physiological function of NF-?B becomes the hot spots in the field.The research emphasis is to discover and identify the all DNA binding sites and target genes of NF-?B in the whole genome.According to these information,we can discover the physiological processes involved by NF-?B and identify the function view of NF-?B.Because NF-?B is a typical inducible type of transcription factor,it's direct target gene spectrums and corresponding regulatory networks are different with different inducers and cell types,thus,it is difficult to study the complete target gene spectrums and regulatory networks of NF-?B.Currently chromatin immunoprecipitation assay(ChIP)is the major experimental techniques that investigates the binding characteristics of transcription factors in vivo.Compared with ChIP-chip,ChIP-seq(ChIP in combination with sequencing)can truly identify the the genome-wide binding characteristics of transcription factors and processes the epigenetic analysis,and then can reveal the regulation pattern of gene expressions in living cells and provide the experimental data for building a network model in regulating gene expression.Because of nonuniformity of chromatin compression,DNA breaking,PCR amplification bias,redundancy of genome sequence and errors in the process of sequencing in ChIP-seq experiment,these can introduce the errors and then cause false positive;moreover,the indirect combination of transcription factors and DNA also can increase the noise;therefore,it must remove the noise by statistical methods and obtains the accurate signal of peaks.ChIP-seq directly sequences DNA fragments of interest without the hybridization of probes on the chip,thus,it has the fewer amplification artifacts,higher resolution,higher coverage and wider range of applications.ChIP-seq is the important advanced technique in the field of functional genomics,but it generates a large amount of data with high complexity and poses grim challenges for bioinformatics researchers.The main research contents of this paper include:1.Appling ChIP-seq,RNAi and gene expression profile techniques identify the target genes of NF-?B in human HeLa cells stimulated by TNF-a.The activated NF-?B first binds to the specific DNA sequence in vivo and then starts to regulate the expression of its target genes.Therefore,the research of NF-?B binding characteristics in the genome is important for identifying target genes and explaining for transcription complexes and diverse transcriptional regulatory mechanisms.We identified 266 activated and 318 repressed direct target genes of NF-?B5 50%and 90%of these genes contained canonical and non-canonical ?B site motifs in the ChIP peaks occupied them.Only a few of target genes indentified in this study overlapped with those indentified in other cell types,further demonstrating the cell-or inducer-specificity of many target genes of NF-?B.These data confirmed that NF-?B had the highly selective regulation for different target genes spectrums with different inducers and cell types.This research discovered that although a large number of genes(13600)were bound by NF-?B in HeLa cells with the stimulation of TNF-a,only 4.3%(584)of these genes' transcriptions were significantly regulated.A large number of potential target genes in the human genome may provide the diverse target genes and selective regulation for target genes of NF-?B,therefore,the function and regulation of NF-?B have great complexity and diversity,and it is important for promoting the diversification and highly selective regulation of NF-?B target genes.Meanwhile it was discovered that NF-?B showed several significant binding characteristics in vivo,such as the inter-and intra-chromosome different binding strength(fold enrichment of ChIP-seq peaks over 20)associated with the state of chromatins,the dominant introns areas binding to vast majority target genes through multiple sites,more extensive occupation of target genes by non-canonical ?B sites than canonical ?B sites,and astonishing genes coverage in the human genome.These binding characteristics in genome in vivo affect the transcriptions of its direct target genes,reflecting some important features of NF-?B which acts as an inducible transcription factor involving in many biological processes and responding to different internal and external stimuli.2.Through manual information retrieval and the data available in our laboratory,the database of NF-?B target genes was established that is now the most complete,accurate NF-?B target genes database containing the specific experimental information and published them in our web server(http://tfdb.seu.edu.cn/nfkb/).NF-?B is a typical inducible transcription factor,expression regulation of the target genes spectrums is different with different inducers and cell types.This database contained 2267 target genes and 1667 distinct target genes after removing the duplications,and simultaneously collected the specific inducers,cell types,expression quantity of target genes,experiment methods,references,the sequences of NF-?B binding domain and the counts of the specific binding motifs.The data provided by this database can help us better understand the eukaryotic gene expression regulation networks,which has important basic research value.3.Through the GO function and KEGG analysis of the collected NF-?B target genes,the results showed that the NF-?B was a central regulator of cancers,cell apoptosis,Jak-STAT,allograft rejection,the stress response and immune response.NF-?B involved in bone disease,life expectancy,chronic obstructive pulmonary disease,systemic lupus erythematosus,multiple sclerosis,myocardial infarction.The classic target genes of NF-?B involved in the occurrence of a variety of cancers,can act as the treatment of disease target.Many pathogens and viruses can activate NF-?B,and it indicated that NF-?B is an evolutionarily conserved immune and inflammatory mediator.NF-?B is also the central regulator of cell metabolism,a total of 608 NF-?B target genes were involved in various cellular metabolic processes.They played important roles in diseases caused by metabolic disorders,such as cardiovascular disease and diabetes mellitus.Because 98 transcription factors were the target genes of NF-?B,NF-?B played an important role in the transcriptional regulation network of organism.The type of inducers generally determines whether the expression of the NF-?B target gene is up-regulated or down-regulated.4.The regulatory network map of the NF-?B target genes in HeLa cells and THP-1 cells and the expression regulation network of NF-?B microRNAs(miRNAs)target genes were established.Although the NF-?B target genes identified in HeLa cells and THP-1 cells have only 39 identical genes,the biological function of NF-?B in HeLa cells and THP-1 cells was similar.miRNAs act as posttranscriptional regulators of gene expression and can regulate many target genes.miRNAs constituted an important layer of regulation of gene expression with profound impacts on biological organisms,and played an important role in NF-?B signaling pathway.Dysregulatiori of miRNAs often associates with tumor development and progression.miRNAs can function as both oncogenes or tumor suppressors in different tumors and cell types,which is cell type specific.Therefore,miRNAs can be used as therapeutic target of cancers.NF-?B played an important role in forming the unique miRNAs expression pattern with cell type specificity of the adaptive and innate immune system of each cell type through the regulation of the expression of NF-?B miRNAs target genes;and played an important role in cell development,function and epigenetics.