RESUMEN
In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3' ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.
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Combinatorial interactions among transcription factors are critical to directing tissue-specific gene expression. To build a global atlas of these combinations, we have screened for physical interactions among the majority of human and mouse DNA-binding transcription factors (TFs). The complete networks contain 762 human and 877 mouse interactions. Analysis of the networks reveals that highly connected TFs are broadly expressed across tissues, and that roughly half of the measured interactions are conserved between mouse and human. The data highlight the importance of TF combinations for determining cell fate, and they lead to the identification of a SMAD3/FLI1 complex expressed during development of immunity. The availability of large TF combinatorial networks in both human and mouse will provide many opportunities to study gene regulation, tissue differentiation, and mammalian evolution.
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Regulación de la Expresión Génica , Redes Reguladoras de Genes , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Evolución Molecular , Humanos , Ratones , Monocitos/citología , Especificidad de Órganos , Proteína smad3/metabolismo , Transactivadores/metabolismoRESUMEN
The Functional ANnoTation Of the Mammalian genome (FANTOM) Consortium has continued to provide extensive resources in the pursuit of understanding the transcriptome, and transcriptional regulation, of mammalian genomes for the last 20 years. To share these resources with the research community, the FANTOM web-interfaces and databases are being regularly updated, enhanced and expanded with new data types. In recent years, the FANTOM Consortium's efforts have been mainly focused on creating new non-coding RNA datasets and resources. The existing FANTOM5 human and mouse miRNA atlas was supplemented with rat, dog, and chicken datasets. The sixth (latest) edition of the FANTOM project was launched to assess the function of human long non-coding RNAs (lncRNAs). From its creation until 2020, FANTOM6 has contributed to the research community a large dataset generated from the knock-down of 285 lncRNAs in human dermal fibroblasts; this is followed with extensive expression profiling and cellular phenotyping. Other updates to the FANTOM resource includes the reprocessing of the miRNA and promoter atlases of human, mouse and chicken with the latest reference genome assemblies. To facilitate the use and accessibility of all above resources we further enhanced FANTOM data viewers and web interfaces. The updated FANTOM web resource is publicly available at https://fantom.gsc.riken.jp/.
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Anotación de Secuencia Molecular , ARN Largo no Codificante/genética , Transcriptoma/genética , Animales , Sitios de Unión , Cromatina/metabolismo , Drosophila/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Genoma , Humanos , Metadatos , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Regiones Promotoras Genéticas , ARN Largo no Codificante/metabolismo , Factores de Transcripción/metabolismo , Interfaz Usuario-ComputadorRESUMEN
Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2-L (211 kb), pSLA2-M (113 kb) and pSLA2-S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of pSLA2-L and pSLA2-M up to 3.1 kb from the ends and have homology to those of typical Streptomyces species. Mutant 2-39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of pSLA2-L from strain 51252, which carries only pSLA2-L, also resulted in terminal deletions in newly obtained mutants. The tap-tpg gene pair, which encodes a telomere-associated protein and a terminal protein for end patching, is located on pSLA2-L and pSLA2-M but has not hitherto been found on the chromosome. These results led us to the idea that the tap-tpg of pSLA2-L or pSLA2-M functions to maintain a linear chromosome in strain 7434AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap-tpg of pSLA2-M.
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Cromosomas Bacterianos/genética , Cromosomas Bacterianos/ultraestructura , ADN Bacteriano/metabolismo , Plásmidos/genética , Streptomyces/genética , Secuencia de Aminoácidos , Replicación del ADN , Datos de Secuencia Molecular , Mutación , Recombinación Genética , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Telómero/genéticaRESUMEN
While some old adults stay healthy and non-frail up to late in life, others experience multimorbidity and frailty often accompanied by a pro-inflammatory state. The underlying molecular mechanisms for those differences are still obscure. Here, we used gene expression analysis to understand the molecular underpinning between non-frail and frail individuals in old age. Twenty-four adults (50% non-frail and 50% frail) from InCHIANTI study were included. Total RNA extracted from whole blood was analyzed by Cap Analysis of Gene Expression (CAGE). CAGE identified transcription start site (TSS) and active enhancer regions. We identified a set of differentially expressed (DE) TSS and enhancer between non-frail and frail and male and female participants. Several DE TSSs were annotated as lncRNA (XIST and TTTY14) and antisense RNAs (ZFX-AS1 and OVCH1 Antisense RNA 1). The promoter region chr6:366,786,54-366,787,97;+ was DE and overlapping the longevity CDKN1A gene. GWAS-LD enrichment analysis identifies overlapping LD-blocks with the DE regions with reported traits in GWAS catalog (isovolumetric relaxation time and urinary tract infection frequency). Furthermore, we used weighted gene co-expression network analysis (WGCNA) to identify changes of gene expression associated with clinical traits and identify key gene modules. We performed functional enrichment analysis of the gene modules with significant trait/module correlation. One gene module is showing a very distinct pattern in hub genes. Glycogen Phosphorylase L (PYGL) was the top ranked hub gene between non-frail and frail. We predicted transcription factor binding sites (TFBS) and motif activity. TF involved in age-related pathways (e.g., FOXO3 and MYC) shows different expression patterns between non-frail and frail participants. Expanding the study of OVCH1 Antisense RNA 1 and PYGL may help understand the mechanisms leading to loss of homeostasis that ultimately causes frailty.
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Fragilidad , ARN Largo no Codificante , Humanos , Masculino , Femenino , Anciano , Anciano Frágil , Fragilidad/genética , Perfilación de la Expresión Génica , ARN Largo no Codificante/genética , ARN sin Sentido/genéticaRESUMEN
Non-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid Polypedilum vanderplanki is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments. We used improved sequencing strategies to assemble a chromosome-level genome sequence for P. vanderplanki for deep comparative analysis of genomic location of genes associated with desiccation tolerance. Using whole genome-based cross-species and intra-species analysis, we provide evidence for the unique functional specialization of Chromosome 4 through extensive acquisition of novel genes. In contrast to other insect genomes, in the sleeping chironomid a uniquely high degree of subfunctionalization in paralogous anhydrobiosis genes occurs in this chromosome, as well as pseudogenization in a highly duplicated gene family. Our findings suggest that the Chromosome 4 in Polypedilum is a site of high genetic turnover, allowing it to act as a 'sandbox' for evolutionary experiments, thus facilitating the rapid adaptation of midges to harsh environments.
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Within the scope of the FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem cells (iPSCs) and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition. From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.3%) show molecular phenotypes. Integrating the molecular phenotypes with chromatin-interaction assays further reveals cis- and trans-interacting partners as potential primary targets. Additionally, cell-type enrichment analysis identifies lncRNAs associated with pluripotency, while the knockdown of LINC02595, CATG00000090305.1, and RP11-148B6.2 modulates colony formation of iPSCs. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit a consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights that molecular phenotyping is more comprehensive in revealing affected pathways.
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Células Madre Pluripotentes Inducidas , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Oligonucleótidos Antisentido , Perfilación de la Expresión Génica/métodos , Células Madre Embrionarias/metabolismoRESUMEN
Gene expression is controlled at the transcriptional and post-transcriptional levels. The TACC2 gene was known to be associated with tumors but the control of its expression is unclear. We have reported that activity of the intronic promoter p10 of TACC2 in primary lesion of endometrial cancer is indicative of lymph node metastasis among a low-risk patient group. Here, we analyze the intronic promoter derived isoforms in JHUEM-1 endometrial cancer cells, and primary tissues of endometrial cancers and normal endometrium. Full-length cDNA amplicons are produced by long-range PCR and subjected to nanopore sequencing followed by computational error correction. We identify 16 stable, 4 variable, and 9 rare exons including 3 novel exons validated independently. All variable and rare exons reside N-terminally of the TACC domain and contribute to isoform variety. We found 240 isoforms as high-confidence, supported by more than 20 reads. The large number of isoforms produced from one minor promoter indicates the post-transcriptional complexity coupled with transcription at the TACC2 locus in cancer and normal cells.
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Empalme Alternativo , Proteínas Portadoras/genética , Neoplasias Endometriales/patología , Exones , Intrones , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Supresoras de Tumor/genética , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Femenino , Humanos , Isoformas de Proteínas , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism's physiological function. Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. The recent technological boost in sequencing approaches unveiled long noncoding RNAs (lncRNAs) as prominent regulatory players of various gene expression levels in a cell-type-specific manner. RESULTS: To investigate the potential roles of lncRNAs in vascular biology, we performed antisense oligonucleotide (ASO) knockdowns of lncRNA candidates specifically expressed either in human lymphatic or blood vascular endothelial cells (LECs or BECs) followed by Cap Analysis of Gene Expression (CAGE-Seq). Here, we describe the quality control steps adopted in our analysis pipeline before determining the knockdown effects of three ASOs per lncRNA target on the LEC or BEC transcriptomes. In this regard, we especially observed that the choice of negative control ASOs can dramatically impact the conclusions drawn from the analysis depending on the cellular background. CONCLUSION: In conclusion, the comparison of negative control ASO effects on the targeted cell type transcriptomes highlights the essential need to select a proper control set of multiple negative control ASO based on the investigated cell types.
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Técnicas de Silenciamiento del Gen/métodos , Oligonucleótidos Antisentido/genética , Especificidad de Órganos/genética , ARN Largo no Codificante/genética , Adulto , Células Endoteliales/metabolismo , Técnicas de Silenciamiento del Gen/normas , Humanos , Sistema Linfático/citología , Sistema Linfático/metabolismo , Oligonucleótidos Antisentido/normas , TranscriptomaRESUMEN
Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.
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Repeticiones de Microsatélite , Redes Neurales de la Computación , Enfermedades Neurodegenerativas/genética , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción Genética , Células A549 , Animales , Secuencia de Bases , Biología Computacional/métodos , Aprendizaje Profundo , Elementos de Facilitación Genéticos , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/metabolismo , Polimorfismo Genético , Regiones Promotoras GenéticasRESUMEN
BACKGROUND: Japanese flounder (Paralichthys olivaceus) is one of the most economically important marine species in Northeast Asia. Information on genetic markers associated with quantitative trait loci (QTL) can be used in breeding programs to identify and select individuals carrying desired traits. Commercial production of Japanese flounder could be increased by developing disease-resistant fish and improving commercially important traits. Previous maps have been constructed with AFLP markers and a limited number of microsatellite markers. In this study, improved genetic linkage maps are presented. In contrast with previous studies, these maps were built mainly with a large number of codominant markers so they can potentially be used to analyze different families and populations. RESULTS: Sex-specific genetic linkage maps were constructed for the Japanese flounder including a total of 1,375 markers [1,268 microsatellites, 105 single nucleotide polymorphisms (SNPs) and two genes]; 1,167 markers are linked to the male map and 1,067 markers are linked to the female map. The lengths of the male and female maps are 1,147.7 cM and 833.8 cM, respectively. Based on estimations of map lengths, the female and male maps covered 79 and 82% of the genome, respectively. Recombination ratio in the new maps revealed F:M of 1:0.7. All linkage groups in the maps presented large differences in the location of sex-specific recombination hot-spots. CONCLUSIONS: The improved genetic linkage maps are very useful for QTL analyses and marker-assisted selection (MAS) breeding programs for economically important traits in Japanese flounder. In addition, SNP flanking sequences were blasted against Tetraodon nigroviridis (puffer fish) and Danio rerio (zebrafish), and synteny analysis has been carried out. The ability to detect synteny among species or genera based on homology analysis of SNP flanking sequences may provide opportunities to complement initial QTL experiments with candidate gene approaches from homologous chromosomal locations identified in related model organisms.
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Mapeo Cromosómico/métodos , Lenguado/genética , Ligamiento Genético , Animales , Femenino , Genoma/genética , Japón , Masculino , Repeticiones de Microsatélite/genética , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genética , Recombinación Genética/genética , Sintenía/genéticaRESUMEN
Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics, lankacidin and lankamycin, and carries three linear plasmids, pSLA2-L (211 kb), -M (113 kb), and -S (18 kb), whose nucleotide sequences were previously reported. The complete nucleotide sequence of the S. rochei chromosome has now been determined using the long-read PacBio RS-II sequencing together with short-read Illumina Genome Analyzer IIx sequencing and Roche 454 pyrosequencing techniques. The assembled sequence revealed an 8,364,802-bp linear chromosome with a high G + C content of 71.7% and 7,568 protein-coding ORFs. Thus, the gross genome size of S. rochei 7434AN4 was confirmed to be 8,706,406 bp including the three linear plasmids. Consistent with our previous study, a tap-tpg gene pair, which is essential for the maintenance of a linear topology of Streptomyces genomes, was not found on the chromosome. Remarkably, the S. rochei chromosome contains seven ribosomal RNA (rrn) operons (16S-23S-5S), although Streptomyces species generally contain six rrn operons. Based on 2ndFind and antiSMASH platforms, the S. rochei chromosome harbors at least 35 secondary metabolite biosynthetic gene clusters, including those for the 28-membered polyene macrolide pentamycin and the azoxyalkene compound KA57-A.
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Cromosomas Bacterianos , Genes Bacterianos , Metabolismo Secundario/genética , Streptomyces/genética , Secuencia de Bases , Mapeo Cromosómico , Familia de Multigenes , Plásmidos/genéticaRESUMEN
CAGE (cap analysis of gene expression) is a method for identifying transcription start sites by sequencing the first 20 or 21 nucleotides from the 5' end of capped transcripts, allowing genome-wide promoter analyses to be performed. The potential of the CAGE as a form of expression profiling was limited previously by sequencing technology and the labor-intensive protocol. Here we describe an improved CAGE method for use with a next generation sequencer. This modified method allows the identification of the RNA source of each CAGE tag within a pooled library by introducing DNA tags (barcodes). The method not only drastically improves the sequencing capacity, but also contributes to savings in both time and budget. Additionally, this pooled CAGE tag method enables the dynamic changes in promoter usage and gene expression to be monitored.
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Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN/instrumentación , Sitio de Iniciación de la Transcripción , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.
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Perfilación de la Expresión Génica , Genoma , Animales , Regulación de la Expresión Génica , Humanos , Ratones , Regiones Promotoras Genéticas , Especificidad de la EspecieRESUMEN
Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) loci are largely predictive of resistance to epidermal growth factor receptor (EGFR) therapy in colorectal cancer (CRC). A highly sensitive detection system for the KRAS gene mutations is urgently needed; however, conventional methods have issues with feasibility and cost performance. Here, we describe a novel detection system using a fluorescence 'Eprobe' capable of detecting low level KRAS gene mutations, via real-time PCR, with high sensitivity and simple usability. We designed our Eprobes to be complementary to wild-type (WT) KRAS or to the commonly mutated codons 12 and 13. The WT Eprobe binds strongly to the WT DNA template and suppresses amplification by blocking annealing of the primer during PCR. Eprobe-PCR with WT Eprobe shows high sensitivity (0.05-0.1% of plasmid DNA, 1% of genomic DNA) for the KRAS mutation by enrichment of the mutant type (MT) amplicon. Assay performance was compared to Sanger sequencing using 92 CRC samples. Discrepancies were analyzed by mutation genotyping via Eprobe-PCR with full match Eprobes for 7 prevalent mutations and the next generation sequencing (NGS). Significantly, the Eprobe system had a higher sensitivity for detecting KRAS mutations in CRC patient samples; these mutations could not be identified by Sanger sequencing. Thus, the Eprobe approach provides for highly sensitive and convenient mutation detection and should be useful for diagnostic applications.
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Neoplasias Colorrectales/genética , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Neoplasias Colorrectales/patología , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Desnaturalización de Ácido Nucleico/genética , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
In DNA amplification, the initial step of copying a target sequence from the template DNA--the so-called intermediate product generation step--is very important. In examining the turn-back primer (TP)-dependent isothermal DNA amplification (TIA) method, we determined the actual time point of intermediate product generation by extrapolating dsDNA amplification curves. Our results indicate that intermediate product creation is the rate-limiting step in TIA, and good TP design is advantageous for improving the intermediate production process.
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Cartilla de ADN/química , Modelos Genéticos , Técnicas de Amplificación de Ácido Nucleico/métodos , Sondas de Ácido Nucleico/química , Algoritmos , Cartilla de ADN/genética , Humanos , Modelos Moleculares , Sondas de Ácido Nucleico/genética , Análisis de Secuencia de ADN/métodosRESUMEN
Gene regulatory networks in living cells are controlled by the interaction of multiple cell type-specific transcription regulators with DNA binding sites in target genes. Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence binding protein (ICSBP), is a transcription factor expressed predominantly in myeloid and lymphoid cell lineages. To find the functional direct target genes of IRF8, the gene expression profiles of siRNA knockdown samples and genome-wide binding locations by ChIP-chip were analyzed in THP-1 myelomonocytic leukemia cells. Consequently, 84 genes were identified as functional direct targets. The ETS family transcription factor PU.1, also known as SPI1, binds to IRF8 and regulates basal transcription in macrophages. Using the same approach, we identified 53 direct target genes of PU.1; these overlapped with 19 IRF8 targets. These 19 genes included key molecules of IFN signaling such as OAS1 and IRF9, but excluded other IFN-related genes amongst the IRF8 functional direct target genes. We suggest that IRF8 and PU.1 can have both combined, and independent actions on different promoters in myeloid cells.
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Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Técnicas Genéticas , Humanos , Modelos Biológicos , Células Mieloides/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
BACKGROUND: Immediate early genes are considered to play important roles in dynamic gene regulatory networks following exposure to appropriate stimuli. One of the immediate early genes, early growth response gene 1 (EGR-1), has been implicated in differentiation of human monoblastoma cells along the monocytic commitment following treatment with phorbol ester. EGR-1 has been thought to work as a modifier of monopoiesis, but the precise function of EGR-1 in monocytic differentiation has not been fully elucidated. RESULTS: We performed the first genome-wide analysis of EGR-1 binding sites by chromatin immunoprecipitation with promoter array (ChIP-chip) and identified EGR-1 target sites in differentiating THP-1 cells. By combining the results with previously reported FANTOM4 data, we found that EGR-1 binding sites highly co-localized with CpG islands, acetylated histone H3 lysine 9 binding sites, and CAGE tag clusters. Gene Ontology (GO) analysis revealed enriched terms, including binding of molecules, in EGR-1 target genes. In addition, comparison with gene expression profiling data showed that EGR-1 binding influenced gene expression. Moreover, observation of in vivo occupancy changes of DNA binding proteins following PMA stimulation indicated that SP1 binding occupancies were dramatically changed near EGR-1 binding sites. CONCLUSIONS: We conclude that EGR-1 mainly recognizes GC-rich consensus sequences in promoters of active genes. GO analysis and gene expression profiling data confirm that EGR-1 is involved in initiation of information transmission in cell events. The observations of in vivo occupancy changes of EGR-1 and SP1 suggest that several types of interplay between EGR-1 and other proteins result in multiple responses to EGR-1 downstream genes.