Mapping DNA-protein interactions in large genomes by sequence tag analysis of genomic enrichment.
Identifying the chromosomal targets of transcription factors is important for reconstructing the transcriptional regulatory networks underlying global gene expression programs. We have developed an unbiased genomic method called sequence tag analysis of genomic enrichment (STAGE) to identify the direct binding targets of transcription factors in vivo. STAGE is based on high-throughput sequencing of concatemerized tags derived from target DNA enriched by chromatin immunoprecipitation. We first used STAGE in yeast to confirm that RNA polymerase III genes are the most prominent targets of the TATA-box binding protein. We optimized the STAGE protocol and developed analysis methods to allow the identification of transcription factor targets in human cells. We used STAGE to identify several previously unknown binding targets of human transcription factor E2F4 that we independently validated by promoter-specific PCR and microarray hybridization. STAGE provides a means of identifying the chromosomal targets of DNA-associated proteins in any sequenced genome.
Kim
Jonghwan
J
Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology & Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712-0159, USA.
Bhinge
Akshay A
AA
Morgan
Xochitl C
XC
Iyer
Vishwanath R
VR
D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D050704:E2F4 Transcription Factor; D005821:Genetic Techniques; D016678:Genome; D006801:Humans; D047468:Immunoprecipitation; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050704:E2F4 Transcription Factor; D014157:Transcription Factors; D004247:DNA