This Article Full Text Full Text (PDF) Supplemental Research Data All Versions of this Article: 529803v1 13/7/1765    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Khambata-Ford, S. Articles by Myers, R. M. Search for Related Content PubMed PubMed Citation Articles by Khambata-Ford, S. Articles by Myers, R. M. Social Bookmarking                   What's this?

Methods

Identification of Promoter Regions in the Human Genome by Using a Retroviral Plasmid Library-Based Functional Reporter Gene Assay

¹ Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA ² Stanford Medical Informatics, Stanford University School of Medicine, Stanford, California 94305, USA ³ Stanford Human Genome Center, Stanford University School of Medicine, Stanford, California 94305, USA ⁴ Department of Computer Science, Stanford University, Stanford, California 94305, USA

Attempts to identify regulatory sequences in the human genome have involved experimental and computational methods such as cross-species sequence comparisons and the detection of transcription factor binding-site motifs in coexpressed genes. Although these strategies provide information on which genomic regions are likely to be involved in gene regulation, they do not give information on their functions. We have developed a functional selection for promoter regions in the human genome that uses a retroviral plasmid library-based system. This approach enriches for and detects promoter function of isolated DNA fragments in an in vitro cell culture assay. By using this method, we have discovered likely promoters of known and predicted genes, as well as many other putative promoter regions based on the presence of features such as CpG islands. Comparison of sequences of 858 plasmid clones selected by this assay with the human genome draft sequence indicates that a significantly higher percentage of sequences align to the 500-bp segment upstream of the transcription start sites of known genes than would be expected from random genomic sequences. We also observed enrichment for putative promoter regions of genes predicted in at least two annotation databases and for clones overlapping with CpG islands. Functional validation of randomly selected clones enriched by this method showed that a large fraction of these putative promoters can drive the expression of a reporter gene in transient transfection experiments. This method promises to be a useful genome-wide function-based approach that can complement existing methods to look for promoters.

⁵ Present address: Pharmacogenomics, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton NJ 08543, USA.

⁶ Corresponding author.
E-MAIL myers{at}shgc.stanford.edu; FAX (650) 725-9689.

Article published online before print in June 2003.

[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GenBank under accession nos. AY270202–AY271252.]

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Yaragatti, C. Basilico, and L. Dailey Identification of active transcriptional regulatory modules by the functional assay of DNA from nucleosome-free regions Genome Res., June 1, 2008; 18(6): 930 - 938. [Abstract] [Full Text] [PDF]

				G. M. Cooper and C. D. Brown Qualifying the relationship between sequence conservation and molecular function Genome Res., February 1, 2008; 18(2): 201 - 205. [Abstract] [Full Text] [PDF]

				G. M. Cooper, E. A. Stone, G. Asimenos, NISC Comparative Sequencing Program, E. D. Green, S. Batzoglou, and A. Sidow Distribution and intensity of constraint in mammalian genomic sequence Genome Res., July 1, 2005; 15(7): 901 - 913. [Abstract] [Full Text] [PDF]