Direct isolation and identification of promoters in the human genome

  1. Tae Hoon Kim1,
  2. Leah O. Barrera1,
  3. Chunxu Qu1,
  4. Sara Van Calcar1,
  5. Nathan D. Trinklein4,
  6. Sara J. Cooper4,
  7. Rosa M. Luna2,
  8. Christopher K. Glass2,
  9. Michael G. Rosenfeld3,
  10. Richard M. Myers4, and
  11. Bing Ren1,2,5
  1. 1 Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093, USA
  2. 2 Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093, USA
  3. 3 Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093, USA
  4. 4 Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA

Abstract

Transcriptional regulatory elements play essential roles in gene expression during animal development and cellular response to environmental signals, but our knowledge of these regions in the human genome is limited despite the availability of the complete genome sequence. Promoters mark the start of every transcript and are an important class of regulatory elements. A large, complex protein structure known as the pre-initiation complex (PIC) is assembled on all active promoters, and the presence of these proteins distinguishes promoters from other sequences in the genome. Using components of the PIC as tags, we isolated promoters directly from human cells as protein-DNA complexes and identified the resulting DNA sequences using genomic tiling microarrays. Our experiments in four human cell lines uncovered 252 PIC-binding sites in 44 semirandomly selected human genomic regions comprising 1% (30 megabase pairs) of the human genome. Nearly 72% of the identified fragments overlap or immediately flank 5′ ends of known cDNA sequences, while the remainder is found in other genomic regions that likely harbor putative promoters of unannotated transcripts. Indeed, molecular analysis of the RNA isolated from one cell line uncovered transcripts initiated from over half of the putative promoter fragments, and transient transfection assays revealed promoter activity for a significant proportion of fragments when they were fused to a luciferase reporter gene. These results demonstrate the specificity of a genome-wide analysis method for mapping transcriptional regulatory elements and also indicate that a small, yet significant number of human genes remains to be discovered.

Footnotes

  • [Supplemental material is available online at www.genome.org.]

  • Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.3430605. Article published online before print in May 2005.

  • 5 Corresponding author. E-mail biren{at}ucsd.edu; fax (858) 534-7750.

    • Accepted March 28, 2005.
    • Received November 3, 2004.
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