## Description

Bidirectional promoters are the regulatory regions that fall between pairs of genes, where the 5' ends of the genes within a pair are positioned in close proximity to one another. This spacing facilitates the initiation of transcription of both genes, creating two transcription forks that advance in opposite directions. The formal definition of a bidirectional promoter requires that the transcription initiation sites are separated by no more than 1,000 bp from one another. Using these criteria we have comprehensively annotated the human and mouse genomes for the presence of bidirectional promoters, using in silico approaches. The identification of these promoters is contingent upon the presence of adjacent, oppositely oriented pairs of genes, because few distinguishing features are available to uniquely identify bidirectional promoters de novo. Genomic annotations used for our identification phase include:

- A) UCSC known genes annotations (items with score=800).
- B) GenBank mRNA annotations (score=600).
- C) spliced ESTs (score=400).

The annotations for protein coding genes (A) are strongly supported and therefore provide a high quality dataset for mapping bidirectional promoters. In contrast, bidirectional promoters supported by spliced ESTs (C) alone have varying levels of evidence, ranging from one characterized transcript to hundreds of them. For this reason, the mRNA annotation (B) from GenBank provides a stringent level of validation for the start sites of the EST transcripts. As a large class of regulatory sequences, bidirectional promoters exemplify a rich source of unexplored biological information in the human genome. When compared to the mouse genome, these promoters are identifiable as truly orthologous locations, being maintained in regions of conserved synteny (including both genes and the intervening promoter region) that have undergone no rearrangements since the last common ancestor of mammals, and in some cases fish. We use this approach to annotate orthologous bidirectional promoters in nonhuman species until genomic annotations become available.

## Methods

### Assigning Orthologous Regions

A multi-stage approach to mapping orthology at bidirectional promoters was developed. Orthology assignments are strongest in coding regions. Therefore we began by mapping single human genes regulated by bidirectional promoters from the Known Genes annotations onto the mouse genome. Orthology assignments were determined using the "chains and nets" data from the UCSC Human Genome Browser mysql tables. Chains in the Genome Browser represent sequences of gapless aligned blocks. Nets provide a hierarchical ordering of those chains. Level 1 chains contain the longest, best-scoring sequence chains that span any selected region. Subsequent levels in the net represent the results of rearrangements, duplications, insertions and deletions that may have disrupted the presence of conserved synteny derived from an ancestral sequence.

### Confirming Orthologous Genes

After determining the orthology assignments using the UCSC chains and nets data, we used the Known Gene annotations or spliced ESTs to search the identity of genes within the corresponding region. Known Genes represent protein-coding genes and therefore can be verified by chains and nets alignments, followed by confirmation of protein identity in both species. Spliced ESTs carry less descriptive information than protein coding genes and therefore were validated in the second species by their presence in an orthologous region, showing conserved synteny of the two genes within a pair, and meeting the criteria of less than 1,000 bp of intergenic distance between those transcripts. Our method for mapping bidirectional promoters in spliced EST datasets is described in more detail in a previous publication. If the program verified evidence for orthology and conserved-syntenic gene arrangement, then the orthologous bidirectional promoter was confirmed. After orthologous assignments were confirmed for pairs of human genes, the reciprocal assignments were analyzed from mouse to human. Currently orthologous bidirectional promoter regions (that have been identified using UCSC known genes) have been mapped in human, chimp, macaque, mouse, rat, dog and cow genomes).

## Credits

These data were produced by Mary Q. Yang in the Elnitski lab at NHGRI, NIH. (contact: elnitski@mail.nih.gov)

## References

Yang MQ, Elnitski L. A computational study of bidirectional promoters in the human genome. Springer Lecture Series: Notes in Bioinformatics 2007.

Yang MQ, Elnitski L. Orthology of Bidirectional Promoters Enables Use of a Multiple Class Predictor for Discriminating Functional Elements in the Human Genome. Proceedings of the 2007 International Conference on Bioinformatics & Computational Biology. pp. 218-228. 2007. ISBN: 1-60132-042-6.

Yang MQ, Koehly L, Elnitski L. Comprehensive annotation of human bidirectional promoters identifies co-regulatory relationships among somatic breast and ovarian cancer genes. PLOS Computational Biology 2007.

Yang MQ, Taylor J, Elnitski L. Comparative analyses of bidirectional promoters in vertebrates. BMC Bioinformatics May 2008.

Piontkivska H, Yang MQ, Larkin DM, Lewin HA, Reecy J, Elnitski L. Cross-species mapping of bidirectional promoters enables prediction of unannotated 5' UTRs and identification of species-specific transcripts. BMC Genomics. 2009 Apr 24;10:189. PMID: 19393065.

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