BACKGROUND
the completed sequence of numerous vertebrate genomes has enabled rapid gene annotation across species using orthologous relationships.
this approach is feasible because purifying selection, acting on the open reading frames of coding exons and aimed at preserving encoded protein sequences, minimizes the sequence divergence that can occur.
the sequences of these protein-coding genes generally change more slowly over millions of years than do non-coding sequences.
similarity at the nucleotide level is reflected in the likeness of structure and function of the gene products produced in different species.
additional features, such as non-coding functional elements, are also maintained as conserved sequences across species through the action of purifying selection  <cit> .
enhancer elements are often predicted from their distinctive sequence conservation.
other functional classes, such as promoters, contain more plasticity in their composition and do not lend themselves to identification in this manner.
given that precise computational methods are not yet developed for predicting promoter regions in newly assembled genomes, their annotation lags behind that of coding genes and enhancers.
we hypothesized that promoter regions could be reliably mapped across species using a unique class of promoter that is flanked by genes on each side.
these promoters, known as bidirectional promoters, would be useful for annotating promoter regions across mammals because the genes on both the left and right sides of the promoter change slowly.
thus, the promoter region is maintained as a recognizable, intergenic, architectural region that is amenable to computational discovery.
furthermore, if no repetitive elements were inserted at the bidirectional promoter region in either species, the intergenic distances should be maintained across species.
to lend support to this hypothesis, takai and jones   <cit>  showed the exclusion of repetitive elements from bidirectional promoters of human chromosomes  <dig>   <dig>  and  <dig>
bidirectional promoters were originally defined as the regulatory regions present in the intergenic space of two oppositely oriented genes whose transcription start sites  were separated by no more than  <dig>  bp  <cit> .
such genes appear in a head-to-head arrangement, i.e.
facing away from one another, and are transcribed from opposite strands of dna.
the closely spaced arrangement of the tsss flanking the bidirectional promoter was recognized as a non-random event, proven by the fact that a greater-than-expected number of genes had this architecture  <cit> .
up to 10% of human protein-coding genes were initially identified with bidirectional promoters.
we subsequently identified thousands of additional, putative, bidirectional promoters by analyzing divergently transcribed, spliced est data  <cit> .
the methodology of mapping bidirectional promoters across species used here treats the genes on each side of a promoter as anchors that delimit the intergenic, orthologous regulatory region.
if the genome of the other species contains conserved gene order and orientation at the orthologous location, then the intergenic promoter region must have evolved from the ancestral sequence at that location.
if the intergenic distance of the annotated transcripts in the other species is also maintained as ≤  <dig>  bp, the orthologous bidirectional promoter is declared validated.
of added benefit, this method is not dependent on the level of nucleotide sequence conservation in the promoter regions, which can vary extensively  <cit> .
the enrichment of bidirectional promoters in the human genome evokes questions about their evolution.
in some cases, chromosomal rearrangements could have conjoined promoter regions of two genes.
those genes would remain united through all subsequent speciation events due to selective pressure against change.
any breakage of the union  could disrupt the normal regulation of both genes, potentially having profound  effects on cellular function.
if true, bidirectional promoters should provide an evolutionary timestamp of rearrangement events across mammalian genomes.
alternatively, some unidirectional promoters could have lost control of their regulated transcription, enabling rna polymerase to load and traverse in the opposite direction  <cit> .
this scenario could serve as a mechanism for generating new genes in the genome, which would occur in a rare and species-specific manner.
building on our previous computational infrastructure, we utilize updated human genome annotations to compare bidirectional promoters in human and bovine genomes to test the hypothesis that long-term evolutionary histories of these promoters could be identified and used to annotate the bovine genome.
we used these data to create a detailed regulatory map of orthologous promoter regions across  <dig> placental mammals .
as an outcome of the analysis, we have shown that the "locked" arrangement of genes around these promoters enables prediction of unannotated 5' utrs using cross-species comparisons.
furthermore, we identified bidirectional promoters that lack orthologous counterparts in all other species, supporting the conclusion that species-specific genes can be identified from rigorous, cross-species comparisons of this dataset.
one human-specific example was from the family of five recq helicase paralogs , all of which have bidirectional promoters that developed independently.
RESULTS
mapping bidirectional promoters in the cow genome
bidirectional promoters were predicted in the cow genome from in silico analyses of gene order, orientation and intergenic distances, analogous to our studies in the human and mouse genomes  <cit> .
the official bovine gene set  contained  <dig>  bidirectional gene pairs.
those predictions were made without the normal requirement for an intergenic distance of ≤  <dig>  bp due to incomplete annotation of 5' utrs in the cow genome.
thus, we labeled the data as "low-stringency" predictions.
in comparison, our previous studies in the human and mouse genomes identified  <dig> – <dig>  bidirectional promoters whose intergenic distances were limited to  <dig>  bp   <cit> , suggesting that the number of bidirectional promoters possible in the cow genome was as high as  <dig> .
we concluded that our low stringency bovine set captured a majority of actual bidirectional promoters, but that limited annotations contributed false positive and false negative predictions.
to further assess the bidirectional promoters in the cow genome, we examined additional transcript evidence.
the ogs v <dig> dataset, which lacked information regarding most 5' utrs of bovine genes, was supplemented with refseq annotations from genbank and spliced est data from the ucsc cow genome browser.
together these datasets identified  <dig>  bovine bidirectional promoters, all of which met the requirement of no more than  <dig>  bp separating any pair of tsss .
we hypothesized that bidirectional promoters form a core regulatory structure in all mammalian genomes.
to assess this hypothesis quantitatively, the intergenic distances for all promoters having a "head-to-head" configuration were examined in the human, chimp, cow, mouse, and rat genomes .
for comparison, we examined "head-to-tail" genes that are arranged in a parallel orientation and "tail-to-tail" genes whose 3' ends are adjacent.
these data showed a significant enrichment for the head-to-head arrangement within  <dig>  bp compared to the same distance in the other datasets .
human bidirectional promoters have orthologs in cow
to address a core set of orthologous bidirectional promoters in mammals, we mapped positions of the regulatory regions in the human and cow genomes.
we noted that less than 25%  of the human bidirectional promoters controlled expression of genes that encode proteins.
the remainder regulated combinations of protein-coding genes with rna gene partners or pairs of rna genes.
we proceeded by mapping only these  <dig>  human promoters from the protein coding set, because of the limited annotations of rna genes available in most mammalian species.
when compared between human and cow, 94% of the regions mapped to an orthologous position at one or both of the genes flanking each promoter.
moreover, the intergenic distances of orthologous bidirectional promoters were found to be similar for the majority of gene pairs .
nevertheless, despite the presence of orthology, some intergenic distances in cow were much larger than the allowable  <dig>  bp, providing an opportunity to assess if the differences represented biological diversity or technical anomalies.
individual gene pairs were examined for transcript evidence at the orthologous locations in cow.
those transcripts were required to uphold the  <dig>  bp intergenic distance rule to validate the location as an orthologous bidirectional promoter.
refseq annotations in cow provided validation for 20% of the  <dig>  human promoters .
an additional 7% of the cross-species predictions were validated from the ogs v <dig> annotations.
supplementing this comparison, the spliced est annotations from cow confirmed another 5% of the orthologous promoter predictions.
in total,  <dig> human promoters were validated in cow through a combined assessment of orthology and transcript annotations.
an additional  <dig> human bidirectional promoters were supported at orthologous locations by the low-stringency predictions in the cow genome.
these data confirmed that conserved synteny was maintained at regions orthologous to human bidirectional promoters; however, validation of those regions as true bidirectional promoters was hindered by the possibility of missing data at the 5' ends of genes in cow.
of the remaining comparisons, the majority were present at orthologous locations, but lacked an annotation for one of the two genes in the pair  or for both genes .
only 6% of the gene pairs had no record of orthology in the cow genome .
cross-species utr prediction
missing utr information in the bovine genome could be addressed using complementary evidence from multiple gene annotations.
over half of the bidirectional promoters predicted using cow ogs v <dig> annotations were the low stringency type, with an intergenic distance greater than  <dig>  bp.
comparison to the high-stringency est data identified 5'utrs for the genes in this data that brought neighboring tsss closer together.
these utrs were located at genomic distances of up to  <dig>  bp upstream of the characterized ogs v <dig> coding exons.
in a pilot experiment, we addressed the large intergenic distances found in cow relative to human by examining ten gene-pairs that had extremely large intergenic distances in cow.
the genes from each pair were spaced greater than  <dig> kb apart at their 5' ends, compared to a  <dig> kb intergenic distance in human .
all ten of the gene pairs were re-annotated to update their 5' utrs  and nine of ten were subsequently validated as bidirectional promoters in the cow genome.
following the addition of the utr annotations, a comparison of the cow and human genes showed uniformly long first introns, averaging  <dig>  bp.
these long distances contrasted sharply with the short distances being maintained between the tsss of each set of genes.
thus, manual curation performed for  <dig> cow genes confirmed that unannotated 5' utrs accounted for the failure of  <dig> of those regions to be validated as orthologous promoters in cow in our original comparison.
similar evidence for missing data at the 5' utrs was found in an analysis of the full dataset .
for example, human promoters were mapped to the cow genome and validated using cumulative evidence from each of the cow gene sets.
given that the reference gene annotations are maintained as separate resources, we found they contain complementary as well as unique information that supported our claims.
the ogs v <dig> data, which has a minimal number of 5' utr annotations, gained utrs from refseq or est data.
despite our best efforts, a large number  of ogs v <dig> gene predictions failed to be validated in bidirectional promoters because the est or refseq datasets lacked entries for those genes, confirming that the bovine transcriptome data is incomplete.
chrom
a reciprocal examination of orthology at bidirectional promoters mapped cow bidirectional promoters to the human genome.
ninety percent of bidirectional promoters in cow mapped to the human genome at orthologous locations.
a small number contained an intergenic distance that was too large to be called a bidirectional promoter in the human gene pair.
six of these regions were chosen for manual curation.
five of the six were confirmed as bidirectional promoters upon re-evaluation of the available transcript information  .
the new annotations were recorded in the  <dig> vega human gene annotations  <cit> .
chrom
a unique bidirectional promoter in cow
genomic rearrangements that displaced one gene from an orthologous bidirectional gene pair occurred in less than 1% of the genes analyzed.
an example of such an event was found when the bidirectional promoter for cow cyb5r <dig> .
did not validate in human.
the human region contained the ortholog for cyb5r <dig>  but not the partner gene from cow .
this partner was expressed in numerous cow expression libraries from the brain  and although it had a minimal open reading frame, it showed strong evidence for rna secondary structure .
the unique appearance of the transcript in cow was explained by a  <dig> mb chromosomal inversion on cow chromosome  <dig> .
the transcript dv <dig> crossed this rearrangement breakpoint.
none of the other sequenced mammalian genomes showed evidence of the  <dig> mb inversion.
a duplicated mir <dig> sine was found flanking the inversion on both sides, with one copy being embedded within exon  <dig> of the dv <dig> transcript.
although the repetitive element was implicated in the inversion, no clear model explained its role mechanistically.
conserved synteny between the human, macaque, chimp, mouse, rat, dog, and pig genomes indicated that no other genomes could reconstitute this transcript because it spanned the unique chromosomal junction.
therefore, the transcript dv <dig> was identified as a bovine-specific transcript being transcribed from a bidirectional promoter that was not bidirectional in any other species.
parallel evolution of bidirectional promoters regulating recq helicases
the presence of the unique bidirectional promoter in cow suggested that novel transcripts could be identified in any mammalian genome using our comparative approach.
we therefore examined the human genome for evidence of novel rna transcripts regulated by bidirectional promoters that were not predicted in other species.
we found that the bidirectional promoter of the bloom syndrome gene  regulated a candidate human-specific transcript.
with limited coding potential, reliable expression evidence, and strong secondary structure  this transcript  was generated from a chromosomal region that aligns at the nucleotide level in several mammals, from human to opossum, suggesting an evolutionary history of at least  <dig> million years  <cit> .
however, evidence for transcription from this site was limited to human.
exons of this transcript exhibited sequence identity between human and chimp, but not in more distant primate genomes.
furthermore, no compensatory mutations were present in the exonic regions of more distant primate sequences, which would have indicated that an rna gene was functional and under selection in those species.
a variant containing a small open reading frame under purifying selection in human and chimp  did not align to the marmoset genome .
we concluded that any coding potential of the transcript was limited within old world monkeys, including human.
these data indicated that the blm partner emerged from an existing promoter in a genomic region that has been stable since the time that marsupials split with the ancestor of modern humans yet has only recently become transcriptionally active in the opposite direction .
in addition to blm, four other recq paralogs exist in the human genome and all were found to have bidirectional promoters.
a phylogenetic tree of the five recq family members supported their early duplication from an ancestral gene early in metazoan evolution  <cit>  without any gene conversion events that would complicate the evolutionary assessment .
we questioned whether bidirectional promoters had emerged at these sites in a parallel manner, or if the original gene pair duplicated into a series of paralogous gene-pairs that carried along their promoters as part of the duplication event .
to investigate the relationships of the recq gene homologs and their partners, the evolutionary distances between human and cow orthologs were computed as ds and dn values .
for each recq gene the rate of synonymous substitutions exceeded that of nonsynonymous substitutions, confirming that purifying selection maintained their respective amino acid sequences .
divergence at the amino acid level  was equivalent to that of most protein coding genes in the human genome.
estimated by the bootstrap method in mega <dig>  <cit> )
in contrast to the relatively high similarity among paralogous recq family members, the partners of those genes showed no similarity with one another within the same genomes.
notably, the rate of synonymous substitutions substantially exceeded the rate of nonsynonymous substitutions for all of these orthologous gene pairs , indicating that purifying selection also maintained these partner sequences .
these results precluded a role for positive selection  as the explanation for all  <dig> paralogs having bidirectional promoters with unrelated partner genes.
thus, we concluded that each of these bidirectional promoters developed independently, with the blm promoter being the youngest example.
subsequent and ongoing analyses are aimed at identifying the components that could have caused this event.
thus far, no significant sequence alignments have been found between the promoters of the recq genes using blastz alignments.
estimated by the bootstrap method in mega <dig>  <cit> )
comparative vertebrate analyses
to further address the level of conservation expected at these promoter regions, we examined phylop scores  <cit> , which provide a measure of sequence constraint or accelerated change compared to a neutral background model.
multi-species alignments generated from  <dig> vertebrates were the source for the phylop scores .
coding sequences, which carry a strong signature of sequence conservation, had the highest scores of any dataset we examined.
bidirectional promoters  scored only slightly higher than promoters in head-to-tail arrangements .
the level of conservation decreased further for tail-to-tail genes and ancestral repeats.
thus, bidirectional promoters are not maintained as highly conserved sequences in mammalian genomes, despite their close positioning between genes that are under negative  selection.
instead, these promoters tolerate sequence divergence, which is consistent with observations of promoters in general  <cit> .
we found that a core set of regulatory factors is significantly enriched in the orthologous promoter set compared to the tail-to-tail regions .
our approach of mapping orthologous bidirectional promoters across species was expanded to produce a 5-species map of validated bidirectional promoters .
the dataset containing  <dig>  bidirectional promoters of human protein-coding genes was mapped to the other genomes and validated using refseq annotations from each of those genomes, keeping the  <dig>  bp maximal intergenic distance requirement.
nearly all regions showed orthology at one or both of the genes, except 10% of regions in chimp, 6% in cow, 5% in mouse and 8% in rat, respectively.
these locations represent complete changes in genomic content between species.
the remaining regions contained orthology but validated at relatively low rates as illustrated by the magenta areas in the heat map.
nevertheless, these promoters were validated across species in numbers consistent with the gene annotations available for each genome:  <dig> promoters in chimp,  <dig> in mouse and  <dig> in rat, respectively .
these data indicate that many 5' utrs remain to be annotated in mammalian genomes, moreover, some protein coding genes are absent from these gene collections.
discussion
bidirectional promoter analyses contribute many benefits to genomic annotations including predictions of unannotated genes and 5' utrs in mammalian genomes.
no other methods exist to predict utrs across species, and conventional techniques to align ests from other species perform poorly in the divergent utr sequences.
in addition to 5' utr annotations, the conserved architecture of bidirectional promoters enables annotation of orthologous promoters as well as identification of missing coding annotations in other mammals.
our previous work reported that similar intergenic distances were present at orthologous positions of bidirectional promoters in pairwise comparisons between human and mouse or human and chimp.
thus, the finding that the human and cow comparison showed fewer bidirectional promoters in the bovine genome was attributed to the early stage of the bovine annotation effort, in which 5' utrs of genes have not been fully characterized.
as the depth and coverage of transcribed regions in the cow genome increase, both the in-species annotations and cross-species validations of bidirectional promoters will benefit.
consistent with this idea, our analyses strongly advocate continued efforts towards defining the 5' ends of genes, in order to expedite the annotation of adjacent regions, which contain promoter sequences.
the resulting information will enable downstream analyses of conservation of regulatory networks that act through the same transcription factor binding sites.
despite the limitations imposed by the shallow depth of transcripts available in the bovine genome, our data provide strong evidence that bidirectional promoters can be mapped across species using conservation of gene synteny to rapidly annotate these functional regions in non-human genomes.
furthermore, the benefit of mapping these promoters independently in other species is that new bidirectional promoters can be reciprocally predicted and validated in the human genome.
we were able to use bidirectional promoters to identify chromosomal rearrangements, which harbor species-specific transcripts with robust evidence and biological intrigue.
for example, recq genes originated via duplication events that created  <dig> paralogs early in the evolution of metazoans, but their bidirectional partners are unrelated to each other.
therefore, the emergence of bidirectional promoters in this gene family was not a passive result of duplication of the original gene pair bringing the promoter along with it.
this conclusion is apparent from the lack of similarity among the partner genes.
moreover, dn/ds analyses show that the partners are under strong purifying selection and have not undergone accelerated change, which would have masked their original similarities if they had been paralogs.
any tendency towards rearrangement by these genes is no longer apparent, as each gene partner has remained stably associated with its recq paralog for more than  <dig> million years.
retrotransposition could be another mechanism bringing recq genes near their partners or vice versa, but does not explain the blm gene pairing.
the partner of the blm gene is unique to catarrhini and has only recently evolved, however, the underlying genomic sequence is orthologous to opossum sequence, precluding a recent introduction of the partner gene into the region.
given these data, we hypothesize that elements in or near the recq promoters are responsible for initiating transcription in the opposing direction.
recent work by core et al.
<dig>  <cit>  and seila et al.
<dig>  <cit>  demonstrates that promoters can load rna pol ii in both the forward and reverse directions to maintain a short region of open chromatin.
we propose that in some cases, this phenomenon could provide a mechanism for generating novel, full-length transcripts that are spliced and polyadenylated in a species-specific manner.
CONCLUSIONS
we have produced a record of orthologous bidirectional promoters in  <dig> placental mammals .
furthermore, we addressed the evolution of new genes that can be identified by mapping bidirectional promoters across species.
continued work on the development of a cross-species regulatory map for these promoters is likely to reveal additional information about transcripts that are not only unique to individual species, but also functionally relevant.

 the completed sequence of numerous vertebrate genomes has enabled rapid gene annotation across species using orthologous relationships. this approach is feasible because purifying selection, acting on the open reading frames of coding exons and aimed at preserving encoded protein sequences, minimizes the sequence divergence that can occur. the sequences of these protein-coding genes generally change more slowly over millions of years than do non-coding sequences. similarity at the nucleotide level is reflected in the likeness of structure and function of the gene products produced in different species. additional features, such as non-coding functional elements, are also maintained as conserved sequences across species through the action of purifying selection. enhancer elements are often predicted from their distinctive sequence conservation. other functional classes, such as promoters, contain more plasticity in their composition and do not lend themselves to identification in this manner. given that precise computational methods are not yet developed for predicting promoter regions in newly assembled genomes, their annotation lags behind that of coding genes and enhancers. we hypothesized that promoter regions could be reliably mapped across species using a unique class of promoter that is flanked by genes on each side. these promoters, known as bidirectional promoters, would be useful for annotating promoter regions across mammals because the genes on both the left and right sides of the promoter change slowly. thus, the promoter region is maintained as a recognizable, intergenic, architectural region that is amenable to computational discovery. furthermore, if no repetitive elements were inserted at the bidirectional promoter region in either species, the intergenic distances should be maintained across species. to lend support to this hypothesis, takai and jones showed the exclusion of repetitive elements from bidirectional promoters of human chromosomes and bidirectional promoters were originally defined as the regulatory regions present in the intergenic space of two oppositely oriented genes whose transcription start sites were separated by no more than bp. such genes appear in a head-to-head arrangement, i.e. facing away from one another, and are transcribed from opposite strands of dna. the closely spaced arrangement of the tsss flanking the bidirectional promoter was recognized as a non-random event, proven by the fact that a greater-than-expected number of genes had this architecture. up to 10% of human protein-coding genes were initially identified with bidirectional promoters. we subsequently identified thousands of additional, putative, bidirectional promoters by analyzing divergently transcribed, spliced est data. the methodology of mapping bidirectional promoters across species used here treats the genes on each side of a promoter as anchors that delimit the intergenic, orthologous regulatory region. if the genome of the other species contains conserved gene order and orientation at the orthologous location, then the intergenic promoter region must have evolved from the ancestral sequence at that location. if the intergenic distance of the annotated transcripts in the other species is also maintained as ≤ bp, the orthologous bidirectional promoter is declared validated. of added benefit, this method is not dependent on the level of nucleotide sequence conservation in the promoter regions, which can vary extensively. the enrichment of bidirectional promoters in the human genome evokes questions about their evolution. in some cases, chromosomal rearrangements could have conjoined promoter regions of two genes. those genes would remain united through all subsequent speciation events due to selective pressure against change. any breakage of the union could disrupt the normal regulation of both genes, potentially having profound effects on cellular function. if true, bidirectional promoters should provide an evolutionary timestamp of rearrangement events across mammalian genomes. alternatively, some unidirectional promoters could have lost control of their regulated transcription, enabling rna polymerase to load and traverse in the opposite direction. this scenario could serve as a mechanism for generating new genes in the genome, which would occur in a rare and species-specific manner. building on our previous computational infrastructure, we utilize updated human genome annotations to compare bidirectional promoters in human and bovine genomes to test the hypothesis that long-term evolutionary histories of these promoters could be identified and used to annotate the bovine genome. we used these data to create a detailed regulatory map of orthologous promoter regions across placental mammals. as an outcome of the analysis, we have shown that the "locked" arrangement of genes around these promoters enables prediction of unannotated 5' utrs using cross-species comparisons. furthermore, we identified bidirectional promoters that lack orthologous counterparts in all other species, supporting the conclusion that species-specific genes can be identified from rigorous, cross-species comparisons of this dataset. one human-specific example was from the family of five recq helicase paralogs, all of which have bidirectional promoters that developed independently.  mapping bidirectional promoters in the cow genome bidirectional promoters were predicted in the cow genome from in silico analyses of gene order, orientation and intergenic distances, analogous to our studies in the human and mouse genomes. the official bovine gene set contained bidirectional gene pairs. those predictions were made without the normal requirement for an intergenic distance of ≤ bp due to incomplete annotation of 5' utrs in the cow genome. thus, we labeled the data as "low-stringency" predictions. in comparison, our previous studies in the human and mouse genomes identified – bidirectional promoters whose intergenic distances were limited to bp, suggesting that the number of bidirectional promoters possible in the cow genome was as high as. we concluded that our low stringency bovine set captured a majority of actual bidirectional promoters, but that limited annotations contributed false positive and false negative predictions. to further assess the bidirectional promoters in the cow genome, we examined additional transcript evidence. the ogs v dataset, which lacked information regarding most 5' utrs of bovine genes, was supplemented with refseq annotations from genbank and spliced est data from the ucsc cow genome browser. together these datasets identified bovine bidirectional promoters, all of which met the requirement of no more than bp separating any pair of tsss. we hypothesized that bidirectional promoters form a core regulatory structure in all mammalian genomes. to assess this hypothesis quantitatively, the intergenic distances for all promoters having a "head-to-head" configuration were examined in the human, chimp, cow, mouse, and rat genomes. for comparison, we examined "head-to-tail" genes that are arranged in a parallel orientation and "tail-to-tail" genes whose 3' ends are adjacent. these data showed a significant enrichment for the head-to-head arrangement within bp compared to the same distance in the other datasets. human bidirectional promoters have orthologs in cow to address a core set of orthologous bidirectional promoters in mammals, we mapped positions of the regulatory regions in the human and cow genomes. we noted that less than 25% of the human bidirectional promoters controlled expression of genes that encode proteins. the remainder regulated combinations of protein-coding genes with rna gene partners or pairs of rna genes. we proceeded by mapping only these human promoters from the protein coding set, because of the limited annotations of rna genes available in most mammalian species. when compared between human and cow, 94% of the regions mapped to an orthologous position at one or both of the genes flanking each promoter. moreover, the intergenic distances of orthologous bidirectional promoters were found to be similar for the majority of gene pairs. nevertheless, despite the presence of orthology, some intergenic distances in cow were much larger than the allowable bp, providing an opportunity to assess if the differences represented biological diversity or technical anomalies. individual gene pairs were examined for transcript evidence at the orthologous locations in cow. those transcripts were required to uphold the bp intergenic distance rule to validate the location as an orthologous bidirectional promoter. refseq annotations in cow provided validation for 20% of the human promoters. an additional 7% of the cross-species predictions were validated from the ogs v annotations. supplementing this comparison, the spliced est annotations from cow confirmed another 5% of the orthologous promoter predictions. in total, human promoters were validated in cow through a combined assessment of orthology and transcript annotations. an additional human bidirectional promoters were supported at orthologous locations by the low-stringency predictions in the cow genome. these data confirmed that conserved synteny was maintained at regions orthologous to human bidirectional promoters; however, validation of those regions as true bidirectional promoters was hindered by the possibility of missing data at the 5' ends of genes in cow. of the remaining comparisons, the majority were present at orthologous locations, but lacked an annotation for one of the two genes in the pair or for both genes. only 6% of the gene pairs had no record of orthology in the cow genome. cross-species utr prediction missing utr information in the bovine genome could be addressed using complementary evidence from multiple gene annotations. over half of the bidirectional promoters predicted using cow ogs v annotations were the low stringency type, with an intergenic distance greater than bp. comparison to the high-stringency est data identified 5'utrs for the genes in this data that brought neighboring tsss closer together. these utrs were located at genomic distances of up to bp upstream of the characterized ogs v coding exons. in a pilot experiment, we addressed the large intergenic distances found in cow relative to human by examining ten gene-pairs that had extremely large intergenic distances in cow. the genes from each pair were spaced greater than kb apart at their 5' ends, compared to a kb intergenic distance in human. all ten of the gene pairs were re-annotated to update their 5' utrs and nine of ten were subsequently validated as bidirectional promoters in the cow genome. following the addition of the utr annotations, a comparison of the cow and human genes showed uniformly long first introns, averaging bp. these long distances contrasted sharply with the short distances being maintained between the tsss of each set of genes. thus, manual curation performed for cow genes confirmed that unannotated 5' utrs accounted for the failure of of those regions to be validated as orthologous promoters in cow in our original comparison. similar evidence for missing data at the 5' utrs was found in an analysis of the full dataset. for example, human promoters were mapped to the cow genome and validated using cumulative evidence from each of the cow gene sets. given that the reference gene annotations are maintained as separate resources, we found they contain complementary as well as unique information that supported our claims. the ogs v data, which has a minimal number of 5' utr annotations, gained utrs from refseq or est data. despite our best efforts, a large number of ogs v gene predictions failed to be validated in bidirectional promoters because the est or refseq datasets lacked entries for those genes, confirming that the bovine transcriptome data is incomplete. chrom a reciprocal examination of orthology at bidirectional promoters mapped cow bidirectional promoters to the human genome. ninety percent of bidirectional promoters in cow mapped to the human genome at orthologous locations. a small number contained an intergenic distance that was too large to be called a bidirectional promoter in the human gene pair. six of these regions were chosen for manual curation. five of the six were confirmed as bidirectional promoters upon re-evaluation of the available transcript information. the new annotations were recorded in the vega human gene annotations. chrom a unique bidirectional promoter in cow genomic rearrangements that displaced one gene from an orthologous bidirectional gene pair occurred in less than 1% of the genes analyzed. an example of such an event was found when the bidirectional promoter for cow cyb5r. did not validate in human. the human region contained the ortholog for cyb5r but not the partner gene from cow. this partner was expressed in numerous cow expression libraries from the brain and although it had a minimal open reading frame, it showed strong evidence for rna secondary structure. the unique appearance of the transcript in cow was explained by a mb chromosomal inversion on cow chromosome. the transcript dv crossed this rearrangement breakpoint. none of the other sequenced mammalian genomes showed evidence of the mb inversion. a duplicated mir sine was found flanking the inversion on both sides, with one copy being embedded within exon of the dv transcript. although the repetitive element was implicated in the inversion, no clear model explained its role mechanistically. conserved synteny between the human, macaque, chimp, mouse, rat, dog, and pig genomes indicated that no other genomes could reconstitute this transcript because it spanned the unique chromosomal junction. therefore, the transcript dv was identified as a bovine-specific transcript being transcribed from a bidirectional promoter that was not bidirectional in any other species. parallel evolution of bidirectional promoters regulating recq helicases the presence of the unique bidirectional promoter in cow suggested that novel transcripts could be identified in any mammalian genome using our comparative approach. we therefore examined the human genome for evidence of novel rna transcripts regulated by bidirectional promoters that were not predicted in other species. we found that the bidirectional promoter of the bloom syndrome gene regulated a candidate human-specific transcript. with limited coding potential, reliable expression evidence, and strong secondary structure this transcript was generated from a chromosomal region that aligns at the nucleotide level in several mammals, from human to opossum, suggesting an evolutionary history of at least million years. however, evidence for transcription from this site was limited to human. exons of this transcript exhibited sequence identity between human and chimp, but not in more distant primate genomes. furthermore, no compensatory mutations were present in the exonic regions of more distant primate sequences, which would have indicated that an rna gene was functional and under selection in those species. a variant containing a small open reading frame under purifying selection in human and chimp did not align to the marmoset genome. we concluded that any coding potential of the transcript was limited within old world monkeys, including human. these data indicated that the blm partner emerged from an existing promoter in a genomic region that has been stable since the time that marsupials split with the ancestor of modern humans yet has only recently become transcriptionally active in the opposite direction. in addition to blm, four other recq paralogs exist in the human genome and all were found to have bidirectional promoters. a phylogenetic tree of the five recq family members supported their early duplication from an ancestral gene early in metazoan evolution without any gene conversion events that would complicate the evolutionary assessment. we questioned whether bidirectional promoters had emerged at these sites in a parallel manner, or if the original gene pair duplicated into a series of paralogous gene-pairs that carried along their promoters as part of the duplication event. to investigate the relationships of the recq gene homologs and their partners, the evolutionary distances between human and cow orthologs were computed as ds and dn values. for each recq gene the rate of synonymous substitutions exceeded that of nonsynonymous substitutions, confirming that purifying selection maintained their respective amino acid sequences. divergence at the amino acid level was equivalent to that of most protein coding genes in the human genome. estimated by the bootstrap method in mega ) in contrast to the relatively high similarity among paralogous recq family members, the partners of those genes showed no similarity with one another within the same genomes. notably, the rate of synonymous substitutions substantially exceeded the rate of nonsynonymous substitutions for all of these orthologous gene pairs, indicating that purifying selection also maintained these partner sequences. these results precluded a role for positive selection as the explanation for all paralogs having bidirectional promoters with unrelated partner genes. thus, we concluded that each of these bidirectional promoters developed independently, with the blm promoter being the youngest example. subsequent and ongoing analyses are aimed at identifying the components that could have caused this event. thus far, no significant sequence alignments have been found between the promoters of the recq genes using blastz alignments. estimated by the bootstrap method in mega ) comparative vertebrate analyses to further address the level of conservation expected at these promoter regions, we examined phylop scores, which provide a measure of sequence constraint or accelerated change compared to a neutral background model. multi-species alignments generated from vertebrates were the source for the phylop scores. coding sequences, which carry a strong signature of sequence conservation, had the highest scores of any dataset we examined. bidirectional promoters scored only slightly higher than promoters in head-to-tail arrangements. the level of conservation decreased further for tail-to-tail genes and ancestral repeats. thus, bidirectional promoters are not maintained as highly conserved sequences in mammalian genomes, despite their close positioning between genes that are under negative selection. instead, these promoters tolerate sequence divergence, which is consistent with observations of promoters in general. we found that a core set of regulatory factors is significantly enriched in the orthologous promoter set compared to the tail-to-tail regions. our approach of mapping orthologous bidirectional promoters across species was expanded to produce a 5-species map of validated bidirectional promoters. the dataset containing bidirectional promoters of human protein-coding genes was mapped to the other genomes and validated using refseq annotations from each of those genomes, keeping the bp maximal intergenic distance requirement. nearly all regions showed orthology at one or both of the genes, except 10% of regions in chimp, 6% in cow, 5% in mouse and 8% in rat, respectively. these locations represent complete changes in genomic content between species. the remaining regions contained orthology but validated at relatively low rates as illustrated by the magenta areas in the heat map. nevertheless, these promoters were validated across species in numbers consistent with the gene annotations available for each genome: promoters in chimp, in mouse and in rat, respectively. these data indicate that many 5' utrs remain to be annotated in mammalian genomes, moreover, some protein coding genes are absent from these gene collections. discussion bidirectional promoter analyses contribute many benefits to genomic annotations including predictions of unannotated genes and 5' utrs in mammalian genomes. no other methods exist to predict utrs across species, and conventional techniques to align ests from other species perform poorly in the divergent utr sequences. in addition to 5' utr annotations, the conserved architecture of bidirectional promoters enables annotation of orthologous promoters as well as identification of missing coding annotations in other mammals. our previous work reported that similar intergenic distances were present at orthologous positions of bidirectional promoters in pairwise comparisons between human and mouse or human and chimp. thus, the finding that the human and cow comparison showed fewer bidirectional promoters in the bovine genome was attributed to the early stage of the bovine annotation effort, in which 5' utrs of genes have not been fully characterized. as the depth and coverage of transcribed regions in the cow genome increase, both the in-species annotations and cross-species validations of bidirectional promoters will benefit. consistent with this idea, our analyses strongly advocate continued efforts towards defining the 5' ends of genes, in order to expedite the annotation of adjacent regions, which contain promoter sequences. the resulting information will enable downstream analyses of conservation of regulatory networks that act through the same transcription factor binding sites. despite the limitations imposed by the shallow depth of transcripts available in the bovine genome, our data provide strong evidence that bidirectional promoters can be mapped across species using conservation of gene synteny to rapidly annotate these functional regions in non-human genomes. furthermore, the benefit of mapping these promoters independently in other species is that new bidirectional promoters can be reciprocally predicted and validated in the human genome. we were able to use bidirectional promoters to identify chromosomal rearrangements, which harbor species-specific transcripts with robust evidence and biological intrigue. for example, recq genes originated via duplication events that created paralogs early in the evolution of metazoans, but their bidirectional partners are unrelated to each other. therefore, the emergence of bidirectional promoters in this gene family was not a passive result of duplication of the original gene pair bringing the promoter along with it. this conclusion is apparent from the lack of similarity among the partner genes. moreover, dn/ds analyses show that the partners are under strong purifying selection and have not undergone accelerated change, which would have masked their original similarities if they had been paralogs. any tendency towards rearrangement by these genes is no longer apparent, as each gene partner has remained stably associated with its recq paralog for more than million years. retrotransposition could be another mechanism bringing recq genes near their partners or vice versa, but does not explain the blm gene pairing. the partner of the blm gene is unique to catarrhini and has only recently evolved, however, the underlying genomic sequence is orthologous to opossum sequence, precluding a recent introduction of the partner gene into the region. given these data, we hypothesize that elements in or near the recq promoters are responsible for initiating transcription in the opposing direction. recent work by core et al. and seila et al. demonstrates that promoters can load rna pol ii in both the forward and reverse directions to maintain a short region of open chromatin. we propose that in some cases, this phenomenon could provide a mechanism for generating novel, full-length transcripts that are spliced and polyadenylated in a species-specific manner.  we have produced a record of orthologous bidirectional promoters in placental mammals. furthermore, we addressed the evolution of new genes that can be identified by mapping bidirectional promoters across species. continued work on the development of a cross-species regulatory map for these promoters is likely to reveal additional information about transcripts that are not only unique to individual species, but also functionally relevant. 
we found evidence supporting the independent emergence of bidirectional promoters from the family of five recq helicase genes, which gained their bidirectional promoters and partner genes independently rather than through a duplication process. this type of regulatory architecture is found more frequently than expected by chance in the human genome, yet many specifics underlying the regulatory design are unknown. bidirectional promoters are shared regulatory regions that influence the expression of two oppositely oriented genes. given that the function of most orthologous genes is similar across species, we hypothesized that the architecture and regulation of bidirectional promoters might also be similar across species, representing a core regulatory structure and enabling annotation of these regions in additional mammalian genomes. a representative set of human bidirectional promoters was mapped to orthologous sequences in other mammals. the two predictions that did not have orthology as bidirectional promoters in the other species resulted from unique events that initiated transcription in the opposite direction in only those species. furthermore, by expanding our comparisons from pairwise to multispecies analyses we developed a map representing a core set of bidirectional promoters in mammals. we show that the orthologous positions of bidirectional promoters provide a reliable guide to directly annotate over one thousand regulatory regions in sequences of mammalian genomes, while also serving as a useful tool to predict 5' utr positions and identify genes that are novel to a single species. by mapping the intergenic distances of genes in human, chimpanzee, bovine, murine, and rat, we show an enrichment for pairs of genes equal to or less than bp between their adjacent 5' ends compared to pairs of genes that fall in the same orientation or whose 3' ends are side-by-side.
 bidirectional promoters are shared regulatory regions that influence the expression of two oppositely oriented genes. this type of regulatory architecture is found more frequently than expected by chance in the human genome, yet many specifics underlying the regulatory design are unknown. given that the function of most orthologous genes is similar across species, we hypothesized that the architecture and regulation of bidirectional promoters might also be similar across species, representing a core regulatory structure and enabling annotation of these regions in additional mammalian genomes.  by mapping the intergenic distances of genes in human, chimpanzee, bovine, murine, and rat, we show an enrichment for pairs of genes equal to or less than bp between their adjacent 5' ends compared to pairs of genes that fall in the same orientation or whose 3' ends are side-by-side. a representative set of human bidirectional promoters was mapped to orthologous sequences in other mammals. we confirmed predictions for 5' utrs in nine of ten manual picks in bovine based on comparison to the orthologous human promoter set and in six of seven predictions in human based on comparison to the bovine dataset. the two predictions that did not have orthology as bidirectional promoters in the other species resulted from unique events that initiated transcription in the opposite direction in only those species. we found evidence supporting the independent emergence of bidirectional promoters from the family of five recq helicase genes, which gained their bidirectional promoters and partner genes independently rather than through a duplication process. furthermore, by expanding our comparisons from pairwise to multispecies analyses we developed a map representing a core set of bidirectional promoters in mammals.  we show that the orthologous positions of bidirectional promoters provide a reliable guide to directly annotate over one thousand regulatory regions in sequences of mammalian genomes, while also serving as a useful tool to predict 5' utr positions and identify genes that are novel to a single species. 