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Background: Being the first noneutherian mammal sequenced, Monodelphis domestica (opossum) offers great potential for enhancing our understanding of the evolutionary processes that take place in mammals. This study focuses on the evolutionary relationships between conservation of noncoding sequences, cis-regulatory elements, and biologic functions of regulated genes in opossum and eight vertebrate species. Results: Analysis of 145 intergenic microRNA and all protein coding genes revealed that the upstream sequences of the former are up to twice as conserved as the latter among mammals, except in the first 500 base pairs, where the conservation is similar. Comparison of promoter conservation in 513 protein coding genes and related transcription factor binding sites (TFBSs) showed that 41% of the known human TFBSs are located in the 6.7% of promoter regions that are conserved between human and opossum. Some core biologic processes exhibited significantly fewer conserved TFBSs in human-opossum comparisons, suggesting greater functional divergence. A new measure of efficiency in multigenome phylogenetic footprinting (base regulatory potential rate [BRPR]) shows that including human-opossum conservation increases specificity in finding human TFBSs. Conclusion: Opossum facilitates better estimation of promoter conservation and TFBS turnover among mammals. The fact that substantial TFBS numbers are located in a small proportion of the human-opossum conserved sequences emphasizes the importance of marsupial genomes for phylogenetic footprinting-based motif discovery strategies. The BRPR measure is expected to help select genome combinations for optimal performance of these algorithms. Finally, although the etiology of the microRNA upstream increased conservation remains unknown, it is expected to have strong implications for our understanding of regulation of their expression. Published: 16 May 2007 Genome Biology 2007, 8:R84 (doi:10.1186/gb-2007-8-5-r84) Received: 6 November 2006 Revised: 29 January 2007 Accepted: 16 May 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/5/R84 Genome Biology 2007, 8:R84 R84.2 Genome Biology 2007, Volume 8, Issue 5, Article R84 Mahony et al. http://genomebiology.com/2007/8/5/R84 Background One of the prime motivating factors driving the sequencing of vertebrate genomes is the expectation that the role played by the functional regions of the human genome may be discerned by finding molecular level commonalities with and differences from other animals. This is especially true of the newly sequenced opossum (Monodelphis domestica), which is the first completed marsupial genome. Being the first noneutherian mammal sequenced, the opossum helps to clarify which sequence changes occurred before and after the divergence of mammalian ancestors from other vertebrates [1], and has already provided new insight into the evolution of mammalian major histocompatibility complex genes [2]. It is also hoped that the opossum genome may yield insights into how gene regulation has evolved in vertebrates. In protein coding genes, gene regulation is primarily controlled by short DNA sequences in the vicinity of the gene's transcription start sites (TSSs), which are targets for transcription factor proteins. A high degree of evolutionary conservation of these promoter regions can be attributed to functional cisregulatory elements. The increased conservation in the biologically more important parts of the promoter region has been explored by various phylogenetic footprinting algorithms, such as PhyloGibbs [3], ConSite [4], rVista [5], and FOOTER [6], to improve the prediction of transcription factor binding sites (TFBSs) in vertebrate genomes. Phylogenetic footprinting is a comparative genomics approach that exploits cross-species sequence conservation in order to predict regulatory genomic elements. In the absence of evolutionary information, TFBSs can be evaluated in terms of sequence similarity scans against frequency matrices derived from alignments of known binding sites for a given transcription factor [7]. However, the typical short length of TFBSs (5 to 20 base pairs [bp]) and their inherent level of sequence degeneracy makes them notoriously difficult to predict with any degree of specificity using similarity searches alone [8]. Phylogenetic footprinting provides a way to reduce the sequence search space to regions that are conserved (and therefore more likely to contain functional elements), thereby improving the specificity of TFBS prediction. In order to improve the performance of phylogenetic footprinting algorithms, the evolutionary aspects of the promoter regions and the TFBSs residing in them must be investigated. Evolutionary distance is an important factor in the effectiveness of phylogenetic footprinting techniques. For example, the divergence between chimpanzee and human is generally insufficient to reduce the sequence search space in any meaningful way; conversely, the divergence between Drosophila and human can be too large for any regulatory sequence conservation to be detected. Recently, the maximum sensitivity of phylogenetic footprinting techniques has been measured via estimations of the rate of TFBS 'turnover' between human and rodent genomes [9-13]. We consider that a TFBS has undergone turnover if the sequence in which it resides is not conserved between the species compared. High or low TFBS turnover rates do not necessarily coincide with the rate of changes in the regulatory mechanism (for instance, replacement TFBSs can arise by chance elsewhere in the promoter region or functional TFBSs may still be present in nonconserved regions). Turnover, however, corresponds to the minimum false-negative rate for detection of TFBSs via phylogenetic footprinting, and thus it serves as a critical bound on the success of such algorithms. Human-rodent TFBS turnover has been estimated at between 28% and 40% [9-13], suggesting that TFBSs are among the most malleable functional elements in the genomic landscape. However, although rodents and primates diverged relatively recently (approximately 90 million years ago [14]), the shorter generational time of rodents has placed a large degree of dissimilarity between the two clades, as is evident in the human-dog comparisons [15]. Therefore, TFBS turnover rates will have to be estimated in other mammals before a clearer picture of the selective pressure on mammalian TFBSs can emerge. Another major mechanism for control of gene expression is provided by microRNA (miRNA) genes. miRNAs are small (22 to 61 bp long), noncoding RNAs that downregulate their target genes via base complementarity to their mRNA molecules [16,17]. Each miRNA can target multiple genes and each gene can be targeted by multiple miRNAs [18-21]. In vertebrates, their expression is tissue specific [22] and has been shown to play an important role during development [23-25]. Although some miRNAs are found in the introns of coding genes and therefore are probably regulated by the promoters of the genes in which they reside [26], others are located in the intergenic parts of the genome. Little is known about the transcriptional regulation of these intergenic miRNAs, although RNA polymerase II appears to be involved in the process [27]. This suggests that they may have active promoter regions that contain cis-regulatory elements, similar to coding genes. The following question then arises; how does the conservation in the upstream regions of the intergenic miRNA genes compare with that of the protein coding genes? In this respect, opossum and the other vertebrate species provide a broad range of evolutionary distances in which this issue may be addressed. In this report we present our findings regarding promoter conservation of all protein coding genes and upstream sequence conservation of intergenic miRNA genes in eight vertebrate genomes as compared with human. To our knowledge, this is the first time that such a comprehensive study has been conducted on potential regulatory regions of both protein coding and miRNA genes in vertebrates. Also, because the opossum genome is placed at an evolutionary midpoint relative to eutherian mammals and nonmammalian vertebrates, using it as an outgroup to the existing eutherian genomes allows for the estimation of the mammalian TFBS turnover rate. Furthermore, the opossum genome provides an opportunity to assess which transcriptional signals and Genome Biology 2007, 8:R84 http://genomebiology.com/2007/8/5/R84 Genome Biology 2007, Volume 8, Issue 5, Article R84 Mahony et al. R84.3