Nitrate elimination.

A systematic approach is described for analysis of evolutionarily conserved cis-regulatory DNA using cis-Decoder, a tool for discovery of conserved sequence elements that are shared between similarly regulated enhancers. Analysis of 2,086 conserved sequence blocks (CSBs), identified from 135 characterized enhancers, reveals most CSBs consist of shorter overlapping/adjacent elements that are either enhancer type-specific or common to enhancers with divergent regulatory behaviors. Our findings suggest that enhancers employ overlapping repertoires of highly conserved core elements. Background Tissue-specific coordinate gene expression requires multiple inputs that involve dynamic interactions between sequence specific DNA-binding transcription factors and their target DNAs. The enhancer or cis-regulatory module is the focal point of integration for many of these regulatory events. Enhancers, which usually span 0.5 to 1.0 kb, contain clusters of transcription factor DNA-binding sites (reviewed by [1-3]). DNA sequence comparisons of different co-regulating enhancers suggest that many may rely on different combinations of transcription factors to achieve coordinate gene regulation. For example, the Drosophila pan-neural genes deadpan, scratch and snail all have distinct central nervous system (CNS) enhancers that drive expression in the same embryonic neuroblasts, yet comparisons of these enhancers reveal that they have few sequences in common [4,5]. Comparative genomic analysis of orthologous cis-regulatory regions reveals that many contain multi-species conserved sequences (MCSs; reviewed by [6-8]). Close inspection of enhancer MCSs reveals that these sequences are made up of smaller blocks of conserved sequences, designated here as 'conserved sequence blocks' (CSBs). EvoPrint analysis of enhancer CSBs reveals that many have remained unchanged for over 160 million years (My) of collective divergence [9] (and see below). CSBs that are over 10 base-pairs (bp) long are likely to be made up of adjacent or overlapping sequencespecific transcription factor DNA-binding sites. For example, DNA-binding sites for transcription factors that play essential roles in the regulation of the previously characterized Drosophila Krüppel central domain enhancer [10-12] are found adjacent to or overlapping one another within enhancer CSBs [9]. Although transcription factor consensus DNA-binding sites are detected within CSBs, searches of 2,086 CSBs (27,996 total bp) curated from 35 mammalian and 99 Drosophila characterized enhancers reveal that well over half of the sequences do not correspond to known DNA-binding sites and, as yet, have no assigned function(s) (this paper). Published: 9 May 2007 Genome Biology 2007, 8:R75 (doi:10.1186/gb-2007-8-5-r75) Received: 29 September 2006 Revised: 18 December 2006 Accepted: 9 May 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/5/R75 Genome Biology 2007, 8:R75 R75.2 Genome Biology 2007, Volume 8, Issue 5, Article R75 Brody et al. http://genomebiology.com/2007/8/5/R75 In order to initiate the functional dissection of novel CSBs and to gain a better understanding of their substructure, we have developed a multi-step protocol and accompanying computer algorithms (collectively known as cis-Decoder; see Figure 1) that allow for the rapid identification of short 6 to 14 bp DNA sequence elements, called cis-Decoder tags (cDTs), within enhancer CSBs that are also present in CSBs from other enhancers with either related or divergent functions. There is no limit to the number of enhancer CSBs examined by this approach, which allows one to build large cDT-libraries. Due to their different copy numbers, positions and/or orientations within the different enhancers, the conserved short sequence elements may otherwise go unnoticed by more conventional DNA alignment programs. Because this approach does not rely on any previously described transcription factor consensus DNA-binding site information or any other predicted motif or the presence of overrepresented sequences, cis-Decoder analysis affords an unbiased 'evo-centric' view of shared single or multiple sequence homologies between different enhancers. The cDT-libraries and cis-Decoder alignment tools enable one to differentiate between functionally different enhancers before any experimental expression data have been collected. cis-Decoder analysis reveals that most CSBs have a modular structure made up of two classes of interlocking sequence elements: those that are conserved only in other enhancers that regulate overlapping expression patterns; and more common conserved sequence elements that are part of divergently regulated enhancers. To demonstrate the efficacy of cis-Decoder analysis in identifying shared enhancer sequence elements, we show how cDTlibrary scans of different EvoPrinted mammalian and Drosophila enhancers accurately identify shared sequences within enhancers involved in similar regulatory behaviors. The cis-regulatory regions of the mammalian Delta-like 1 (Dll1) and Drosophila snail genes, which contain closely associated neural and mesodermal enhancers, were selected to highlight cis-Decoder's ability to differentiate between enhancers with different regulatory functions. We show how a cDT-library generated from both mammalian and Drosophila enhancer CSBs can be used to identify enhancer typespecific elements that have been conserved during the evolutionary diversification of metazoans. Finally, we show how cis-Decoder analysis can be used to examine novel putative enhancer regions. Results and discussion Generation of EvoPrints and CSB-libraries Our analysis of mammalian cis-regulatory sequences included 14 neural and 21 mesodermal enhancers whose regulatory behaviors have been characterized in developing mouse embryos. A full list of enhancers used in this study and the references describing their embryonic expression patterns is given in Table 1. In most cases, their EvoPrints included orthologs from placental mammals (human, chimp, rhesus monkey, cow, dog, mouse, rat) or also included the opossum; these species afford enough additive divergence (≥200 My) to resolve most enhancer MCSs [13]. When possible, chicken and frog orthologs were also included in the EvoPrints. Except when EvoDifference profiles [9] revealed sequencing gaps or genomic rearrangements in one or more species that were not present in the majority of the different orthologous DNAs, pair-wise reference species versus test species readouts from all of the above BLAT formatted genomes [14] were used to generate the EvoPrints. Using the EvoPrint-Parser program, both forward and reverse-complement sequences of each enhancer CSB of 6 bp or greater were extracted, named and consecutively numbered. Based on their enhancer regulatory expression pattern, CSBs were grouped into two different CSB-libraries, neural and mesodermal (Tables 1 and 2). Although there exists a distinction between expression in either neural or mesodermal tissues, each of the CSB-libraries represent a heterogeneous population of enhancers that drive gene expression in different cells and/or different developmental times in these tissues. For this study, CSBs of 5 bp or less were not included in the analysis. Although these shorter CSBs, particularly the 5 and 4 bp CSBs, are most likely important for enhancer function, the use of CSBs of 6 bp or larger (representing greater than 80% of the conserved MCS sequences) is sufficient to resolve sequence element differences between enhancers that regulate divergent expression patterns (see cis-Decoder methodology for identification of conserved sequence elem nts shared among diffe ent enhancers Figure 1 cis-Decoder methodology for identification of conserved sequence elements shared among different enhancers. The cis-Decoder methodology allows one to discover short 6 to 14 bp sequence elements within conserved enhancer sequences that are shared by other functionally related enhancers or are common to many enhancers with divergent regulatory behaviors. These shared sequence elements or cDTs can be used to identify and differentiate between cis-regulatory enhancer regions that regulate different tissue-specific expression patterns. cisDecoder analysis involves the sequential use of the following webaccessed computer algorithms: EvoPrinter → EvoPrint-parser → CSB-aligner → cDT-scanner → Full-enhancer scanner → cDT-cataloger. 1. EvoPrinter Detects MCSsand optimizes choiceof test speciesDNA using EvoDifference prints. 4. cDT-scanner Scans an EvoPrint with different cDT-libraries to identify shared conserved sequence elements. 2. EvoPrint-parser Curates Conserved Sequence Blocks (CSBs) to generate CSB-libraries from functionally related enhancers. 5. Full-enhancer scanner Identif ies repeated cDTs and/or CSBs in less conserved sequences flanking enhancer CSBs. 3. CSB-aligner Identif ies shared sequence elements in related or unrelated enhancer CSBs to generate different cDT-libraries. 6. cDT-cataloger Lists enhancer CSBs with shared sequence elements. Genome Biology 2007, 8:R75 http://genomebiology.com/2007/8/5/R75 Genome Biology 2007, Volume 8, Issue 5, Article R75 Brody et al. R75.3