Comparative Genomics of Gene Regulation—Conservation and Divergence of Cis-Regulatory Information Antonio CA Meireles-Filho and Alexander Stark

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Comparative genomics of gene regulation—conservation and divergence of cis-regulatory information Antonio CA Meireles-Filho and Alexander Stark

We recently witnessed a tremendous increase in genomics and regulator binding sites. Combined with detailed studies on gene regulation and in entirely sequenced genomes dissections of individual regulatory sequences, this has from closely related species. This has triggered analyses that answered old questions but created new ones and – in suggest a wide range of evolutionary dynamics of gene particular – fuelled the controversy over the evolutionary regulation, from rapid turnover of transcription-factor binding conservation of regulatory sequences. In this review, we sites to conservation of enhancer function across large will discuss current work on the conservation and diver- evolutionary distances. Many examples show that enhancers gence of regulatory sequences. can evolve beyond recognizable sequence similarity while retaining function. However, bioinformatics approaches are Main text increasingly able to detect conserved regulatory elements Gene-regulatory information through characteristic evolutionary sequence signatures. The genome encodes gene-regulatory information in the Cis-regulatory changes are also a major source of form of modular cis-regulatory sequences or enhancers, morphological evolution, which might be facilitated by many each of which contributes certain parts of a gene’s overall biochemically functional elements that are selectively neutral expression pattern in an additive fashion (e.g. [1]). and by the buffering function of redundant enhancers and Enhancers have been found thousands of base pairs ‘shadow’ enhancers. upstream or downstream of the regulated gene, in the gene’s or in neighboring genes’ introns, and even work Address across intervening genes (e.g. [2,3]). While this indicates Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, that the relative location of enhancers can be highly A-1030 Vienna, Austria variable, some enhancers’ positions are constrained, such Corresponding author: Stark, Alexander ([email protected]) as a distal enhancer of brinker in Drosophila and mosqui- toes [3], or the Hox gene cluster (see discussion in [3]).

Current Opinion in Genetics & Development 2009, 19:565–570 Each enhancer contains binding sites for specific sets of This review comes from a themed issue on TFs and is thus only active in cell types where certain Genomes and evolution factors are present. It integrates and thresholds regulatory Edited by Martha Bulyk and Michael Levine cues and is typically assumed to have a binary on/off Available online 11th November 2009 output. The TFs’ binding preferences can be summarized in the form of short regulatory motifs [4], which usually 0959-437X/$ – see front matter contain specified positions that are highly discriminative # 2009 Elsevier Ltd. All rights reserved. and positions that are more degenerate. While the basic DOI 10.1016/j.gde.2009.10.006 nature of enhancers as binding platforms for TFs is estab- lished, their more detailed structural requirements remain less clear. Two models represent the two extremes of structural requirements: the enhanceosome and the bill- Introduction board models (Figure 1;[5]). The enhanceosome model is The basis for multicellular life is the ability to create based on the interferon-b enhancer, which serves as a rigid functional specialization and distinct cell types through assembly platform for defined protein complexes and has differentially regulated gene expression. The information strict requirements on binding-site composition, order, that underlies this regulation is encoded in the genome and orientation, and spacing [6]. Enhanceosomes thus display is ‘read’ by sequence-specific transcription factors (TFs). near-perfect conservation across their entire lengths [6], To date, we only know a small fraction of any animal’s which is generally rare for enhancers [7]. A billboard regulatory sequences and deciphering the sequences’ spa- enhancer in its extreme form merely integrates regulatory tio-temporal activity has remained one of the greatest – and input from individual factors that can be bound in any most fascinating – challenges of today’s biology. order, orientation, and spacing. Even the individual factors themselves might change provided that the sum of activat- The past few years have seen tremendous progress in the ing and repressing inputs remains constant [5]. genome-wide assessment of gene expression and of transcription-factor DNA binding. Similarly, the sequencing of Most enhancers lack a perfect conservation of motif entire genomes from closely related species has enabled organization and lie between these extremes: neuro-ecto- comparative studies of known regulatory sequences dermal enhancers in fly and mesodermal enhancers in www.sciencedirect.com Current Opinion in Genetics & Development 2009, 19:565–570 566 Genomes and evolution

Figure 1

Enhancer models and their expected evolutionary characteristics. Enhancers evolve according to their functional constraints over short (e.g. within mammals or Drosophilidae) and long evolutionary time-spans (e.g. human–fish/chicken or D. melanogaster–mosquito/sepsidae. Rigid enhanceosome- type enhancers display extended sequence conservation to maintain the exact organization of TFBS (left; [6]). Billboard-type enhancers have only minimal functional constraints and allow binding-site substitutions provided that the sum of TF input activities remains constant (right; [5]). Over large evolutionary time-spans, they appear shuffled extensively, often beyond detectable sequence similarity when using standard methods. Many enhancers are probably between these extremes and show both fixed TF arrangements and more flexible parts (center). Thin vertical or diagonal black and grey dashed lines connect orthologous motifs vs. novel motifs for the same TF (motif turnover), respectively; lack of lines indicates that a motif is replaced by a novel motif for a different, functionally equivalent TF. Horizontal black lines represent sequence with conserved orthologous TFBS, while horizontal grey dashed lines represent divergent sequence or novel sites. The conservation track (bottom) shows a likely outcome of standard conservation measures, where black indicates detectable conservation and grey minimal conservation of short elements that is possibly missed. ciona displayed characteristic motif spacing but different studying 10 kb non-coding sequence surrounding each of overall motif organization [8,9]. Enhancers can maintain 4000 orthologous gene pairs, up to 90% of each factor’s function through stabilizing selection of compensatory target genes differed between the two organisms. Even changes as revealed through the study of chimeric enhan- when the factors were bound to the promoters of ortho- cers [10]. Known fly enhancers are selected against logous genes, a majority of the binding sites did not align deletions and clustered binding sites and certain motif- [15]. This difference was determined almost exclusively arrangements are preferentially conserved [11,12,13], by changes in cis, as the factors’ binding sites on human suggesting some level of constraint on enhancer archi- chromosome 21 in mouse hepatocytes resembled those in tecture. Indeed, altered spacing in enhancers from related human cells [16]. species can have functional consequences, fine-tuning enhancer responses to morphogen gradients [8], and These findings suggest that gene regulation might have making enhancer sequences even from closely related diverged substantially, providing an attractive expla- species not always functionally equivalent [14]. This nation for the apparent lack of sequence conservation might stem from cooperative DNA binding of some of many functional elements in the human genome TFs, but lack of understanding makes it difficult to [17,18] and TFs binding sites (TFBS) in Drosophila reliably identify enhancers or to estimate the impact of [19]. Alternatively, many of the elements detected by sequence changes on their activity. ChIP might be non-functional or biochemically active yet selectively neutral, such that evolutionary conservation Conservation of cis-regulatory connections and might in fact help to identify the relevant ones sequences? [11,1719]. Indeed, it remained unclear to what extent A series of two recent papers on direct target genes of four the divergent binding sites in [15,16] corresponded to liver-specific TFs showed that TF binding diverged functional enhancers required for hepatocyte differen- substantially between human and mouse [15,16]: when tiation [20].

Current Opinion in Genetics & Development 2009, 19:565–570 www.sciencedirect.com Comparative genomics of gene regulation—conservation and divergence of cis-regulatory information Meireles-Filho and Stark 567

Figure 2 example, the deep conservation ofa hairy binding site across the sequenced Drosophila species [11,33] that allowed its comparative prediction at very high conﬁdence [26]. Sequence conservation also improved the identiﬁcation of enhancers via clustering of TFBS [34,35,36].

Detecting constrained cis-regulatory elements computationally—accounting for evolutionary signatures Frequent alterations of regulatory connections is also unexpected given many reports in the literature of enhancers, whose function is conserved across large evolutionary distances such as between mammals and ﬁsh (e.g. [30,37,38], or between Drosophila, Anopheles,andSepsis (e.g. [3,39,40].

Some of these studies concluded that standard measures to assess genome sequence conservation tend to ‘overlook regulatory sequences’ [38]: five commonly used Comparative prediction of individual TFBS. Comparative genomics in 12 measures of evolutionary sequence constraint detected Drosophila genomes using the Branch-Length-Score (BLS) framework only roughly half of the functional phox2b enhancers in [26] predicted the physiologically relevant binding site of the Drosophila human–fish comparisons [38], similar to an earlier result TF hairy in the upstream region of achaete [65]. The hairy motif (PWM) is from Transfac [66] and we created the sequence logo with the WebLogo using other methods including BLAT (see below; [37]). program [67]. The thin and thick blue lines represent the achaete non- However, human and fish enhancers still shared features coding and coding regions, respectively, the transcription start is such as nucleotide composition and motif-content that indicated by the blue arrow, and the conservation track shows sequence discriminated them from non-functional sequence. Lack conservation in insects (modified after a UCSC Genome Browser screenshot [68]. of extensive nucleotide identity over large evolutionary distances has also been found between Drosophila and scavenger flies (Sepsidae;[39]): despite their near identical Rapid divergence of crucial core regulatory connections expression pattern in transgenic Drosophila embryos, sep- would be unexpected given highly similar developmental sid enhancer sequences appeared strongly diverged, partly programs regulated by orthologous TFs with conserved rearranged, and seemed to have lost a substantial fraction of expression patterns. Some factors and their regulatory con- the binding sites required in Drosophila [39]. Residual nections are very deeply conserved among animals, most similarities were restricted to short blocks, which were notably the so-called ‘gene-regulatory network kernels’ strongly enriched in TFBS [39] and partly occurred in such as the heart specification kernel that is shared between conserved order, distance, and orientation [41,42]. flies and vertebrates (e.g. [21]). Further, the similarity of transcriptional profiles between homologous cell types Interestingly, in all cases conservation of the respective (‘molecular fingerprints’, e.g. [22]) suggests conservation enhancer sequences had been detected in more closely of the defining regulators and their target genes. Regulatory related species, e.g. between zebrafish and fugu, human connections related to metabolism and growth can also be and non-primate mammals [37], and within the Drosophila highly conserved, such as ribosome biogenesis genes that species [39]. In addition, short blocks of shared sequences contain promoter-proximal E(CG) binding sites for the or conserved TF binding motifs in these regions could be Myc:Max heterodimer in many species [23]. detected and served to predict conserved enhancers across large evolutionary distances (e.g. [30,40]), indicat- Many studies suggest that regulatory sequences are con- ing that there seems to be a discrepancy between func- served, especially at close evolutionary distances. For tional conservation and detectable sequence conservation example, experimentally determined TFBS in Drosophila of enhancers. Some of the studies above relied on melanogaster contain TF binding motifs that are conserved methods that require extended sequence identity beyond in other Drosophila species [24,25]. Detailed comparison the lengths of individual TF motifs (e.g. BLAT; [43]). withsequence-matchedcontrols showedthat TFmotifsare Further, individual TFBS evolve according to the motifs more highly conserved than the overall enhancer sequence degeneracy profile, such that, for example, positions with [26]. Many TF binding motifs also show significantly low information content appear unconstrained [12,44]. increased genome-wide conservation levels that allow their de novo discovery (e.g. [11,27–29]) and the prediction of Several different strategies have recently been developed individual TFBS and targetgenes inyeast,worms, flies,and to account for the evolutionary properties of regulatory mammals (e.g. [26,30,31,32]). Figure 2 illustrates, for sequences. For example, Asthana et al. aimed specifically www.sciencedirect.com Current Opinion in Genetics & Development 2009, 19:565–570 568 Genomes and evolution

at detecting short elements under evolutionary constraint The functional divergence of enhancers is probably facili- [45], and Garber et al. at detecting biased substitutions tated by redundancies that can buffer loss-of-function characteristic of degenerate positions within TFBS [46]. situations: several genes have multiple enhancers (e.g. An entirely novel approach focused on the three-dimen- ‘shadow enhancers’ [59]) that drive expression in over- sional structure (topology) of the DNA helix that probably lapping or identical patterns. Further, neutral binding affects regulator binding [47]. While DNA topology can be sites might be a rich source for the de novo creation of inferred from the sequence, the relationship is not simple enhancers from non-functional sequence [17], which has and roughly twice as many bases had conserved topology probably happened during evolution given clearly differ- rather than sequence. All three approaches detected novel ent paralogous enhancers with similar functions [60]. elements that were enriched in regulatory sequences such Additionally, an old theory that transposable elements as DNase I hypersensitive sites. These examples empha- might contribute substantially to regulatory re-wiring [61] size the notion of evolutionary signatures that reflect the gained recent support: they harbour binding sites for functional constraints of diverse classes of functional ele- several TFs [62,63], consistent with their exaptation ments and are more suited for their comparative discovery as functional enhancers [64]. than raw nucleotide-level sequence conservation [11]. Conclusions Assessing motif conservation and predicting cis- Over the course of evolution, orthologous regulatory regulatory motifs sequences diverge by accumulating sequence changes that An important aspect in phylogenetic analyses is the under- are selected negatively if they disrupt important functions lying sequence alignments. Even for protein sequences, leading to phenotypic defects or otherwise evolve neu- multiple sequence alignments are the source of several trally. This is analogous to synonymous changes in protein- problems and can result in erroneous conclusions [48], coding DNA sequences, which do not alter the protein especially regarding the placement of gaps [49]. Multiple sequence and thus occur frequently. As we currently genome alignments generally suffer from similar problems, cannot assess the functional impact of regulatory sequence which strongly influence enhancer analyses and predic- changes, we rely on more or less direct measures of nucleo- tions: the conservation of TF motif instances across 12 tide-level sequence conservation. These often ignore the Drosophila genomes, for example, differed substantially enhancers’ functional and evolutionary properties (evol- (up to 50%) between different alignments [26]. utionary signatures; [11]) and may thus not detect conserved regulatory sequences (Figure 1). Such errors can lead to apparent motif-divergence or -loss, such that local adjustments during the motif search (e.g. We foresee that regulatory genomics datasets (e.g. ChIP- rMonkey; [50]), or motif-aware alignments [51–53] might seq and RNA-seq) from individual species within a phy- be more sensitive in detecting conservation. As an alterna- logeny will soon allow the distinction of functional con- tive, we chose to scan the motifs in the individual informant servation, neutral divergence, and species-specific sequences separately and used the alignment only to map regulation. Novel bioinformatics approaches will then identified motifs to a common reference. Depending on help to determine the minimal requirements for con- the motifs’ information content, we were able to allow for served enhancer function, an important step towards substantial motif-movement in the alignment, which – understanding the regulatory code. together with a framework for scoring conservation according to the phylogenetic tree (Branch-Length-Score, BLS) – Conflict of interest enabled sensitive and specific predictions (Figure 2;[26]). The authors declare no conflict of interest. This framework has recently been applied for microRNA target prediction in mammals [54] and extended to include Acknowledgements a probabilistic term to measure the likelihood of motif- We thank members of the Stark group (IMP) and Roger Revilla (IMP) for helpful discussions and apologize to all authors that we could not cite presence in each species [31]. because of the limited number of allowed citations and the requirement to restrict them to recent work. The Stark group is supported by the Austrian Divergence of cis-regulatory elements Ministry for Science and Research through the GEN-AU Bioinformatics Integration Network III. When discussing enhancer conservation, it should not be overlooked that cis-regulatory mutations are seen as the References and recommended reading major source of evolutionary innovations [55,56,57 ], Papers of particular interest, published within the annual period of meaning that enhancer function has probably evolved review, have been highlighted as: if the corresponding phenotypic traits have diverged. For of special interest example, mutations in a tan enhancer lead to the diver- of outstanding interest gence of body pigmentation between Drosophila yakuba and santomea [2] and divergence of shavenbaby enhancers 1. Visel A, Akiyama JA, Shoukry M, Afzal V, Rubin EM, correlates with changes of trichome patterns between D. Pennacchio LA: Functional autonomy of distant-acting human melanogaster and D. sechellia [58]. enhancers. Genomics 2009, 93:509-513.

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