Phylogeny Disambiguates the Evolution of Heat-Shock cis -Regulatory Elements in Drosophila Sibo Tian, Robert A. Haney*, Martin E. Feder Department of Organismal Biology and Anatomy, University of Chicago, Chicago, Illinois, United States of America Abstract Heat-shock genes have a well-studied control mechanism for their expression that is mediated through cis-regulatory motifs known as heat-shock elements (HSEs). The evolution of important features of this control mechanism has not been investigated in detail, however. Here we exploit the genome sequencing of multiple Drosophila species, combined with a wealth of available information on the structure and function of HSEs in D. melanogaster, to undertake this investigation. We find that in single-copy heat shock genes, entire HSEs have evolved or disappeared 14 times, and the phylogenetic approach bounds the timing and direction of these evolutionary events in relation to speciation. In contrast, in the multi- copy gene Hsp70, the number of HSEs is nearly constant across species. HSEs evolve in size, position, and sequence within heat-shock promoters. In turn, functional significance of certain features is implicated by preservation despite this evolutionary change; these features include tail-to-tail arrangements of HSEs, gapped HSEs, and the presence or absence of entire HSEs. The variation among Drosophila species indicates that the cis-regulatory encoding of responsiveness to heat and other stresses is diverse. The broad dimensions of variation uncovered are particularly important as they suggest a substantial challenge for functional studies. Citation: Tian S, Haney RA, Feder ME (2010) Phylogeny Disambiguates the Evolution of Heat-Shock cis-Regulatory Elements in Drosophila. PLoS ONE 5(5): e10669. doi:10.1371/journal.pone.0010669 Editor: Cathal Seoighe, National University of Ireland Galway, Ireland Received February 5, 2010; Accepted April 23, 2010; Published May 17, 2010 Copyright: 2010 Tian et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by the National Science Foundation (IOB06-41278). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: robert.a.haney@gmail.com Introduction Although cis-acting sequences are clearly vital to the regulation of gene expression and its evolution [1], the decoding of the organizational basis for this regulation and evolution is still a work in progress despite considerable effort, numerous research studies, and substantial genomic data. The difficulty of this task resides in the nature of the code itself: it is irregular, contains numerous synonyms and exceptions, and no rule appears to go unviolated [2]. We suggest, as have others [3] that a way forward may be in the combination of two aspects: a cis-regulatory sequence whose mechanism of controlling gene expression is understood in detail, and a robust phylogeny of successively more distantly related species. The former feature links explicit consequences for gene expression to variation in the cis-regulatory sequence. A phyloge- netic approach has two advantages beyond ‘‘phylogenetic footprinting’’; i.e., the identification of putative cis-regulatory sequence through conservation [4]. First, it poses specific hypotheses about where in evolution changes have arisen; these hypotheses may in turn anchor other hypotheses about specific genetic mechanisms of evolutionary change and the adaptive consequences of the change. Second, it highlights which features of the cis-regulatory sequence are free to evolve and which are stabilized by selection, providing corroboration for functional studies but also possibly suggesting other, unrecognized features of the functional mechanism for further testing [5–7]. Here we exemplify these points in the cis-regulatory sequences governing the heat-shock response, heat-shock elements (HSEs). HSEs are among the best-characterized and simplest of cis- regulatory elements (CREs) in metazoans (Figure 1); this and the nature of the heat-shock response itself provide an exceptionally firm foundation for analysis. In brief, heat, other proteotoxic stresses, and diverse other signals change gene expression dramatically. In Drosophila, the transcription of most active genes is halted [8] and a restricted set of heat-shock genes, many encoding molecular chaperones and including HSEs in their proximal promoter regions, are coordinately upregulated during or after heat-stress [9]. Unlike in many other promoters, experimentally-verified HSEs of Drosophila are rarely beyond ,500 bp of the transcription start site, and this relatively short proximal promoter suffices for full-strength transcription [10]. HSEs are binding sites for HSFs (heat-shock factors, Figure 1), transcription factors whose trimerization mediates the heat-shock response in eukaryotes [11,12] in combination with numerous co- regulators [13]. In many higher eukaryotes HSFs are multiple, with family members varying in stimuli for their activation, affinity for HSEs, and downstream targets [14,15]. Fortuitously for the present study, HSF is singular in Drosophila [16] and highly conserved [17]. [This contrasts with the co-evolution of CRE binding sites and TFs both in other HSFs and for other TFs (eg. [18])] The DNA binding domain of HSF contains an antiparallel b sheet and a cluster of three a helices, one of which (helix 3) functions in DNA recognition in the context of a helix-turn-helix structure [19]. This domain is itself highly conserved [17], implying that variation in HSEs is primarily if not exclusively responsible for variation in HSF-HSE interaction in Drosophila. PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10669