Figure 1. Binding of Heat-Shock Factor (HSF) trimers to Heat Shock Elements (HSEs) mediate the transcription of heat-shock genes. This cartoon, greatly simplified from [33], Fig. 11 of [13], and Fig. 4 of [31], depicts two HSEs, each comprising three consecutive 5 bp sequences of nGAAn and nTTCn in alternation and each binding an HSF trimer. The HSF monomers (mHSF) interact with one another via a trimerization domain and the ‘‘wing’’ of the winged helix-loop-helix motif. In HSF, Helix 3, whose sequence is conserved in eukaryotes, is responsible for the specificity of interaction with the HSEs. Once bound, HSF trimers interact with the transcriptional complex, including RNA polymerase II (Pol II) to result in gene expression. See Introduction for additional explanation. Inset: symbolic representation of the promoter organization, as will appear in subsequent figures. Aspects omitted from the cartoon include HSEs longer than three 5 bp units, which support cooperative binding of HSF trimers, variation in sequence within HSEs, variation in position and spacing of HSEs, GAGA factor and its binding sites, other cis-regulatory elements, nucleosomes and their acetylation sites, and numerous proteins involved in transcriptional regulation. doi:10.1371/journal.pone.0010669.g001 Well-defined features of the HSE sequence seem critical for HSF-HSE binding. In flies as in other taxa, the canonical HSE comprises inverted repeats of the pentanucleotide sequence 59- NGAAN-39, where N is any nucleotide [20], and typically is within 400 bp upstream of the transcriptional start site. HSEs often contain at least 3 continuous 5 bp subunits alternating between NGAAN or NTTCN or vice versa, with each 5 bp HSE subunit capable of binding one monomer of the HSF trimer [20–22]. The alternating multiple subunits promote cooperative binding of HSF trimers in the major groove of the DNA helix [23], which correlates with more stable protein-protein and DNA- protein interactions and stronger heat shock responses [24,25]. In general, the number of consecutive 5 bp units relates to the magnitude of the heat-shock response. However, in yeast, HSEs of equal length (4 subunits) and homologous to the consensus have appreciably different binding properties depending on which subunit variant (NTTCN or NGAAN) occupies the most distal position [26]. This difference is due to the ability of the variant beginning with NTTCN to bind an additional HSF trimer [26], and is not evident in experimental studies of D. melanogaster [21]. The three central nucleotides, GAA or TTC, are highly conserved in D. melanogaster [10], implying they are functionally constrained. Indeed, the 2nd position of an NGAAN motif is critical for binding, with the 3rd and 4th positions having a lesser effect and the 5th position no effect [23]. Mutations in the first position also affect HSF binding [23], however, although the compositional bias is less than for positions 3 or 4. These data suggest the consensus motif should again [see, for example, [22,27–30] as former episodes of redefinition] be redefined as AGAAN [23]. As both HSF activation and the heat-shock response are multistep processes, variation in these processes cannot unilaterally be attributed to variation in HSEs, however. Finally, HSEs are often multiple. Although HSF-HSE complexes that form near the transcription start site (TSS) can engage the transcriptional apparatus through physical proximity, even more distal HSEs can similarly engage through the bending of the Phylogeny Orders HSE Evolution promoter DNA [31]. The approximation of distal HSEs to the TSS is a function of both their position within the promoter and the interaction of GAGA factor and its binding sites, positioned nucleosomes and their acetylation state, and other co-regulators [13,31,32]. As with other aspects of the cis-regulatory code, HSEs deviate from the canon. In vitro, HSEs with only 2 subunits can form stable interactions with HSF [21,33]. Furthermore, the 5 bp inverted repeats need not be consecutive. Gapped HSEs, for example, contain internal 5 bp blocks with little or no homology to the canonical motif flanked by canonical sequences in the proper orientation. These HSEs have binding affinity comparable to the minimal functional binding sequence as long as they exceed 4 subunits [20]. In yeast, gapped versions constitute an entire class of HSEs [34]. HSF’s affinity for HSEs varies with their sequence and position [13,35]. HSF also appears to bind promoters that lack either typical or gapped HSEs, and in genes not previously associated with the heat-shock response [34,36–38]. In some cases, recognizable motifs for other transcription factors are present, suggesting that HSF binding to these promoters is mediated differently than when HSEs are present [38]. Here we use this knowledge to elucidate the evolution of cis- regulatory sequence in Drosophila heat-shock proximal promoters derived from 12 species whose genomes have been sequenced [39], and. We also include data from the island endemic species D. santomea, which has diverged substantially from its sister species, D. yakuba, in thermal tolerance [40], a phenotype potentially explicable by regulatory divergence in heat-shock promoters. As suggested above, the combination of detailed mechanistic understanding of HSE structure-function and a phylogenetic context reveals hitherto unrecognized features of heat-shock promoters that apparently are under functional constraint. Moreover, where heat-shock promoters have evolved, this same combination defines the nature and timing of the underlying evolutionary events. Results Phylogeny implicates function The 13 species of Drosophila exhibit at least 419 computationally identifiable HSEs in the 8 heat-shock genes under study (Table S1, Table S2, Figures 2–9). This estimate includes 223 in usually single-copy genes (Hsp22, Hsp23, Hsp26, Hsp27, DnaJ-1, Hsp68, and Hsp83), with the balance in Hsp70, which is always multi-copy. These HSEs vary in occurrence, number, position, and conformity with the canonical sequence. For example, HSEs range from two 5 bp subunits, [in Hsp23, Hsp26, DnaJ-1, and Hsp70 (Figures 3, 4, 6, 8)] to eleven subunits [in Hsp68 (Figure 7)]. Importantly, phylogenetic conservation in the 13 species suggests that certain features are functionally significant and are therefore subject to selection. These features include: (a) Putative HSEs that previously had been overlooked. In Hsp26, for example, a third (and putative) HSE consisting of 2–3 5 bp units occurs in all melanogaster subgroup (Figure 4) species. This sequence was observed previously in D. melanogaster but reasonably declared not an HSE at that time [41]. Its conservation in 6 species for .10 MY suggests that this conclusion should be revisited. (b) Tail-to-tail configurations, which are preferred and persist in evolution. Identified HSEs are both head-to-head (an HSE with a nGAAn-type motif at its most distal site) and tail-to-tail (an HSE in which nTTCn is the most distal subunit) [26]. The tail-to-tail configuration is preferred, however. Of the 393 HSEs in the analysis, 236 began with nTTCn while only 157 began with PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10669