Evolution of Hsp70 Gene Expression: A Role for Changes in AT-Richness within Promoters

Abstract

In disparate organisms adaptation to thermal stress has been linked to changes in the expression of genes encoding heat-shock proteins (Hsp). The underlying genetics, however, remain elusive. We show here that two AT-rich sequence elements in the promoter region of the hsp70 gene of the fly Liriomyza sativae that are absent in the congeneric species, Liriomyza huidobrensis, have marked cis-regulatory consequences. We studied the cis-regulatory consequences of these elements (called ATRS1 and ATRS2) by measuring the constitutive and heat-shock-induced luciferase luminescence that they drive in cells transfected with constructs carrying them modified, deleted, or intact, in the hsp70 promoter fused to the luciferase gene. The elements affected expression level markedly and in different ways: Deleting ATRS1 augmented both the constitutive and the heat-shock-induced luminescence, suggesting that this element represses transcription. Interestingly, replacing the element with random sequences of the same length and A+T content delivered the wild-type luminescence pattern, proving that the element's high A+T content is crucial for its effects. Deleting ATRS2 decreased luminescence dramatically and almost abolished heat-shock inducibility and so did replacing the element with random sequences matching the element's length and A+T content, suggesting that ATRS2's effects on transcription and heat-shock inducibility involve a common mechanism requiring at least in part the element's specific primary structure. Finally, constitutive and heat-shock luminescence were reduced strongly when two putative binding sites for the Zeste transcription factor identified within ATRS2 were altered through site-directed mutagenesis, and the heat-shock-induced luminescence increased when Zeste was over-expressed, indicating that Zeste participates in the effects mapped to ATRS2 at least in part. AT-rich sequences are common in promoters and our results suggest that they should play important roles in regulatory evolution since they can affect expression markedly and constrain promoter DNA in at least two different ways.

Activity of Heat Shock Genes’ Promoters in Thermally Contrasting Animal Species

“…Thus, the hsp70 promoters in the fly Liriomyza sativae harbor AT-rich sequence elements (ATRS) that are absent in the congeneric species. Deletions of the ATRS from promoters augmented both the constitutive and heat-shock-inducible expression of the luciferase reporter [ 44 ]. Intriguingly, the authors detected sites for the binding of the Zeste transcription factor within these ATRSs, which may play the role of GAF in this Diptera species, somehow enhancing the strength of the hsp70 promoter [ 45 ].…”

Analysis of genetic diversity of the heat shock protein 70 gene on the basis of abundant sequence polymorphisms in chicken breeds

“…Mutations in gene sequences that change gene expression by altering the morphology and physiology have been reported (Chen et al, 2007;Graze et al, 2009). Chen et al (2011) reported that changes in the AT content of the promoter markedly affect HSP70 expression of HSP70, which may play an important role in regulatory evolution. Therefore, other changes in the chicken HSP70 gene sequence might also explain the evolution of this gene.…”

Variation and constraints in species-specific promoter sequences

“…Despite this mathematical definition, such sequences are expected to be associated with structural properties and functional processes of high biological importance and complexity. For instance, it has been proved that they are endowed with functional properties by controlling gene expression (Eddy et al, 2011;Vinces et al, 2009;Chen et al, 2011), by enhancing non-specific TF-DNA binding affinity (Gemayel et al, 2010;Sela and Lukatsky, 2011) and by influencing chromatin organization (Segal and Widom, 2009). On the basis of these remarks, we have decided to investigate the role played by such regular components in determining the bias described by h n ðlÞ.…”