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.
Highlights
Phenotypic differences between species can be due to genetic changes that alter gene products as well as their expression level [1,2,3,4]
We identified two AT-rich sequence stretches in the hsp70 promoter that are present in L. sativae and may explain much of the differences in hsp expression level and heat inducibility observed between this species and L. huidobrensis
Because AT-rich sequences account for a large proportion of the promoter region of these hsps, the results suggest that changes in the AT-rich sequences of promoter regions may contribute often to the differentiation of gene expression level and inducibility in closely related species, so that such changes may participate frequently in the evolution of gene expression
Summary
Phenotypic differences between species can be due to genetic changes that alter gene products as well as their expression level [1,2,3,4]. Identifying the cis-regulatory changes underlying expression differences between species, remains challenging both experimentally and bioinformatically [4,6,7]. This is true for the AT-rich sequences frequently found in eukaryotic promoter regions [8,9,10] that vary substantially across taxa with respect to abundance and organization. No essential sequence motifs have been characterized in such regions that are shared by many taxa and whose presence was linked with regulatory consequences for gene expression [11,12,13]
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