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Evolution of hsp70 gene expression: a role for changes in AT-richness within promoters.

Chen B, Jia T, Ma R, Zhang B, Kang L - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

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.

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Transient luciferase luminescence and the Zeste binding sites predicted within ATRS2.(A) Putative binding sites for transcription factor Zeste in ATRS2 of Lsahsp70. Mutated sequences (MT) in each putative Zeste motif are shown below the wild-type sequences (WT). (B) The effects of mutating the putative Zeste binding sites on the luminescence. (C) Luminescence effects of Zeste overexpression. To overexpress Zeste, expression vectors for Zeste or blank control vectors (Zeste “wild-type”) were co-transfected together with Lsahsp70 wild-type reporter plasmids. Bars indicate ± one SD. Asterisks (*) means significance at the 0.05 level and (**) at the 0.01 level.
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pone-0020308-g004: Transient luciferase luminescence and the Zeste binding sites predicted within ATRS2.(A) Putative binding sites for transcription factor Zeste in ATRS2 of Lsahsp70. Mutated sequences (MT) in each putative Zeste motif are shown below the wild-type sequences (WT). (B) The effects of mutating the putative Zeste binding sites on the luminescence. (C) Luminescence effects of Zeste overexpression. To overexpress Zeste, expression vectors for Zeste or blank control vectors (Zeste “wild-type”) were co-transfected together with Lsahsp70 wild-type reporter plasmids. Bars indicate ± one SD. Asterisks (*) means significance at the 0.05 level and (**) at the 0.01 level.

Mentions: Homology search in databases for transcription-factor binding sites identified several putative sites for transcription factors in ATRS2. Among them were two binding sites for the Zeste transcription factor (which is involved in transvection in Drosophila; [32], [33]). The presence of two such sites in a relatively short region prompted us to study the possible role of these sites in hsp regulation. The two putative Zeste-binding sites are located between positions -664 and -668 (Zeste 1) and between positions -690 and -694 (Zeste 2). These sites are relatively GC-rich compared to the flanking DNA (Figure 4A).


Evolution of hsp70 gene expression: a role for changes in AT-richness within promoters.

Chen B, Jia T, Ma R, Zhang B, Kang L - PLoS ONE (2011)

Transient luciferase luminescence and the Zeste binding sites predicted within ATRS2.(A) Putative binding sites for transcription factor Zeste in ATRS2 of Lsahsp70. Mutated sequences (MT) in each putative Zeste motif are shown below the wild-type sequences (WT). (B) The effects of mutating the putative Zeste binding sites on the luminescence. (C) Luminescence effects of Zeste overexpression. To overexpress Zeste, expression vectors for Zeste or blank control vectors (Zeste “wild-type”) were co-transfected together with Lsahsp70 wild-type reporter plasmids. Bars indicate ± one SD. Asterisks (*) means significance at the 0.05 level and (**) at the 0.01 level.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105046&req=5

pone-0020308-g004: Transient luciferase luminescence and the Zeste binding sites predicted within ATRS2.(A) Putative binding sites for transcription factor Zeste in ATRS2 of Lsahsp70. Mutated sequences (MT) in each putative Zeste motif are shown below the wild-type sequences (WT). (B) The effects of mutating the putative Zeste binding sites on the luminescence. (C) Luminescence effects of Zeste overexpression. To overexpress Zeste, expression vectors for Zeste or blank control vectors (Zeste “wild-type”) were co-transfected together with Lsahsp70 wild-type reporter plasmids. Bars indicate ± one SD. Asterisks (*) means significance at the 0.05 level and (**) at the 0.01 level.
Mentions: Homology search in databases for transcription-factor binding sites identified several putative sites for transcription factors in ATRS2. Among them were two binding sites for the Zeste transcription factor (which is involved in transvection in Drosophila; [32], [33]). The presence of two such sites in a relatively short region prompted us to study the possible role of these sites in hsp regulation. The two putative Zeste-binding sites are located between positions -664 and -668 (Zeste 1) and between positions -690 and -694 (Zeste 2). These sites are relatively GC-rich compared to the flanking DNA (Figure 4A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

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.

Show MeSH