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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 driven by constructs carrying hsp70 promoter with altered A+T content.ATRS1 or ATRS2 were replaced with random sequences of the same size but different A+T content; “WT”: construct with wild-type promoter of Lsahsp70 gene. Methods are as in Figure 2. Luminescence is expressed as in Figure 2. Bars indicate ± one SD. Asterisk (*) indicates a difference significant at the 0.05 level.
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pone-0020308-g003: Transient luciferase luminescence driven by constructs carrying hsp70 promoter with altered A+T content.ATRS1 or ATRS2 were replaced with random sequences of the same size but different A+T content; “WT”: construct with wild-type promoter of Lsahsp70 gene. Methods are as in Figure 2. Luminescence is expressed as in Figure 2. Bars indicate ± one SD. Asterisk (*) indicates a difference significant at the 0.05 level.

Mentions: To investigate whether the A+T content of ATRS1 and ATRS2 contributes to their regulatory activity, we replaced ATRS1 or ATRS2 in Lsahsp70 with random sequences of the same length and various A+T contents, and then assessed the effects on gene expression. Under non-heat-shock conditions, no difference in luciferase luminescence was observed when replacing ATRS1 with sequences having 50% or 75% A+T content but after heat shock the luminescence was significantly reduced with the 50% AT sequence (P<0.05). In contrast and remarkably, swapping ATRS1 with a sequence of identical A+T content delivered the wild-type transcriptional level and heat inducibility (Figure 3). Therefore ATRS1 sequence replacements with near-native A+T content can replace the ATRS1 element at least as far as heat-shock inducibility in transfected cells is concerned.


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 driven by constructs carrying hsp70 promoter with altered A+T content.ATRS1 or ATRS2 were replaced with random sequences of the same size but different A+T content; “WT”: construct with wild-type promoter of Lsahsp70 gene. Methods are as in Figure 2. Luminescence is expressed as in Figure 2. Bars indicate ± one SD. Asterisk (*) indicates a difference significant at the 0.05 level.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105046&req=5

pone-0020308-g003: Transient luciferase luminescence driven by constructs carrying hsp70 promoter with altered A+T content.ATRS1 or ATRS2 were replaced with random sequences of the same size but different A+T content; “WT”: construct with wild-type promoter of Lsahsp70 gene. Methods are as in Figure 2. Luminescence is expressed as in Figure 2. Bars indicate ± one SD. Asterisk (*) indicates a difference significant at the 0.05 level.
Mentions: To investigate whether the A+T content of ATRS1 and ATRS2 contributes to their regulatory activity, we replaced ATRS1 or ATRS2 in Lsahsp70 with random sequences of the same length and various A+T contents, and then assessed the effects on gene expression. Under non-heat-shock conditions, no difference in luciferase luminescence was observed when replacing ATRS1 with sequences having 50% or 75% A+T content but after heat shock the luminescence was significantly reduced with the 50% AT sequence (P<0.05). In contrast and remarkably, swapping ATRS1 with a sequence of identical A+T content delivered the wild-type transcriptional level and heat inducibility (Figure 3). Therefore ATRS1 sequence replacements with near-native A+T content can replace the ATRS1 element at least as far as heat-shock inducibility in transfected cells is concerned.

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