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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 an hsp70 promoter fused to the luciferase gene.The constructs were transfected and expressed in L. sativae primary cells with and without heat-shock treatment. Transfected cells were incubated at 25° for 12 h, placed at either 37° (“heat shock”) or 25° (“non-heat-shock”) for 60 min, transferred to a 25° cell incubator for 60 min, and then used for luminescence assays. Luminescence values are the ratio of firefly to Renilla luminescence. Constructs with wild-type promoters of the LhuHsp70 or LsaHsp70 gene are labeled “WT”; “ΔATRS1” labels a construct with a LsaHsp70 promoter lacking the ATRS1 element (see Figure 1); “ΔATRS2” labels one without ATRS2; “ΔATRS1&2” labels constructs with neither ATRS1 nor ATRS2. The number above each two-column group represents induction fold of luminescence under heat-shock over non-heat-shock condition. The average luminescence over five independent experiments is plotted (mean± one standard deviation (SD)). Different letters above the error bar indicate a significant difference at the 0.05% level within treatments (One-way ANOVA and Turkey's post-hoc test).
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pone-0020308-g002: Transient luciferase luminescence driven by constructs carrying an hsp70 promoter fused to the luciferase gene.The constructs were transfected and expressed in L. sativae primary cells with and without heat-shock treatment. Transfected cells were incubated at 25° for 12 h, placed at either 37° (“heat shock”) or 25° (“non-heat-shock”) for 60 min, transferred to a 25° cell incubator for 60 min, and then used for luminescence assays. Luminescence values are the ratio of firefly to Renilla luminescence. Constructs with wild-type promoters of the LhuHsp70 or LsaHsp70 gene are labeled “WT”; “ΔATRS1” labels a construct with a LsaHsp70 promoter lacking the ATRS1 element (see Figure 1); “ΔATRS2” labels one without ATRS2; “ΔATRS1&2” labels constructs with neither ATRS1 nor ATRS2. The number above each two-column group represents induction fold of luminescence under heat-shock over non-heat-shock condition. The average luminescence over five independent experiments is plotted (mean± one standard deviation (SD)). Different letters above the error bar indicate a significant difference at the 0.05% level within treatments (One-way ANOVA and Turkey's post-hoc test).

Mentions: We first examined the transcriptional activity of the wild-type promoter of Lhuhsp70 and Lsahsp70 using the luciferase assay. In L. sativae primary-culture cells, both native promoters showed clear constitutive transcriptional activity, i.e., are luminescent at 25°C, albeit the Lsahsp70 promoter delivered less luminescence than the Lhuhsp70 promoter (P<0.05; Figure 2). Next, to determine if the two AT-rich insertions affect transcription, we measured the luminescence delivered by constructs carrying a wild-type promoter of Lsahsp70 out of which both ATRS1 and ATRS2 had been deleted experimentally. The double deletion increased luminescence (P<0.05), restoring it to that delivered by the wild-type promoter of Lhuhsp70 (Figure 2). The deletion of ATRS1 alone resulted in elevated luminescence (P<0.05), suggesting that ATRS1 down-regulates transcription. In contrast, deleting ATRS2 decreased luminescence sevenfold (P<0.01) relative to that of the native Lsahsp70 promoter. These results suggest that ATRS2 acts to up-regulate transcription at the distal promoter (Figure 2).


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 an hsp70 promoter fused to the luciferase gene.The constructs were transfected and expressed in L. sativae primary cells with and without heat-shock treatment. Transfected cells were incubated at 25° for 12 h, placed at either 37° (“heat shock”) or 25° (“non-heat-shock”) for 60 min, transferred to a 25° cell incubator for 60 min, and then used for luminescence assays. Luminescence values are the ratio of firefly to Renilla luminescence. Constructs with wild-type promoters of the LhuHsp70 or LsaHsp70 gene are labeled “WT”; “ΔATRS1” labels a construct with a LsaHsp70 promoter lacking the ATRS1 element (see Figure 1); “ΔATRS2” labels one without ATRS2; “ΔATRS1&2” labels constructs with neither ATRS1 nor ATRS2. The number above each two-column group represents induction fold of luminescence under heat-shock over non-heat-shock condition. The average luminescence over five independent experiments is plotted (mean± one standard deviation (SD)). Different letters above the error bar indicate a significant difference at the 0.05% level within treatments (One-way ANOVA and Turkey's post-hoc test).
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Related In: Results  -  Collection

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pone-0020308-g002: Transient luciferase luminescence driven by constructs carrying an hsp70 promoter fused to the luciferase gene.The constructs were transfected and expressed in L. sativae primary cells with and without heat-shock treatment. Transfected cells were incubated at 25° for 12 h, placed at either 37° (“heat shock”) or 25° (“non-heat-shock”) for 60 min, transferred to a 25° cell incubator for 60 min, and then used for luminescence assays. Luminescence values are the ratio of firefly to Renilla luminescence. Constructs with wild-type promoters of the LhuHsp70 or LsaHsp70 gene are labeled “WT”; “ΔATRS1” labels a construct with a LsaHsp70 promoter lacking the ATRS1 element (see Figure 1); “ΔATRS2” labels one without ATRS2; “ΔATRS1&2” labels constructs with neither ATRS1 nor ATRS2. The number above each two-column group represents induction fold of luminescence under heat-shock over non-heat-shock condition. The average luminescence over five independent experiments is plotted (mean± one standard deviation (SD)). Different letters above the error bar indicate a significant difference at the 0.05% level within treatments (One-way ANOVA and Turkey's post-hoc test).
Mentions: We first examined the transcriptional activity of the wild-type promoter of Lhuhsp70 and Lsahsp70 using the luciferase assay. In L. sativae primary-culture cells, both native promoters showed clear constitutive transcriptional activity, i.e., are luminescent at 25°C, albeit the Lsahsp70 promoter delivered less luminescence than the Lhuhsp70 promoter (P<0.05; Figure 2). Next, to determine if the two AT-rich insertions affect transcription, we measured the luminescence delivered by constructs carrying a wild-type promoter of Lsahsp70 out of which both ATRS1 and ATRS2 had been deleted experimentally. The double deletion increased luminescence (P<0.05), restoring it to that delivered by the wild-type promoter of Lhuhsp70 (Figure 2). The deletion of ATRS1 alone resulted in elevated luminescence (P<0.05), suggesting that ATRS1 down-regulates transcription. In contrast, deleting ATRS2 decreased luminescence sevenfold (P<0.01) relative to that of the native Lsahsp70 promoter. These results suggest that ATRS2 acts to up-regulate transcription at the distal promoter (Figure 2).

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
Related in: MedlinePlus