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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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Schematic structure of the promoter region of Lhuhsp70 and Lsahsp70 gene.Lhuhsp70 and Lsahsp70 are the hsp70 orthologs of L. sativa and L. huibrobensis. Two AT-rich sequence elements ATRS1 (495 bp) and ATRS2 (98 bp) are present only in Lsahsp70. The arrow indicates the transcription start site. The position numbers flag each element's first most downstream base relative to the transcription start site. “HSEn”: Heat-shock element (n = 1–4); “TATA”: TATA box; “ATRS1 and ATRS2”: AT-rich elements; The unlabeled box: GAGA element.
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pone-0020308-g001: Schematic structure of the promoter region of Lhuhsp70 and Lsahsp70 gene.Lhuhsp70 and Lsahsp70 are the hsp70 orthologs of L. sativa and L. huibrobensis. Two AT-rich sequence elements ATRS1 (495 bp) and ATRS2 (98 bp) are present only in Lsahsp70. The arrow indicates the transcription start site. The position numbers flag each element's first most downstream base relative to the transcription start site. “HSEn”: Heat-shock element (n = 1–4); “TATA”: TATA box; “ATRS1 and ATRS2”: AT-rich elements; The unlabeled box: GAGA element.

Mentions: The hsp70 transcription start site (initiator) was determined by comparing sequences of the promoter region and the full-length cDNA transcript (GenBank accession number AY842476.2 for L. huidobrensis and AY842477 for L. sativae; [27]). Both TATA boxes are located 30 bp upstream from the initiator sequence. Heat-shock response elements (HSE) are conserved sequences in the promoter regions of Hsps and their binding by the Heat-shock factor (HSF) is central to the heat-shock-induced activation of the genes [29], [30]. We identified four putative HSEs in the hsp70 promoters by looking for the conserved dimer of the 10-bp NTTCNNGAAN sequence characteristic of Drosophila HSEs [31]. The sequences of these HSEs are listed in Table S1. The two HSEs at the proximal promoter, HSE1 and HSE2, have conserved sequence composition and position relative to the transcription start site, whereas the two distal ones, HSE3 and HSE4, occupy very different positions in the two species, being dispersed more further upstream in Lsahsp70 than in Lhuhsp70 (Figure 1).


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)

Schematic structure of the promoter region of Lhuhsp70 and Lsahsp70 gene.Lhuhsp70 and Lsahsp70 are the hsp70 orthologs of L. sativa and L. huibrobensis. Two AT-rich sequence elements ATRS1 (495 bp) and ATRS2 (98 bp) are present only in Lsahsp70. The arrow indicates the transcription start site. The position numbers flag each element's first most downstream base relative to the transcription start site. “HSEn”: Heat-shock element (n = 1–4); “TATA”: TATA box; “ATRS1 and ATRS2”: AT-rich elements; The unlabeled box: GAGA element.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105046&req=5

pone-0020308-g001: Schematic structure of the promoter region of Lhuhsp70 and Lsahsp70 gene.Lhuhsp70 and Lsahsp70 are the hsp70 orthologs of L. sativa and L. huibrobensis. Two AT-rich sequence elements ATRS1 (495 bp) and ATRS2 (98 bp) are present only in Lsahsp70. The arrow indicates the transcription start site. The position numbers flag each element's first most downstream base relative to the transcription start site. “HSEn”: Heat-shock element (n = 1–4); “TATA”: TATA box; “ATRS1 and ATRS2”: AT-rich elements; The unlabeled box: GAGA element.
Mentions: The hsp70 transcription start site (initiator) was determined by comparing sequences of the promoter region and the full-length cDNA transcript (GenBank accession number AY842476.2 for L. huidobrensis and AY842477 for L. sativae; [27]). Both TATA boxes are located 30 bp upstream from the initiator sequence. Heat-shock response elements (HSE) are conserved sequences in the promoter regions of Hsps and their binding by the Heat-shock factor (HSF) is central to the heat-shock-induced activation of the genes [29], [30]. We identified four putative HSEs in the hsp70 promoters by looking for the conserved dimer of the 10-bp NTTCNNGAAN sequence characteristic of Drosophila HSEs [31]. The sequences of these HSEs are listed in Table S1. The two HSEs at the proximal promoter, HSE1 and HSE2, have conserved sequence composition and position relative to the transcription start site, whereas the two distal ones, HSE3 and HSE4, occupy very different positions in the two species, being dispersed more further upstream in Lsahsp70 than in Lhuhsp70 (Figure 1).

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