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Thus, an additional candidate ChoRE sequence on the Txnip gene promoter was identified; for convenience, we dubbed the previously identified ChoRE (∼80 bp) as ChoRE-a and the newly identified ChoRE (∼170 bp) as ChoRE-b (Figure 2C).
In fish (fugu, tetraodon, zebrafish and medaka) Txnip promoters, the nucleotide sequences at the ChoRE-a position actually deviate from a canonical ChoRE; on the contrary, the fish ChoRE-b better mimics a canonical ChoRE sequence than does human ChoRE-b (Figure S4).
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We aligned the Txnip promoter sequences and found that the two ChoRE sequences, a CCAAT box, an inverted CCAAT box and a forkhead box O (FOXO) binding site were all well conserved among these species (Figure S2).
We conclude that both the earlier defined ChoRE and nucleotide sequences near the −170 positions are critical for the induction of Txnip expression by adenosine-containing molecules.
Our view is that the ChoRE-b nucleotide sequences should be GAGCACACCGTGTCCACGCG, especially when the fish Txnip promoters were considered (Figure S4).
The sequences of both the ChoRE sites on the frog Txnip promoter are moderately degenerate thus lying in between the fishes and mammals (Figure S2, S3, and S4).
We examined the Txnip promoter sequences (184 63 bp) and found that, apart from the known ChoRE (CACGAGggcagCACGAG; ∼80 bp upstream of the transcription start site), the nucleotide sequences around the −170 region (CACACCgtgtcCACGCG; Figure 2C) mimicked a degenerate ChoRE, which is defined as two E-boxes (CACGTG) separated by 5 nucleotides [38].
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This suggests that the earlier defined ChoRE alone cannot support optimal induction of Txnip expression by glucose, and that some nucleotide sequences upstream of this ChoRE is also critical for the responsiveness of Txnip promoter to glucose.
The nucleotide sequences between two ChoREs contain a CCAAT box, an inverted CCAAT box and a FOXO binding site (Figure 2C); these three sites are highly conserved in Txnip promoters from different species (Figure S2).
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