Multiple bHLH proteins regulate CIT2 expression in Saccharomyces cerevisiae

Abstract

The basic helix-loop-helix (bHLH) proteins comprise a eukaryotic transcription factor family involved in multiple biological processes. They have the ability to form multiple dimer combinations and most of them also bind a 6 bp site (E-box) with limited specificity. These properties make them ideal for combinatorial regulation of gene expression. The Saccharomyces cerevisiae CIT2 gene, which encodes citrate synthase, was previously known to be induced by the bHLH proteins Rtg1p and Rtg3p in response to mitochondrial damage. Rtg1p-Rtg3p dimers bind two R-boxes (modified E-boxes) in the CIT2 promoter. The current study tested the ability of all nine S. cerevisiae bHLH proteins to regulate the CIT2 gene. The results showed that expression of CIT2-lacZ reporter was induced in a ρ 0 strain by the presence of inositol via the Ino2p and Ino4p bHLH proteins, which are known regulators of phospholipid synthesis. Promoter mutations revealed that inositol induction required a distal E-box in the CIT2 promoter. Interestingly, deleting the INO2, INO4 genes or the cognate E-box revealed phosphate induction of CIT2 expression. This layer of expression required the two R-boxes and the Pho4p bHLH protein, which is known to be required for phosphate-specific regulation. Lastly, the data show that the Hms1p and Sgc1p bHLH proteins also play important roles in repression of CIT2-lacZ expression. Collectively, these results support the model that yeast bHLH proteins coordinate different biological pathways.

Derepression ofINO1Transcription Requires Cooperation between the Ino2p-Ino4p Heterodimer and Cbf1p and Recruitment of the ISW2 Chromatin-Remodeling Complex

“…In recent years we have reported that multiple bHLH proteins regulate the transcription of single genes in yeast (e.g., CIT2 and ENO1) (14,16). We can now add INO1 to this target list, since we showed here that Cbf1p acted in concert with the two known activators of INO1 transcription, Ino2p and Ino4p ( Fig.…”

“…The Ino2p and Ino4p bHLH proteins are known regulators of INO1 transcription (2,67,73). We recently reported that the expression of two well-studied bHLH target genes, CIT2 and ENO1, is regulated by multiple bHLH factors (14,16). Thus, we tested whether other bHLH proteins, besides Ino2p and Ino4p, also regulate INO1 transcription.…”

“…Previous studies in our laboratory have shown that multiple yeast bHLH proteins can regulate a single gene (14,16). This study began with the goal of determining if bHLH proteins other than Ino2p and Ino4p are also involved in the regulation of INO1.…”

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

“…An attractive hypothesis is that the Tye7 protein represses Ty1 antisense transcription by binding to one or more of the three E-boxes. In support of a potential repressive role of TYE7 , a previous study has reported that it could repress the transcription of the E-box-containing CIT2 gene (78). In a first model, Ty1 sense and antisense transcription could interfere in cis .…”

Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

“…This sequence has been shown to contain the two UAS regions required for promoter regulation [71]. The CIT2 p sequence used corresponds to the largest promoter sequence used in a previous study of CIT2 regulation and consisted of the 992 bp region 5′ of the CIT2 CDS, from the −1000 to the −9 position relative to the translational start site [72]. The primers used to amplify this region were SV010 (5′-GCCGCCGAATTCGACCAATGTTAATGA-3′) and SV011 (5′-GCCGCCACTAGTTTACTAGTATTATTAAAACA-3′).…”