Photoregulated gene expression may involve ubiquitous DNA binding proteins.

Several promoter elements have previously been shown to influence the expression of the cab-E gene in Nicotiana plumbaginifolia. Here we demonstrate, by electrophoretic mobility shift and methylation interference assays, that a complex pattern of protein-DNA interactions characterizes this promoter. Among the multiple proteins identified, we focused on five different factors which either occupied important regulatory elements and/or were present in relatively large amounts in nuclear extracts. All of these proteins were distinguished on the basis of their recognition sequence and other biochemical parameters. One, GBF, interacted with a single sequence within the cab-E promoter homologous to the G-box found in many photoregulated and other plant promoters. A second factor, GA-1, bound to the GATA element which is located between the CAAT and TATA boxes of the cab-E and all other LHCII Type I CAB promoters. GA-1 also interacted in vitro with the I-boxes of the Arabidopsis rbcS-1A promoter and the as-2 site of the CaMV 35S promoter. Two other factors, GC-1 and AT-1, bound to multiple recognition sites localized within the GC-rich and AT-rich elements, respectively. GT-1, a protein which interacts with promoters of other light-regulated genes, bound to seven distinct sites distributed throughout the cab-E promoter.     

Co-regulation with genes of phospholipid biosynthesis of the CTR/HNM1-encoded choline/nitrogen mustard permease in Saccbaromyces cerevisiae

An 815 by region of the promoter of the Saccharomyces cerevisiae gene CTR/HNM1, encoding choline permease was sequenced and its regulatory function analysed by deletion studies in an in-frame promoter-lacZ construct. In addition to the TATA box, a 10 by motif (consensus 5′-CATGTGAAAT-3′) was found to be mandatory for CTR/HNM1 expression. This ‘decamer’ motif is located between nucleotides ?262 and ?271 and is identical in 9 of 10 by with the regulatory motif found in the S. cerevisiae INO1 and CHO1 genes. Constructs with the 10 by sequence show high constitutive expression, while elimination or alterations at three nucleotide positions, of the decamer motif in the context of an otherwise unchanged promoter leads to total loss of β-galactosidase production. Expression of the CTR/HNM1 gene in wild-type cells is regulated by the phospholipid precursors inositol and choline; no such influence is seen in cells bearing mutations in the phospholipid regulatory genes INO2, INO4, and OPI1. There is no regulation by INO2 and OPI1 in the absence of the decamer motif. However constructs not containing this sequence (promoter intact to positions ?213 or ?152) are still controlled by INO4. Other substrates of the choline permease, i.e. ethanolamine, nitrogen mustard and nitrogen half mustard do not regulate expression of CTR/HNM1.     

Multiple histone deacetylases are recruited by corepressor Sin3 and contribute to gene repression mediated by Opi1 regulator of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae

Yeast genes of phospholipid biosynthesis are negatively regulated by repressor protein Opi1 when precursor molecules inositol and choline (IC) are available. Opi1-triggered gene repression is mediated by recruitment of the Sin3 corepressor complex. In this study, we systematically investigated the regulatory contribution of subunits of Sin3 complexes and identified Pho23 as important for IC-dependent gene repression. Two non-overlapping regions within Pho23 mediate its direct interaction with Sin3. Previous work has shown that Sin3 recruits the histone deacetylase (HDAC) Rpd3 to execute gene repression. While deletion of SIN3 strongly alleviates gene repression by IC, an rpd3 null mutant shows almost normal regulation. We thus hypothesized that various HDACs may contribute to Sin3-mediated repression of IC-regulated genes. Indeed, a triple mutant lacking HDACs, Rpd3, Hda1 and Hos1, could phenocopy a sin3 single mutant. We show that these proteins are able to contact Sin3 in vitro and in vivo and mapped three distinct HDAC interaction domains, designated HID1, HID2 and HID3. HID3, which is identical to the previously described structural motif PAH4 (paired amphipathic helix), can bind all HDACs tested. Chromatin immunoprecipitation studies finally confirmed that Hda1 and Hos1 are recruited to promoters of phospholipid biosynthetic genes INO1 and CHO2.     

The INO1 promoter of Saccharomyces cerevisiae includes an upstream repressor sequence (URS1) common to a diverse set of yeast genes.

The INO1 promoter of Saccharomyces cerevisiae includes a copy of an upstream repression sequence (URS1; 5'AGCCGCCGA 3') observed in the promoters of several unrelated yeast genes. Expression of INO1-lacZ and CYC1-lacI'Z, activated by the INO1 UASINO, is significantly decreased by the INO1 URS1.