The chromatin structure at the promoter of a glyceraldehyde phosphate dehydrogenase gene from Saccharomyces cerevisiae reflects its functional state.

Abstract

The chromatin structure of TDH3, one of three genes encoding glyceraldehyde phosphate dehydrogenases in Saccharomyces cerevisiae, was analyzed by nuclease digestion. A large hypersensitive region was found at the TDH3 promoter extending from the RNA initiation site at position -40 to position -560. This hypersensitive domain is nucleosome free and includes all putative cis-acting regulatory DNA elements. It is equally present in cells grown on fermentable as well as nonfermentable carbon sources. In a mutant which lacks the trans-activating protein GCR1 and which as a consequence expresses TDH3 at less than 5% of the wild-type level, the chromatin structure is different. Hypersensitivity between -40 and -370 is lost, due to the deposition of nucleosomes on a stretch that is nucleosome free in wild-type cells. Hypersensitivity is retained, however, further upstream (from -370 to -560). A similarly altered chromatin structure, as in a gcrl mutant, is found in wild-type cells when they approach stationary phase. This is the first evidence for a growth-dependent regulation of the TDH3 promoter.Saccharomyces cerevisiae promoters are composed of multiple sequence elements (for reviews, see references 10-12, 32). The TATA box and the RNA initiation site determine the accuracy of transcription initiation, but for transcription to actually take place, upstream elements are also required. These upstream elements are called upstream activating sequences. They seem to be functionally equivalent to enhancers in higher eucaryotes (12). Through the interaction with regulatory proteins, the upstream elements are also responsible for the regulatory properties of a specific promoter.For some time, our interest has been in the role that the chromatin structure plays in gene regulation. Eucaryotic DNA is normally packaged in chromatin, with the nucleosome being a basic subunit (for reviews, see references 9, 9a, 28, 33, 37). It is composed of 146 base pairs (bp) of DNA wrapped tightly around a histone octamer consisting of two copies each of the histones H2A, H2B, H3, and H4. Functionally important DNA elements have, however, been found to reside in many cases in nucleosome-free regions, which makes these sequences highly susceptible to nucleases used to probe the structure of chromatin. For this reason these regions are usually called hypersensitive sites.We have previously shown that an upstream activating sequence element from the promoter of PHO5, a strongly regulated acid phosphatase gene from S. cerevisiae, is contained within a short hypersensitive region under conditions of PHOS repression (1). The remaining part of the PH05 promoter is organized in positioned nucleosomes under these conditions. Upon PHOS activation, these nucleosomes are removed and the entire promoter turns hypersensitive (2).In an effort to address the question of whether a chromatin transition at a promoter is the cause or the result of gene activation, we extended our analyses to another strong promoter in S. cerevisiae. We chose TDH3, a heavily * Cor...

A complex regulatory element from the yeast gene ENO2 modulates GCR1-dependent transcriptional activation.

“…In this regard, phased nucleosomes were detected within the 5' regulatory sequences of the yeast glyceraldehyde-3-phosphate dehydrogenase gene TDH3 in strains carrying a gcrl null mutation. These same regulatory sequences were devoid of nucleosomes in a strain carrying the wild-type GCR1 allele (44). We suggest that GCR1 plays a role in a hierarchy of binding events leading to transcriptional activation of EN02.…”

Preferential Accessibility of the Yeast his3 Promoter Is Determined by a General Property of the DNA Sequence, Not by Specific Elements

“…In several derivatives, transcription from both the his3 and pet56 promoters was virtually eliminated yet HinfI cleavage at the Gcn4 site occurred at a level comparable to that of the wildtype chromosomal locus. In a similar vein, Swi-Snf-or activator-dependent alterations of chromatin structure (1,7,12,29,33,46) or nuclease hypersensitivity (23,39) can occur in the absence of a functional TATA element and transcriptional activity of the promoter.…”

“…First, yeast promoters often contain transcription-independent regions of nuclease hypersensitivity (6,7,18,23,25,27,33,39,46). Second, in several of these studies, micrococcal-nuclease mapping suggests that hypersensitivity reflects "nucleosomefree" regions, although it is unclear whether these regions are truly devoid of nucleosomes or have an alternative structure.…”

Sequences within an upstream activation site in the yeast enolase gene ENO2 modulate repression of ENO2 expression in strains carrying a null mutation in the positive regulatory gene GCR1.

“…Pavlovic' and Horz (16) showed that chromatin structure within the 5'-flanking region of the yeast glyceraldehyde-3-phosphate dehydrogenase gene TDH3 is dependent on the GCRI gene. In GCR1+ strains, the upstream regulatory region of TDH3 is devoid of nucleosomes.…”