Abstract
Metabolic reactivation (incubating spheroplasts with galactose and casamino acids) causes disruption of nucleosomes from the upstream regions of the yeast GAL1, GAL10, and GAL80 genes. The disruption is specific. It depends on the transcription activator Gal4; it only occurs in galactose-reactivated chromatin from galactose-grown cells; it only affects upstream region, gene-proximal nucleosomes. Due to this specificity and because some of the same regions have shown induction-dependent changes by in vivo analyses, we suggest that the nucleosome-disrupted structure produced by reactivation is the authentic chromatin structure for these regions under conditions of galactose-induced GAL1-10 and GAL80 expression. It is necessary to carry out a spheroplast reactivation treatment in order to observe this disrupted structure in nuclear chromatin because nucleosomes are redeposited onto these regions during the preliminary steps of nuclear isolation (cell harvest/spheroplast preparation) probably in response to the nonphysiological conditions associated with these steps. However, during the same isolation procedures in cells lacking Gal80 protein, there is no nucleosome deposition on these regions, and the in vivo disrupted structure remains present in the nuclear chromatin. Therefore, the nucleosome deposition process that operates in wild-type cells is dependent on Gal80 protein, defining another activity of this negative regulator.
Highlights
The UASG elements on GAL1–10 and GAL80 lie within constitutively nonnucleosomal, chromatin hypersensitive regions [4, 6, 8]
Why Is the Chromatin Structure of the GAL1–10 Intergenic Region Not Expression-sensitive in Nuclear Chromatin as It Is in Vivo?—GAL1–10 intergenic chromatin consists of an ϳ170-bp nonnucleosomal, UASG-containing hypersensitive region [4, 8], which is surrounded by positioned nucleosomes (8 –11)
Shows generally strong cleavage within the hypersensitive region (4, track 3), except for the UASG elements, which are protected by Gal4p; the nucleosomal regions, upstream of GAL10 and between the HR border and the GAL1 TATA, are relatively protected (E, track 3)
Summary
The UASG elements on GAL1–10 and GAL80 lie within constitutively nonnucleosomal, chromatin hypersensitive regions [4, 6, 8]. In vivo analysis detected structural changes in two of the GAL1–10 intergenic nucleosomal regions upon galactoseinduction of expression [5]. We describe experiments that allow us to detect intergenic nucleosome changes in nuclear chromatin, reconciling the in vivo and nuclear results These experiments yield some unexpected insights on nucleosome disruption/deposition processes taking place on gene control regions. GAL80 expression occurs at a low basal level in glycerol but is induced 5–10-fold higher, via Gal4p, in galactose [2]. This gene family provides excellent opportunities to study the relationship of chromosome structure to gene regulation. For example, that under conditions of induced expression (galactose) Gal4p strongly protects the ma-
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