Fig. 8.

(A) TUP1 repressed upstream regions associate with UAS centred Hda1p. Relative distribution of Hda1p over gene regulatory regions probed by ChIP in wt strains, charted for whole genome (left), and the gene set > 5 × de-repressed in a tup1 mutant (N = 90, right). Hda1p presence over the − 320 to − 260 upstream region (‘UAS’) is 81% increased in this set (p = 0.005) compared with whole genome, while it is lower in the − 90 to − 30 proximal region (‘TSS’) and the 3’ end ‘ORF’ region. ORFs scoring in two regions are plotted in the areas between major categories. Only ORFs scoring above the FDR are included in the pie chart, while detectable Hda1p was 20% increased overall (p = 0.05). (B) TUP1 de-repressed upstream regions have a high incidence of nucleosome depletion. Average of nucleosomal occupancy differences between tup1 and wt strains is plotted over the upstream 1000 bp region. Genes > 5 × de-repressed (N = 90) in a tup1 mutant show a lower average trace than non de-repressed genes (N = 90), or a set of random genomic 1000 bp regions (N = 90). Neither control shows significant changes in nucleosome occupancy between wt and mutant. De-repressed gene changes (tup1) show a wide variance ranging from wt occupancy to depletion occurring with high incidence across a wide upstream region (error bars, 1 SD). (C) Model for the role of Cyc8–Tup1 in FLO1 gene repression. In wt, Cyc8–Tup1 binds to a distinct site at the FLO1 promoter and is associated with an array of strongly positioned, deacetylated nucleosomes covering the promoter and upstream region. The histone deacetylases (HDACs), Rpd3p and Hda1p bind the promoter in a Cyc8–Tup1 dependent manner and contribute to nucleosome positioning, histone deacetylation and gene repression.

In the absence of Cyc8–Tup1 (cyc8 mutant), there is a gross remodelling of FLO1 promoter and upstream chromatin involving nucleosome acetylation, rearrangement and eviction which accompanies FLO1 gene de-repression. The occupancy of Rpd3p and Hda1p (to a lesser extent) is reduced. Swi–Snf is recruited to the site previously occupied by Cyc8–Tup1 and potentially directs nucleosome disruption. The increased chromatin acetylation in the cyc8 mutant suggests that histone acetyltransferase (HAT) recruitment and activity occur in this region.

In the absence of the HDACs Rpd3p and Hda1p (rpd3 hda1 double mutant), Tup1p occupancy persists. Swi–Snf occupancy, nucleosome rearrangement and eviction also occur at the FLO1 promoter and upstream chromatin but at a reduced level compared to cyc8, and FLO1 is only partially de-repressed (compared to cyc8). Histone acetylation over the region is also lower than in the cyc8 mutant suggesting decreased HAT occupancy or activity in this strain.