Chromatin opening and transactivator potentiation by RAP1 in Saccharomyces cerevisiae

Abstract

Transcriptional activators function in vivo via binding sites that may be packaged into chromatin. Here we show that whereas the transcriptional activator GAL4 is strongly able to perturb chromatin structure via a nucleosomal binding site in yeast, GCN4 does so poorly. Correspondingly, GCN4 requires assistance from an accessory protein, RAP1, for activation of the HIS4 promoter, whereas GAL4 does not. The requirement for RAP1 for GCN4-mediated HIS4 activation is dictated by the DNA-binding domain of GCN4 and not the activation domain, suggesting that RAP1 assists GCN4 in gaining access to its binding site. Consistent with this, overexpression of GCN4 partially alleviates the requirement for RAP1, whereas HIS4 activation via a weak GAL4 binding site requires RAP1. RAP1 is extremely effective at interfering with positioning of a nucleosome containing its binding site, consistent with a role in opening chromatin at the HIS4 promoter. Furthermore, increasing the spacing between binding sites for RAP1 and GCN4 by 5 or 10 bp does not impair HIS4 activation, indicating that cooperative protein-protein interactions are not involved in transcriptional facilitation by RAP1. We conclude that an important role of RAP1 is to assist activator binding by opening chromatin.

FIG. 1

Perturbation of nucleosome positioning elicited by GCN4 via a nucleosomal binding site is poorer than that elicited by GAL4. (A) Schematic diagram of plasmids TAGCN1Δ80 and TA17Δ80. Positioned nucleosomes I and II are shown as ellipses. (B) Induction of GCN4 by 3-AT results in minimal perturbation of nucleosome positioning in TAGCN1Δ80. MNase cleavage sites were mapped clockwise from the EcoRV site, as indicated, in naked DNA (lanes 1 and 2) or in chromatin from cells lacking GCN4 or from GCN4⁺ cells induced with 3-AT (lanes 3 to 10). Note that the cleavage seen in the region of nucleosome II (especially lanes 4 to 6, denoted by an asterisk) corresponds to a site cleaved very strongly in naked DNA; we observed some variability in this cleavage in different experiments (see Fig. 6, lanes 4 and 5). (C) Comparison of nucleosome perturbation in TAGCN1Δ80 by GCN4 expressed from the DED1 promoter (lanes 13 and 14) or endogenous GCN4 induced with 3-AT (lanes 15 and 16) with perturbation in TA17Δ80 by GAL4 expressed from a multicopy plasmid bearing the GAL4 gene (lane 18). Lane 17 contains chromatin from cells grown in glucose medium and containing only the endogenous GAL4 gene. Lanes 11 to 18 were run on the same gel. Samples were digested with MNase at 0 U/ml (lanes 3 and 7), 0.5 U/ml (lane 4), 1 U/ml (lanes 1, 5, 8, and 11), 2 U/ml (lanes 6 and 9), 4 U/ml (lanes 2 and 12), 5 U/ml (lanes 10, 13, and 15), or 20 U/ml (lanes 14 and 16 to 18). The locations of nucleosomes I and II are indicated by ellipses.

FIG. 2

The RAP1 binding site is required for HIS4 expression mediated by GCN4 but not by GAL4 or Bicoid. Yeast strains containing integrated HIS4 promoters (diagrammed at the top), differing in the presence of a wild-type (wt) or mutated (mut) RAP1 site and in the activator binding site, were streaked from raffinose medium containing histidine onto galactose medium lacking histidine. GCN4 was expressed from the DED1 promoter, GAL4 was expressed from the ADH1 promoter, and Bicoid was expressed from a modified GAL1 promoter.

FIG. 3

HIS4 expression mediated by the GCN4 activation domain through a GAL4 binding site does not require the RAP1 binding site. Cells containing the GAL4 binding site (UAS_GAL4) with a wild-type (wt) or mutated (mut) RAP1 site in the HIS4 promoter, and expressing GAL4-GCN4 from the DED1 promoter, were streaked from SC-Leu/glucose onto SC-His-Leu/glucose, as were cells containing the GCN4 binding site (UAS_GCN4) with a wild-type or mutated RAP1 binding site.

FIG. 4

HIS4 expression mediated by GAL4 through a weak binding site depends on the RAP1 binding site. Cells containing the strong GAL4 binding site (UAS_GAL4) or the weak GAL4 binding site (UAS_GAL4W) with a wild-type (wt) or mutated (mut) RAP1 binding site were streaked from rich medium onto SC-His/galactose. GAL4 was expressed from the endogenous GAL4 promoter.

FIG. 5

Overexpression of GCN4 partially overcomes the requirement for a RAP1 binding site for GCN4-mediated HIS4 expression. GCN4 was overexpressed by using the hormone-dependent activator GAL4-ER-VP16 to activate the GAL1pr-GCN4 promoter (top). Cells containing the GCN4 binding site (UAS_GCN4) and a wild-type (wt) or mutated (mut) RAP1 binding site, and harboring the GAL1pr-GCN4 plasmid and an expression vector for GAL4-ER-VP16, were streaked onto SC-His-Ura-Leu/glucose plates containing no β-estradiol or containing 100 nM β-estradiol, as indicated.

FIG. 6

Perturbation of nucleosome positioning by RAP1 via a nucleosomal binding site. MNase cleavage sites in plasmids TAGCN1Δ80 and TAR/GCN1Δ80, schematized at the top, as well as TAR_mut/GCN1Δ80, were mapped clockwise from the EcoRV site, as indicated. Cleavage sites were mapped in naked DNA (D) or in chromatin (C) from cells grown in glucose media. Lane 1 contains ΦX/HaeIII marker DNA. Locations of positioned nucleosomes I and II are indicated by ellipses. The closed circles between lanes 4 and 5 and lanes 12 and 13 indicate cleavages enhanced in chromatin relative to DNA, and the star indicates a site protected in chromatin. Each pair of lanes, beginning with lanes 2 and 3, differs only in the concentration of MNase used. Lanes 10 to 13 were derived from a gel separate from lanes 1 to 9.

FIG. 7

Altering the spacing between the RAP1 and GCN4 sites does not impair HIS4 transactivation. Yeast strains (Table 2) contain integrated HIS4 promoters with either a wild-type (wt) or mutated (mut) RAP1 site and have either wild-type spacing between the RAP1 and GCN4 sites or 5 or 10 bp inserted in the UAS (UAS_GCN4+5 and UAS_GCN4+10). Cells were streaked from SC-Leu/glucose onto SC-His-Leu/glucose. GCN4 was expressed from the DED1 promoter.