Gcn4 occupancy of open reading frame regions results in the recruitment of chromatin-modifying complexes but not the mediator complex

Abstract

Eukaryotic transcriptional activators usually recognize short DNA motifs, which are not only located within promoter regions, but also scattered throughout the genome. Assuming that the function of activators at non-promoter regions is wasteful and perhaps harmful, one can ask whether such binding is somehow prevented or if transcription is blocked at a downstream step. Here, we show that the yeast transcriptional activator Gcn4 is associated in vivo with several non-promoter euchromatic sites. This association results in the recruitment of the SAGA (Spt3/Ada/Gcn5/acetyltransferase) complex and the consequent activity of the Gcn5 histone acetyltransferase. The functional recruitment of the Swi/Snf nucleosome-remodelling complex was also evident at sites located in positioned nucleosomes. We show that this assemblage of coactivator complexes is not productive because of the absence of core promoter elements, other than the TATA box, that are required for stable mediator recruitment.

Figure 1

Recruitment of the Gcn4 transcriptional activator and coactivators at non-promoter chromosomal regions. (A) In vivo occupancy by Gcn4 of the TRP3 promoter and target sites located at the indicated regions and after growth in minimal (Min) or histidine starvation (AT) media. Levels shown on the y axis reflect the enrichment of the DNA relative to a region of the PHO5 open reading frame after correction for the ratios of amplification achieved using input DNA. (B) Occupancy of the same sites by the SAGA (Spt3/Ada/Gcn5/acetyltransferase) complex, monitored using a MYC-tagged Spt3 component, in relation to the presence (WT) or absence (gcn4) of Gcn4. (C) Occupancy of Swi/Snf in relation to the presence or absence of Gcn4, probed through a MYC-tagged Snf2 component. (D) Occupancy of the mediator complex in relation to the presence or absence of Gcn4, probed using a MYC-tagged Srb4 component. In the experiments presented in (B–D), strains were grown in conditions of histidine starvation.

Figure 2

The high level of occupancy of the PHO8 site by Gcn4 does not contribute to the regulation of PHO8 expression, or that of the upstream KRE2 gene. (A) The messenger RNA levels of the PHO8 gene from cells grown in either high- or low-phosphate media (HP and LP, respectively) are not affected by the presence of either low (WT/M) or high (WT/AT) levels of Gcn4, or by the absence (gcn4) of this protein. (B) mRNA levels of the KRE2 gene in wild-type (WT) and gcn4 strains grown in either minimal (M) or histidine starvation (AT) media. Actin mRNA (ACT1) was used as loading control in both experiments.

Figure 3

Functional consequences of coactivator recruitment. (A) Gcn5-dependent acetylation of nucleosomal histones located at the regions indicated was measured at low (M) or high (AT) Gcn4 concentrations by chromatin immunoprecipitation using antibodies raised against a diacetylated form of histone H3. Numbers indicate the enrichment of the DNA relative to a telomeric region (TEL) and corrected for the level of amplification achieved using input DNA. TEL (Vogelauer et al., 2000) was used as a control as it is heterochromatic and therefore under-acetylated. (B) The diagram in the left panel shows the mapped positions of nucleosomes around the Gcn4-response element (GCRE) site of the PHO8 gene. Gcn4-dependent remodelling of the nucleosomes positioned on either side of the GCRE located in the PHO8 open reading frame is shown in the left panel. Remodelling was monitored in wild-type (WT) or snf2 strains by determining the sensitivity to micrococcal nuclease and indirect end-labelling. M and AT indicate cells with low and high amounts of Gcn4, respectively. N is the micrococcal-nuclease cleavage pattern of naked DNA. By contrast, similar experiments for the POL2 site (right panel) showed the absence of positioned nucleosomes, as indicated by the identical patterns of micrococcal nuclease cleavages in nuclear (M and AT) or naked (N) DNA.

Figure 4

An engineered non-promoter site defines the requirements for coactivator recruitment. (A) The top left panel shows a representation of the PHO5 promoter with a Gcn4-response element (GCRE) site introduced into the linker between nucleosomes −2 and −1. Also indicated are the ClaI cleavage site used for monitoring nucleosome remodelling, the TATA box and the transcription initiation site (arrow). The right panel shows that in cells growing under histidine limitation (high levels of Gcn4), the wild-type promoter is occupied by Gcn4, the mediator complex (Srb4), SAGA (Spt3) and TATA-binding protein (TBP). The bottom left panel shows that Snf2 is required for remodelling of nucleosome −2. Remodelling was assayed by determining the accessibility to ClaI of cells containing low (M) or high (AT) amounts of Gcn4 in wild-type (WT) or snf2 strains. P indicates the protected DNA; U indicates one fragment that results from cleavage by the enzyme used and secondary digestions. (B) Mutating the TATA element (top left panel) affects only the recruitment of TBP. (C) The absence of all promoter elements and downstream open reading frame sequences (top left panel) allows the recruitment of SAGA.