Different roles for abf1p and a T-rich promoter element in nucleosome organization of the yeast RPS28A gene

Abstract

In vivo mutational analysis of the yeast RPS28A ribosomal protein (rp-)gene promoter demonstrated that both the Abf1p binding site and the adjacent T-rich element are essential for efficient transcription. In vivo Mnase and DNaseI digestion showed that the RPS28A promoter contains a 50-60 bp long nucleosome-free region directly downstream from the Abf1p binding site, followed by an ordered array of nucleosomes. Mutating either the Abf1p binding site or the T-rich element has dramatic, but different, effects on the local chromatin structure. Failure to bind Abf1p appears to cause nucleosome positioning to become disorganized as concluded from the complete disappearance of Mnase hypersensitive sites. On the other hand, mutation of the T-rich element causes the downstream nucleosomal array to shift by approximately 50 bp towards the Abf1p site, resulting in loss of the nucleosome-free region downstream of Abf1p. We conclude that Abf1p is a strong organizer of local chromatin structure that appears to act as a nucleosomal boundary factor requiring the downstream T-rich element to create a nucleosome-free region.

Figure 1

Effect of mutations in the Abf1p binding site and T-rich element of the RPS28A promoter on transcription of the GUS reporter gene [with respect to the E.coli β-glucuronidase gene (32)]. (A) Overview of the reporter used. Vector sequences, Integrative or Episomal, are denoted by an asterisk. (B) Sequences of the various mutants. (C) Northern analysis of the transcript levels of the integrated GUS-reporter constructs. Quantification of mRNA levels is indicated in percentages relative to the wild-type construct IRS3, using actin mRNA as loading control.

Figure 2

Effect of Dat1p on transcription of the RPS28A rp-gene. A dat1 deletion strain (see Materials and Methods) and the isogenic wild-type DAT1 strain were monitored for the expression of the RPS28A and RPL25 rp-genes. When the expression of RPS28A in the dat1 deletion strain was quantified from independent blots, an increase of 10–20% was found relative to the wild-type control. Actin mRNA was used as loading control.

Figure 3

In vivo mapping of nucleosome positions on wild-type RPS28A promoters and promoters containing a mutated Abf1p site. (A) Spheroplasts prepared from single copy integrants of the reporter construct IRS3 (wild-type) and IRS* (unable to bind Abf1p) were permeabilized with nystatin and treated with Mnase. Purified DNA was digested with MluI and HindIII (Fig. 1A) and subjected to indirect end-labeling using a probe flanking the MluI site. The XbaI site corresponds to the position of the Abf1p site. As control, deproteinized DNA from the IRS3 integrant was digested with Mnase (Naked DNA). (B) As in (A), using cells carrying either multicopy plasmids ERS3 (wild-type) or ERS* (unable to bind Abf1p). Naked DNA digests on genomic DNA from both ERS3 and ERS* are included. Distances are from the translational start codon ATG. Protected regions of 160–170 bp are interpreted as positioned nucleosomes (closed elipses). The region between the two strongest Mnase hypersensitive sites (open box) spans 130 bp of the RPS28A–GLN4 intergenic region.

Figure 4

Comparison of in vivo nucleosome positions on the wild-type RPS28A promoter and a promoter containing a mutated T-rich element. Nystatin-permeabilized spheroplasts prepared from wild-type (ERS3) and mutant cells (ETS6) were treated with either Mnase (A) or DNaseI (B). Distances are from the translational start codon ATG. Protected regions of 160–170 bp are interpreted as positioned nucleosomes (closed elipses). The 130 bp Mnase-protected region that covers the Abf1p site (XbaI) in the wild-type promoter (ERS3) has decreased to 85 bp when the T-rich element is mutated (ERS6). Concomitantly, downstream nucleosomes are shifted towards the Abf1p binding site.

Figure 5

High resolution analysis of in vivo Mnase accessibility of the wild-type RPS28A promoter (ERS3) and a RPS28A promoter that contains a mutated T-rich element (ETS6). Primer GUS3028rev was used for unidirectional, linear amplification (five cycles) on DNA obtained after in vivo Mnase digests (Figs 3B and 4). Lane 1–3, chromatin DNA of ERS3 (time samples 0, 1 and 2.5 min); lane 4, naked DNA of ERS3; lane 5–7, chromatin DNA of ETS6 (time samples 0, 1 and 2.5 min); lane 8, naked DNA of ETS6. Sequencing reactions using the same primer are indicated by A, C, G and T. The position of the Abf1p binding site, T-rich element and the multiple translational positions of the mapped nucleosomes are indicated. Distances are from the translational start codon ATG.