for maintenance of the PHO8 NDR1, basal expression of PHO8 but not for RSC
recruitment
The RSC complex contains two subunits (Rsc3 and Rsc30) that can bind a specific DNA sequence
motif in vitro [138]. Such putative binding sites commonly locate to within promoter NDRs and
overlap with sites of increased nucleosome occupancy in a rsc3‐ts strain [138]. This suggested that
such RSC binding motifs could play a role in RSC’s ability to position nucleosomes. We scanned the PHO8 promoter for Rsc3 binding sites using the position weight matrix (PWM) obtained by Badis et
al. [138] and identified three putative binding sites at positions ‐214, ‐151 and ‐10 relative to the PHO8 ORF (Fig. 6 and 49A). The site at ‐10 lies within nucleosome N+1, the site at ‐151 centrally
within the PHO8 NDR2, and the site at ‐214 right at the NDR2/N‐1 border.
Figure 49. The PHO8 promoter harbours three predicted and conserved Rsc3 binding sites.
(A) Sequence alignment of the predicted Rsc3 binding sites at the PHO8 promoter in the indicated Saccharomyces species.
The CGCGC motif that mostly defines the consensus sequence is highlighted in bold and cross‐species conservation is
indicated by black dots. “S. cer. mutated” gives the sequence after mutagenesis of the corresponding Rsc3 binding site. (B)
Same as (A), but for the indicated loci. Promoters for which no putative Rsc3 biding was identified are listed in the bottom
box.
A sequence alignment of this region from closely related Saccharomyces species revealed strong
conservation of the sites at ‐151 and ‐214 in all yeasts except for the more distantly related S. castelli
(Fig. 49B). The site at ‐10, in contrast, was conserved only in the two closest S. cerevisiae relatives.
We mutated all three sites individually (∆‐10, ∆‐151 and ∆‐214) as well as in combination (∆all), both
in the chromosome locus as well as in plasmid pP8apain, and analysed positioning in vivo by DNaseI
indirect endlabelling. The DNaseI pattern for the ∆‐10 and the ∆‐214 PHO8 promoter mutants was as
for the wildtype whereas the ∆‐151 and the ∆all mutants showed strongly reduced DNaseI
accessibility at the PHO8 NDR1 (Fig. 50A). The effect at NDR1 was similar to that seen with the arp9‐
ts, rsc3‐ts and sth1‐td strains, but there was no effect on nucleosome N‐3 upon removal of the
putative Rsc3 site at ∆‐151. Again we confirmed these effects by restriction enzyme accessibility
analysis (Fig. 43B). In keeping with the DNaseI pattern, all Rsc3‐mutants displayed similar HpaI (cuts
the ∆‐151 and ∆all mutants (26% and 43%) compared to the wildtype (69%) or the ∆‐10 and ∆‐214
mutants. Almost identical results were obtained when mutating the respective Rsc3 binding sites on
the pP8apin shuttle vector (Fig. 43C and S2) arguing that the effects were independent of the larger
chromatin context. Collectively, the putative Rsc3 site at ∆‐151 seemed to be required for excluding
nucleosomes from the PHO8 NDR1.
Next, we checked whether mutation of any of the putative Rsc3 binding sites (and the resulting
changes in occupancy over the PHO8 NDR1) was reflected as changes in PHO8 expression. The PHO8
gene codes for an alkaline phosphatase that is the major contributor to the total cellular alkaline
phosphatase activity (Fig. 50B ‐ compare activity of pho8 strain to the wildtype). We measured
alkaline phosphatase activity both under repressing (+Pi) and inducing (‐Pi) conditions to discriminate between a role of the putative binding sites in basal and activated transcription (Fig. 50B). Alkaline
phosphatase activity under repressing conditions was greatly reduced in the ∆‐151 mutant (2.5 units
compared to 15.7 units for the wildtype promoter; p<0.00016; two‐sided paired t‐test). In contrast,
the basal alkaline phosphatase activity for the ∆‐10 promoter was slightly but significantly elevated
(p<0.001; two‐sided paired t‐test). For the ∆‐214 promoter however there was no significant
difference to the wildtype. Mutation of all three putative binding sites resulted in a reduced
phosphatase level under repressive conditions similar to the level seen for the ∆‐151 promoter (Fig.
50B). In contrast, the induced level of alkaline phosphatase activity was not significantly affected in
any Rsc3 binding site mutant (p>0.05; two‐sided paired t‐test). Hence, the putative Rsc3 binding site
at ‐151 is required for basal transcription but not for maximal activity upon induction. Interestingly,
Figure 50. The Rsc3 binding site at position ‐151 was required for nucleosome depletion over the PHO8 promoter NDR and basal expression of PHO8.
(A) DNaseI indirect endlabelling analysis of the PHO8 promoter region in strains with mutations in none (wt, CY337 background), one (Δ‐10, Δ‐151 or Δ‐214) or all (Δall) of the three predicted Rsc3 binding sites at the PHO8 promoter. Bars in between lanes mark the position of the PHO8 promoter NDR. Their thickness corresponds to the extent of DNaseI accessibility, i.e. thin bars highlight increased nucleosome occupancy. (B) Measurement of alkaline phosphatase activity of wt (CY337) and snf2 (CY408) strains with mutations in none (wt), one (Δ‐10, Δ‐151 or Δ‐214) or all (Δall) of the three predicted Rsc3 binding sites at the PHO8 promoter or with pho4 or pho8 deletion. Cells were grown logarithmically in phosphate containing medium (+Pi, repressive conditions) or
placed overnight in phosphate free medium (‐Pi, inducing
conditions). The mean and standard deviation of three to five independent biological replicates are shown. (C) DNaseI indirect endlabelling analysis of the PHO8 promoter region in wt and snf2 strains with mutations in none (wt), one (Δ‐10) or all (Δall) of the three predicted Rsc3 binding sites as indicated. Except for the wildtype sample on the left (+Pi), all nuclei
were prepared after overnight growth in phosphate free medium (‐Pi). (B‐C) Schematics, ramps and makers as in Figure
42.
basal transcription did not depend on the transactivator Pho4, albeit induced activity levels were
greatly reduced in the pho4 mutant (Fig. 50B).
The Rsc30 subunit of the RSC complex recognises an almost identical motif as the Rsc3 subunit [138]
and hence the effects seen upon removal of the "Rsc3 binding sites" could equally be attributed to
loss of Rsc30 binding. However, a rsc30 deletion mutant did not show any increased occupancy over
the PHO8 promoter and no reduction in alkaline phosphatase activity under repressing conditions
(Fig. 59, S3 and data not shown). Together these results argue that the putative Rsc3 binding site at ‐151 is required for basal transcription because of its role in keeping NDR1 nucleosome depleted. We
wondered if the strict Snf2‐dependency of PHO8 promoter remodeling [201] was due to some aspect
of the chromatin structure as shaped by Rsc3‐recruited RSC. However, inducing the ∆all or the ∆‐10
mutant promoter in the snf2 background did not lead to any more remodeling as for a wild type
promoter (Fig. 50C). We also checked alkaline phosphatase activity of the various Rsc3‐site mutants
in the snf2 background. Under non‐inducing (+Pi) conditions the relative levels of the mutant as compared to the wt‐promoter were very
similar to those seen in the wildtype
(SNF2) background. However under
inducing conditions (‐Pi), alkaline phosphatase levels for the ∆‐151 and
the ∆‐214 mutant were significantly
lower (p=0.004 and 0.037 respectively;
two‐sided paired t‐test) than for the
wildtype promoter. Moreover, levels in
the ∆all mutant were even lower than
for the ∆‐151 and ∆‐214 single mutants.
Evidently, the two putative sites at ∆‐151 and ∆‐214 play a role in PHO8
expression under inducing conditions in
the absence of SWI/SNF but this
influence appears to be independent of
effects on nucleosome positions.
Given the aforementioned effects observed in the ∆‐151 mutant, we decided to test if the CGCGC
motif at ‐151 acts through recruitment of RSC. We measured RSC occupancy by ChIP at three
different sites along the PHO8 promoter (Fig. 51A) in a wildtype strain and in the ∆‐151 and ∆‐all
mutants. At all three sites along the wildtype PHO8 promoter we observed an approximate three fold
enrichment of Sth1 (the ATPase subunit of RSC) relative to telomere confirming the presence of RSC
at the PHO8 promoter (Fig. 51B). As there was no reduction in RSC occupancy neither in the ∆‐151
Figure 51. RSC is not recruited to the PHO8 promoter via any of the putative Rsc3 binding sites. (A) Schematics as in Figure 7. Location and type of the PHO8 promoter amplicons used in (B) as indicated. (B) RSC occupancy was determined via ChIP using an anti‐myc antibody (9E11) for strains carrying the STH1‐9MYC allele (FT4 STH1‐9MYC::TRP) in combination with either the wildtype PHO8 promoter (wt) or mutations in one (∆‐151) or all (∆all) putative Rsc3 binding sites (see Fig. 49). Used amplicons are shown in (A) and for the HTA1 promoter, the PGK1 coding region and for the telomere region on the short arm of chromosome 6 as described in Methods (chapter 4.2.3.). Sth1‐Myc occupancy was normalized against the telomere. Error bars correspond to the variation of two biological replicates.
nor the ∆‐all strain we concluded that the putative Rsc3 binding sites play no role in the recruitment
to or binding of RSC to the PHO8 promoter region.