5. ANÁLISIS DE LAS EXPERIENCIAS
5.1 Síntesis de las experiencias más significativos
dependency
In vitro reconstitution of nucleosome positioning at the PHO5 and PHO8 promoters suggested that
differences in nucleosome stability may cause differentially stringent requirements for chromatin
cofactors [175]. In vitro reconstitution of proper positioning at PHO5 required a maximal assembly
degree and maximal amount of WCE ([175]; data not shown), whereas proper positioning at the PHO8 promoter was reconstituted even at sub‐saturating concentrations of histones or WCE ([175];
Fig. 15). This differential in vitro‐stability of the promoter architecture correlated with a differential
co‐factor requirement for nucleosome eviction. Both PHO5 and PHO8 are activated upon phosphate
starvation by binding of the Pho4 transactivator, recruitment of various cofactors (such as Gcn5,
SWI/SNF and Asf1) and subsequent eviction of promoter nucleosomes. Remodeling and removal of
the nucleosomes at the PHO5 promoter occurs in the absence of SWI/SNF (although kinetically
delayed) whereas remodeling at the PHO8 promoter is absolutely dependent on SWI/SNF [201]. The
correlation of high in vitro stability and strong co‐factor dependency in vivo previously led to the
postulation of a causal link, i.e. nucleosomes with high intrinsic stability require specific "strong"
remodeling/removal factors for their eviction, whereas a redundant set of co‐factors exists for
nucleosomes with low(er) intrinsic stability [175].
In addition to PHO5 and PHO8, nucleosome positions and transcriptional regulation was also
characterized for another promoter of the PHO regulon: the PHO84 promoter. Here, the main
feature is two positioned nucleosomes, one upstream and one downstream of a short DNaseI
hypersensitive site (Figs. 6, 10 and 16) that are both remodelled and removed upon promoter
induction. Interestingly, the PHO84 promoter, with respect to remodeler dependency, behaves like a
hybrid between the PHO5 and PHO8 promoter [169]. Remodeling of the downstream nucleosome
occurred in the absence of both the SWI/SNF and Ino80 remodeling enzymes whereas the upstream
nucleosome was not removed in the absence of SWI/SNF or, under sub‐maximal induction
conditions, also not in the absence of Ino80. Thus, the upstream nucleosome had similar remodeling
Figure 14. Reconstitution of in vivo‐like nucleosome positioning at the PHO8 promoter with recombinant Xenopus laevis histones.
pP8apin plasmids were assembled by salt gradient dialysis with recombinant Xenopus laevis histones, incubated with WCE and ATP and analyzed by DNaseI indirect endlabelling. Schematics, ramps and markers as in Figure 8B.
requirements as the PHO8 promoter nucleosomes whereas the downstream nucleosome could be
redundantly remodelled just like the PHO5 promoter nucleosomes. So we tested also here if the
remodeler dependency of these two nucleosomes correlated with their intrinsic stability.
Figure 15. Reduction of the amount of WCE in the reconstitution reaction leads to a progressive
decrease in the number of templates with properly positioned nucleosomes.
Titration of the WCE extract in reconstitution reactions containing pre‐assembled pUC19‐PHO8‐short plasmids. Numbers
above the ramps indicate the relative amount of WCE present in each reconstitution reaction. Dots mark the position of
three bands set up by activities within the WCE (see "1x" lane) and the square the position of a band that is removed by the
activities of the WCE (see "1/128X" lane). Intensities of the dots/squares reflects the intensity of the corresponding bands.
The dot in brackets marks the position of a l band within the lacZ ORF of the bacterial vector backbone that is co‐generated
along with proper positioning (see Fig.8B). Schematics, ramps and markers as in Figure 8B.
As shown in Figure 10, salt assembly already correctly positioned both the upstream and
downstream nucleosome indicating that both nucleosomes might be largely positioned by strong
nucleosome positioning sequences (cis factors), which may lead to high intrinsic stability. In addition,
assembly with a histone:DNA ratio of 0.6 gave the same results, i.e. both nucleosomes remained
properly positioned (Fig. 16A, Lane "0.6 ‐WCE ‐ATP"). However, when we added WCE to the under‐
assembled chromatin, the downstream nucleosome was lost or shifted further downstream as an
extra strong band appeared that overlapped with the position of the downstream nucleosome
(Figure 16A; compare samples for 1:1 and 0.6:1). We confirmed this by measuring restriction enzyme
accessibility at various sites across the promoter including two enzymes that cut within the position
of the upstream (HhaI) and downstream nucleosome (TaqI) (Fig. 16B). Accessibilities for the 1:1 ratio
chromatin were generally very low but the data for the 0.6:1 ratio chromatin was conclusive. The
HhaI site remained much less accessible (36%) upon addition of WCE/ATP than the two sites that cut
in the neighbouring linkers (66% and 73% for BsrBI and MfeI respectively). The TaqI site in contrast
became as accessible (69%) as the neighbouring linkers (73% and 60%) arguing for lower stability of
the downstream compared to the upstream nucleosome.
As further support, we carried out an in silico analysis of the nucleosome forming probability of the
three PHO promoters using the N‐score model developed by Yuan et al. [132]. This model was
trained on S. cerevisiae in vivo nucleosome occupancy data and should therefore reflect the average
nucleosome stability in the presence of all relevant factors in vivo. The N‐Score profiles for the PHO5, PHO8 and PHO84 promoter were kindly provided by G. C. Yuan and supported the correlation
between nucleosome stability and remodeler dependency (Fig. 6). The N‐Score profile for the PHO5
Figure 16. Downstream nucleosome at the PHO84 promoter had a lower intrinsic stability in vitro. (A) In vitro reconstitution of nucleosome positioning at the PHO84 promoter with sub‐maximally assembled chromatin
templates. pUC19‐PHO84 plasmids were pre‐assembled at the indicated histone:DNA mass ratios and incubated in the
presence or absence of WCE and ATP and analyzed by DNaseI indirect endlabelling. Free DNA samples, markers and ramps
as in Figure 10. Vertical bars highlight the extent of the DNaseI accessible region downstream of the upstream nucleosome.
Schematics between lanes 6 and 7 and on the right indicate the approximate positions of the "up"‐stream and "down"‐
stream nucleosome. (B) Same samples as in (A) were analyzed by restriction enzyme accessibility. Accessibility is plotted
relative to the position of the restriction site (in bp from the PHO84 ORF start): BsrBI (‐718), HhaI (‐564), MfeI (‐387), TaqI (‐
360) and PacI (‐239). The average and variation of two biological replicates is shown (BsrBI and PacI only one replicate).
Error bars were in some cases within symbol size. Schematics below the graph show the in vivo positions of the
nucleosomes at the PHO84 promoter, Pho4 binding sites, TATA box and the TSS as in Figure 7.
major peaks in the profile correspond to sites that are linker regions in vivo indicating that the actual
occupied positions in vivo might be at odds with average in vivo sequence preferences. The N‐Score
profile for PHO8 displays generally very high values and the position of the two main troughs
correspond to the positions of the NDRs upstream of the PHO8 and KRE2 ORF while the locations of
the nucleosomes generally overlap with peaks in the N‐Score profile. So the nucleosomes of the PHO8 promoter occupy DNA with high nucleosome forming probability.
At the PHO84 promoter, the upstream nucleosome is centred right over the only major N‐Score
peak. Upstream of this peak is a long region with extremely low N‐Score values corresponding to a
nucleosome depleted region, both in vivo and in vitro (Figure 10, 16, 18 and 19). Downstream of the
major peak, the N‐score profile is flat and displays rather average values. Evidently, the correlation
between the three N‐Score profiles and the actual nucleosome positions strongly support the
hypothesis that remodeler‐dependent nucleosomes are more stable than remodeler‐independent
nucleosomes. Nonetheless, all these correlations, i.e. between the remodeler‐dependency in vivo,
the stabilities in vitro and the predicted stabilities in silico, do not necessarily argue for a causal
relationship between intrinsic stability and remodeler dependency of a nucleosome.
Therefore, we rationally manipulated the intrinsic stability of the upstream nucleosome in order to
test if this affected its dependency on SWI/SNF for remodeling. Homopolymorphic dA:dT stretches
have low intrinsic affinity for nucleosome formation ([122] and references therein). We mutated
stretches of 10 or 19 residues within the upstream nucleosome to adenine in the PHO84 locus‐
containing pCB84a shuttle vector. The corresponding vectors pCB84a‐10A and pCB84a‐19A were
used for analysis of the chromatin architecture in vivo, and a "19A" derivative of the pUC19‐PHO84
plasmid ("pUC19‐PHO84‐19A") for analysis in vitro. A comparison of the N‐Score profile of the
wildtype promoter with its "10A" and "19A" derivatives predicted that these mutations should
stability, we assembled the pUC19‐PHO84‐19A vector in vitro both with a saturating (histone:DNA
mass ratio of 1:1) and sub‐saturating (0.6:1) amount of histones and analysed the positioning in the
presence and absence of WCE/ATP.
Introduction of the "19A" stretch destabilised the
upstream nucleosome, as the region normally
protected by the upstream nucleosome became
accessible to DNaseI and HhaI for both assembly
degrees (Figure 18A and B). This effect was already apparent in the absence of WCE/ATP indicating
that the "19A" mutation not only destabilised the nucleosome in the presence of WCE but already
sufficiently lowered the intrinsic affinity of this stretch of DNA during salt assembly. The integrity of
the downstream nucleosome was mostly unaffected by the altered sequence within the upstream
nucleosome (Figure 18A and B, compare TaqI values of wt and 19A).
Figure 18. Insertion of polyA stretches into the upstream nucleosome leads to a local decrease in N‐Score .
Top: Schematics of the PHO84 promoter (as in Figure 7). Below: N‐Score profiles [132] for the wildtype promoter and for promoters with 10 or 19 nucleotides mutated (arrows) within the upstream nucleosome to adenine. Dashed circles overlayed onto the N‐Score profiles give the nucleosome positions as in the top panel.
Figure 17 . A polyA stretch destabilizes the upstream nucleosome in vitro.
(A) In vitro reconstitution of nucleosome positioning at the PHO84 promoter after mutation of a 19bp stretch to adenine (Fig. 17). pUC19‐PHO84‐19A plasmids were pre‐
assembled at the indicated histone:DNA mass ratios, incubated with or without WCE and ATP and analyzed by DNaseI indirect endlabelling. Free DNA samples, markers and ramps as in Figure 16. Arrows mark the approximate location of the TaqI and HhaI site. (B) same as (A) except that also pUC19‐PHO84 ("wt") was pre‐assembled and samples were incubated with WCE in the presence ("+ATP") or absence ("‐ATP") of ATP. Samples were analyzed by HhaI (upstream nucleosome) and TaqI (downstream nucleosome) accessibility.
Finally, we assessed the effect of destabilizing the upstream nucleosome on its remodeling
requirements in vivo. Positioning on the pCB84a‐10A and ‐19A shuttle vectors was analysed in a
wildtype and an snf2 strain before and after
induction by phosphate starvation. Based on the in vitro data, the initial concern was that the
upstream nucleosome might already have been
displaced under non‐inducing (+Pi) rendering
subsequent experiments inconclusive. However,
the upstream nucleosome was still mostly present
on both the pCB84a‐10A and ‐19A variant
promoter under repressive conditions as
monitored by protection from DNaseI (Figure
19A: +Pi pattern). Stability of the upstream
nucleosome on the pCB84a‐10A vector was
confirmed by measuring HhaI accessibility. At the
chromosomal locus, the HhaI accessibility was
29% and 14% (Fig. 19B) for the wt and snf2 strain
respectively and for the "10A" variant the
accessibilities were even lower (15% and 9%).
Together with the DNaseI data this showed that
the upstream nucleosome at the “10A” variant
was not already destabilised under non‐inducing
conditions. The HhaI accessibility for "19A"
variant in contrast was elevated (Fig. 19B: 40%
and 48%) in line with the high instability of this
construct seen in vitro. This increased accessibility
of the upstream nucleosome was not seen with
DNaseI, probably due to the low digestion degree
integral to the indirect endlabeling protocol.
Nonetheless, even the elevated HhaI values were
well below the values seen after removal of the
nucleosome under fully inducing conditions
(Fig. 19B: 78% (wt pCB84a‐19A ‐Pi)).
As expected, both the upstream and downstream
nucleosomes at the chromosomal locus were
removed in the wildtype strain upon phosphate
starvation as seen from loss of protection from
DNaseI at both sites, whereas only the
downstream nucleosome was removed in the snf2
strain (Fig. 19A). This differential behaviour was
well reflected by the HhaI accessibilities. While
the HhaI accessibility in the wildtype increased from 29% to 83%, accessibility in the snf2 mutant was
unchanged (Fig. 19B). Stunningly, this dependency on SWI/SNF was strongly reduced for both the
10A and 19A construct. HhaI accessibility in the snf2 mutant increased from 9% to 45% on the
pCB84a‐10A vector upon phosphate removal and from 48% to 73% on the pCB84‐19A vector. This
Figure 19. Lowering the intrinsic stability of
the upstream nucleosome reduces the Snf2‐
dependency for its remodelling.
(A) In vivo DNaseI indirect endlabelling analysis of the PHO84 promoter at its chromosomal locus and on
pCB84a‐10A and pCB84‐19A plasmids in a wildtype
(CY337) or an snf2 strain (CY407). Nuclei were prepared
from cells grown in +Pi (repressing conditions) and ‐Pi
(inducing conditions) medium. Arrows indicate the
approximate position of the HhaI site. Ramps and
markers as in Figure 16. (B) HhaI accessibility of the
strains described in (A). The average and variation of two
biological replicates is shown. Error bars were in some
cases within symbol size.
increased accessibility was also reflected in the DNaseI pattern where protection of the region
normally occupied by the upstream nucleosome was reduced (10A) or lost (19A) (Fig. 19A). In other
words, lowering the intrinsic stability by introduction of a stretch of 10A had no impact on
nucleosome integrity under non‐inducing conditions but partially alleviated the Snf2‐dependency of
the upstream nucleosome. Similarly, the 19A variant displayed a partially impaired upstream
nucleosome under non‐inducing conditions that could be completely remodelled even in the absence
of Snf2. In conclusion, the requirement for SWI/SNF to remove the upstream nucleosome upon
induction was not just correlated but, at least in part, caused by the higher intrinsic stability of this
nucleosome.