The results reported in this paper shed more light onto the mechanism by which the high efficiency of transcription of yeast rp-genes is attained. Using a fusion between the promoter of the RPS28A gene and the GUS reporter gene we have performed a mutational analysis that establishes a correlation between the transcriptional activation by the common elements within the rp-gene promoter and the effect of these elements on the local chromatin structure.
The data presented in Figure demonstrate that the Abf1p binding site and the T-rich element of the RPS28A
promoter are equally important for full transcriptional activity, confirming earlier conclusions (3
). However, substitution of the individual segments of the bipartite T-rich element causes a much smaller reduction in transcriptional activity, showing that even a short T-stretch still has a substantial stimulatory capacity. The extent of the stimulatory activity shows an approximate correlation with the size of the segment; in mutant ITS1, in which the remaining portion of the T-stretch is 9 residues long, transcriptional activity is ~65% of the wild-type level versus 55% in mutant ITS3 having a six residue long T-stretch. Changing the bipartite T-rich element into a 21 residue long homopolymeric T sequence (mutant ITS2), however, did not significantly increase transcription over the wild-type level. This contrasts with the results of Iyer and Struhl (17
), who did observe an increase in transcriptional activity with increasing size of the homopolymeric T-stretch. Thus, the bipartite structure of the T-rich element in the RPS28A
promoter might have functional significance.
Two observations prompted us to analyze the effect of the various mutations in the RPS28A
promoter elements on the local chromatin structure. First, we found that deletion of the DAT1
gene, encoding the only protein so far known to influence transcription by binding specifically to T-rich elements (17
), had very little effect on RPS28A
promoter-driven transcription of the GUS
reporter (Fig. ). Secondly, as shown in Figure , replacement of the two (dA:dT) stretches of the T-rich element, which are thought to form a rigid DNA structure (27
), by similarly rigid stretches of dC:dG (mutant ITc
) suggests the requirement for structural rigidity of the DNA downstream of Abf1p in order to activate transcription. These two observations make it unlikely that the T-rich element functions as a recognition site for specific transcriptional activator proteins.
The chromatin structure of the wild-type promoter shows a well-defined organization consisting of a protected region, delineated by two hypersensitive sites, that spans the Abf1p binding site. Several lines of evidence indicate that this protected region of 130 bp does not correspond to a positioned nucleosome: (i) the relatively small size of the region; (ii) the finding that the protected region in DNaseI digests is much smaller, ~90 bp (Fig. ); and (iii) the 130 bp protected region present in the wild-type promoter (ERS3) has decreased to a region of ~85 bp when the T-rich element is mutated (Figs and ). Hence, the 130 bp protected region should be considered as the result of two independent protections on either side of the Abf1p binding site. Analysis of the nucleosome organization of the IRS* and ERS* mutants, carrying a non-functional Abf1p binding site, revealed the complete loss of this highly ordered chromatin structure (Fig. ). Thus, we conclude that after binding to its recognition site in the RPS28A promoter, Abf1p acts as a strong organizer of chromatin structure both upstream and downstream of the binding site.
Whereas the organization of nucleosomes into an ordered array is due to the binding of Abf1p, the creation of the nucleosome-free region adjacent to the Abf1p binding site depends upon the presence of the T-rich element. In the ETS6 mutant, which lacks a functional T-rich element (Fig. ), the positioning of the nucleosomal array upstream of the Abf1p binding site, extending into the GLN4 gene, remains the same as in the wild-type. Downstream of the Abf1p site, however, the array of positioned nucleosomes has shifted towards the Abf1p binding site by ~60 bp, thus completely obliterating the nucleosome-free region present in the wild-type RPS28A promoter. Apparently, Abf1p is not able to create a nucleosome-free region but merely acts as a nucleosomal boundary element.
High-resolution mapping (Fig. ) shows that the nucleosome-free region extends 35–55 bp beyond the T-rich element. Since sequences from 18 bp downstream of the T-rich element are A-rich, having one A4
and two A3
stretches, the observed shift of the nucleosome beyond this region may be attributed to nucleosome exclusion properties of both the T-rich element and the A-rich region. Such nucleosome exclusion properties are known for stretches of As and Ts (see Introduction). However, the strong nucleosomal protection of both the core promoter and the A-rich region upon replacement of the T-rich element (Figs –), suggests that the nucleosome exclusion properties of the A-rich region are very minor indeed. An alternative explanation for the extended nucleosome-free region at the RPS28A
promoter may be that yet unidentified proteins bind downstream of the T-rich element and form a physical boundary for nucleosomes. Previous in vitro
DNase footprinting (28
) and methylation interference experiments (T.M.Doorenbosch, unpublished data) carried out in our laboratory, indicate the existence of a protein(s), called GDUF, that binds to the region of the RPS28A
promoter downstream of the T-rich element between positions –108 and –87. Since the latter position is very close to the upstream boundary of the nucleosomal array at position –83, this factor(s) may form a physical boundary for nucleosomes in vivo
. So far, however, both the identity of these proteins and their possible role in transcriptional activation remains unclear. Nevertheless, the observation that the nucleosome-free region is completely lost when the T-rich element is mutated consequently suggests that the T-rich element enables binding of these proteins to downstream sequences. This strongly correlates with the observations that homopolymeric (dA:dT) elements increase the accessibility of neighbouring binding sites in vivo
). The idea that the T-rich element functions through a similar mechanism to these homopolymeric (dA:dT) elements is further supported by our finding that structurally rigid poly(dC:dG) can functionally replace the poly(dA:dT) sequences of the T-rich element (ITc
in Fig. ), suggesting that the T-rich element also functions by virtue of its intrinsic DNA structure and in this manner increases the accessibility of adjacent sequences to additional proteins.
Even though a T-stretch encompassing as few as six residues can still act in synergism with the Abf1p site to cause substantial transcriptional activation, the fully active T-rich element consists of two stretches of thymidines separated by an A-rich region (Fig. ). This type of alternating sequence is expected to cause bending of the DNA (29
), rather than form the ‘rod-like’ structure characteristic of homopolymeric sequences. Gel electrophoretic analysis of DNA fragments containing the various wild-type and mutant promoter sequences depicted in Figure have indeed revealed differences in mobility that suggest differences in intrinsic DNA bending (data not shown). Abf1p binding, however, also causes the DNA to bend (31
). Altering the phasing between these adjacently positioned DNA-bending elements by insertion of 5 or 10 bp between Abf1p binding site and T-rich element did not significantly affect transcription, neither did insertion of 5 or 10 bp downstream of these elements (data not shown). Apparently, the respective bends in the DNA are not interdependent and are not likely to be involved in, for instance, DNA looping. Alternatively, since any anomalous intrinsic DNA structure may affect the curved path of the DNA around a nucleosome, both the intrinsic DNA-bending properties and the structural rigidity of the thymidine stretches of the T-rich element may contribute in locally affecting DNA–nucleosome interactions, similar to the proposed nucleosome destabilizing action of structurally rigid sequences (17
In conclusion, the data presented in this paper demonstrate that the Abf1p binding site and the T-rich element each play a distinct role in organizing the chromatin structure of an rp-gene promoter and the adjacent coding region. We favour a model in which Abf1p is able to organize chromatin by forming a physical boundary for nucleosomes and the T-rich element subsequently enables binding of other proteins to downstream sequences, creating a nucleosome-free region of ~65 bp downstream of the Abf1p binding site.