A central role for chromatin in the repression of genes in S. cerevisiae
has been postulated for a number of loci. In contrast to genes where local, promoter-specific, chromatin structures have been observed, such as genes SUC2
), and ADH2
), larger domains of organized chromatin have been found at subtelomeric regions (44
), at the recombination enhancer (93
), and for a
-cell-specific genes (74
). Where examined in detail, these domains have consisted of continuous arrays of precisely positioned nucleosomes, delimited by the Matα2p-Mcm1p binding site and the 3′ end of the transcription unit for the a
-specific genes (59
) or by two transcribed gene promoters flanking the recombination enhancer (93
). Based on currently available evidence, particularly the results of histone H4 amino-terminal tail mutations (35
) and interactions of proteins known to be necessary for HM silencing with histones (26
), the ~3-kb silent-mating-type loci also represent regions of transcriptional repression where chromatin structure is important for regulation. In striking contrast to the continuous chromatin organization of other domains, chromatin at HML
is discontinuous. While arrays of nucleosomes abut the E and I silencers, the arrays are punctuated by a 120-bp nucleosome-free region that encompasses the promoter of the divergent α1 and α2 genes (Fig. ).
Adjacent to the silencers and flanking the promoter region, precisely positioned nucleosomes are located at HML
. Each of these regions contains a binding site for Rap1p, the E and I silencers also have an ACS binding site for the ORC complex, and the I silencer contains an Abf1p binding site (6
). Several of these proteins interact with proteins of the Sir group, and Sir3p and Sir4p interact with the amino-terminal regions of histones H3 and H4. The proposal has been made that Rap1p and/or the ORC complex bind to specific DNA sequences, recruit the Sir group, and then organize chromatin structure by interactions with histones. This scenario bears striking similarities to repression of a
-cell-specific genes, where Matα2p and Mcm1p bind to specific DNA sequences, recruit the Ssn6p-Tup1p complex (which interacts with the amino-terminal regions of H3 and H4), and presumably organize chromatin structure (14
). Defining the similarities between these two systems that both appear to produce organized chromatin should advance our understanding of how repressive nucleoprotein structures are established in eukaryotic cells.
In agreement with a current model for silencing in which one or more Sir proteins physically spread from the silencer over the silenced locus (26
), the chromatin between HML-I and the promoter is disrupted in sir
mutants. In contrast, the chromatin organization in the region near HML-E is not altered by any sir
mutation. Organized chromatin near E does not depend on the presence of any individual Sir protein, and its establishment is not only nucleated towards the repressed locus, since positioned nucleosomes can be found flanking E on both sides. It seems likely that some of these features may arise from the proximity of E to the silenced telomere that is separated from HML
by only 10 kb of untranscribed DNA (21
). In the absence of transcribed genes, organized chromatin could be propagated from the telomere of chromosome III, where it is established in a Sir-dependent manner, to the vicinity of HML
. This nucleosomal organization is likely to be independent of Sir proteins, since Sir3 was shown to only spread about 3 kb on a different telomere (62
). It has been shown that placing either HML
α or HML-E and/or HML-I near heterologous genes on chromosome III or on a plasmid alters the level of silencing (3
) and that silencing is generally greater in the proximity of silenced regions such as telomeres. The proximity of a transcriptionally active chromosomal region to HML-I could increase the severity of single sir
mutations, reflecting a context-dependent Sir protein role in the maintenance of highly organized chromatin.
In summary, in their native context, HML-E and HML-I seem functionally different, despite being equally competent at maintaining repression individually (47
). HML-I has binding sites for both Abf1 and Rap1, while HML-E only has a Rap1 site. Abf1 and Rap1 can act as transcriptional activators when present at a promoter site (9
); possibly the Sir proteins prevent their activating function when recruited to the silencer. Destabilization of the silencing complex at a silencer due to the absence of one of the Sir proteins may consequently be more severe if two activators rather than a single one are present. While comparison of E and I at HML
suggests differences in the role of Sir proteins in the establishment of organized chromatin, more in-depth indications of functional differences among the silencers should result from an ongoing characterization of chromatin near the HMR-E element that can silence this locus independently (59a
In contrast to the parallel pathways for HML
- and Matα2p-mediated chromatin assembly suggested above, the precise architecture around promoter elements differs strikingly for the two situations. At a
-cell-specific promoters for STE6
, a positioned nucleosome places the TATA box near the pseudodyad of the nucleosome core (59
); inaccessibility of this critical element to the transcription machinery has been proposed as one mechanism that could lead to repression (70
). Surprisingly, at HML
α, much of the 200-bp intergenic region between the divergently transcribed α1 and α2 genes, including the single shared UAS, is highly accessible to micrococcal nuclease digestion. No repressor binding site (other than that for Rap1p, which also serves as an activator) has been identified in the intergenic region, and both activators and the transcriptional machinery are readily available to transcribe both genes from an identical promoter at MAT
α. Hence, transcriptional repression at HML
α seems likely to be regulated structurally.
Several possibilities arise for such structural regulation. First, the transcription initiation sites for both genes are located in positioned nucleosomes. Although the TATA boxes are not blocked by histone-DNA interactions, assembly of the basal transcription machinery requires significantly greater lengths of DNA than that contacted directly by the TBP (72
), and sequences that would be involved in such interactions are sequestered in the positioned nucleosomes. At MAT
α, the entire region between the two TATA boxes is relatively protected, but the transcription initiation sites are susceptible to micrococcal nuclease cleavage, possibly reflecting TBP and associated factor binding and formation of the transcription initiation complex.
Second, the geometry of chromatin at and around the intergenic region at HML
α could preclude formation of the transcription initiation complex. The two TATA sites are separated by 105 bp, exactly 10 helical turns of DNA in solution. Since TBP creates an ~80° bend when it binds to DNA and an 18-Å lateral displacement between upstream and downstream DNA when it binds to the TATA box (37
), the two nucleosomes which flank the intergenic region have the potential to be involved in a steric clash if TBP is bound to both TATA boxes. Rap1p binding to DNA also bends DNA by more than 50° (20
), so it is likely to affect this possible interaction. If Rap1p serves to anchor chromatin to a karyoskeletal element, the system becomes too complex to make mechanistic predictions based on known structures of proteins and the DNA involved.
Third, a higher-order structure which precludes transcription could be formed by the chromatin at HML
α. Looping of DNA from the HM
loci has been shown to occur readily in vitro; loops between E and I silencers and between the silencers and the promoter region were observed and were shown to require Rap1p (30
). Rap1 was initially isolated from a karyoskeletal fraction, and HML-E and HML-I were found to be associated with a “nuclear scaffold” fraction (30
). While probably reflecting telomere location and therefore only indirectly the location of the nearby HM
loci, immunofluorescence studies show colocalization of Rap1p, Sir3p, and Sir4p with telomeric DNA in discrete foci around the nuclear periphery (13
). Proximity of silenced loci to telomeres has been shown to be necessary for effective silencing (49
). A recent study with topological measurements on circles containing all or parts of HML
α excised in vivo (5
) showed a linking-number difference of ~2 between samples from a wild type versus a sir3
background; the wild type had two more negative supercoils than did the mutant. While a number of reasons could lead to the linking-number deficit in the mutant strain, loss of a double loop of DNA, looped from E to UAS and from UAS to I, in the mutant strains is certainly consistent with this experimental result. Targeting of a LexA-Sir4p chimera to a plasmid by inclusion of LexA binding sequences led to partitioning of the plasmid on cell division, suggesting interaction of the plasmid-bound protein with a nuclear element that partitions equally between mother and daughter cells (2
). Interestingly, partitioning was dependent on Rap1p, suggesting that this protein might form the anchor on the nuclear skeletal element which held the Sir4p-bound plasmid. One can envision Rap1p anchoring HML
α to a karyoskeletal element at three sites, interacting with a Sir protein complex that somehow organizes chromatin and thereby creating a substrate refractory to transcription initiation as well as sequestering the locus to a potentially repressive nuclear location. Differences in effects on chromatin structure along the length of the locus of the sir
mutations, greatest at I and at the promoter and less near E, suggest that the structure is not homogeneous from end to end.
While the chromatin organization of HMLα seems intimately connected with transcriptional silencing, the locus is fully capable of participation in recombination. This is also true of loci involved in mammalian immunoglobulin gene recombination. Resolving the apparent paradox of transcriptional silencing coexisting with recombinational competence provides a healthy experimental challenge.