INTRODUCTION

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Transcriptional repression of the silent-mating-type loci is fundamental for the haploid yeast life cycle. The a or  mating type is determined by expression of master regulatory genes of the active MAT locus near the centromere of chromosome III. Identical genes present at the HM (haploid mating) loci near the telomeres of the same chromosome, HML carrying  information and HMR bearing a information, are not transcribed, thus preserving the unique mating type. The HM loci serve as donors during the gene interconversion event that allows a homothallic haploid cell to switch mating type, ensuring a diploid population in the wild. In addition to transcriptional repression, the DNA of the silenced HM loci is protected from HO endonuclease, which makes a double-strand break at the MAT HO site to initiate mating-type switching (40, 46, 55, 75).

Silencing of the HM-mating-type loci in Saccharomyces cerevisiae is remarkably similar to long-term, epigenetic inactivation of specific genomic domains in complex eukaryotes. X-chromosome inactivation (33) and gene imprinting (82) in mammalian cells, telomeric silencing (22) in yeast, and position effect variegation (reviewed by Henikoff [28]) in Drosophila melanogaster are examples of such parallel situations. Epigenetic states are thought to be achieved by chromosomal condensation into heterochromatin. The molecular events leading to such position-dependent, gene-independent transcriptional repression of a chromosomal region are not well understood. Silencing mechanisms in yeast are likely to involve a repressive chromatin structure equivalent to that of heterochromatin (87). The genetics of yeast silencing have been intensively studied (for a review, see reference 45), and a large number of cis-DNA elements and trans-acting proteins involved in the establishment and maintenance of repression at the silent-mating-type loci have been identified. Studies of histone mutations and modifications are consistent with the involvement of chromatin organization in gene silencing (34, 35, 50, 58). However, chromatin structure at the silent mating type loci has not been analyzed in detail.

Two cis-acting elements are necessary for repression at the silent-mating-type loci. The E and I silencers, which flank both HMR and HML are essential or important for silencing (1, 7, 47, 68). Each silencer consists of either a Rap1p and/or an Abf1p binding site (9, 71) and a binding site for the origin recognition complex (ORC), termed an autonomously replicating sequence consensus site (ACS) (4). The silencers of both the HMR and HML loci are functionally similar, yet their efficiencies in conferring gene silencing are different (69), and functional cooperativity between two distant silencers can enhance repression (6). Transcriptional repression of the genes located between the E and I silencers is independent of their sequence, chromosomal origin, and orientation. Transplacement of the mating-type genes outside the silent locus causes activation of their transcription, while heterologous RNA polymerase II or III genes inserted into the HM loci become silenced (7, 29, 68).

Among the trans-acting factors, the four Sir (silent information regulator) proteins, initially identified by genetic screens for loss of repression at the HM loci (25, 39, 63, 64), function without directly binding to DNA. Null mutations of sir2, sir3, and sir4 result in complete derepression of the HM loci, whereas only partial derepression was observed in the absence of Sir1p (31, 63). Sir1p binds to the Orc1p subunit of ORC, which binds the ACS of the silencers. A role for Sir1p in the establishment of silencing via binding to ORC was suggested (12, 89). While passage through S phase is required for the establishment of silencing, the role of ORC is independent of replication initiation at the silencers (18). Sir3p and Sir4p have been shown to form homo- and heterodimers in vivo and also to interact with the carboxy-terminal region of Rap1p in vitro (23, 51, 52). ORC and at least one of Rap1p or Abf1p bind to the silencer (9, 15). Subsequently, Sir3p and Sir4p can be tethered to the silencers by virtue of their interactions with the initiation complex and establish a matrix (24) which could support a repressive chromatin structure across the entire locus. H3 and H4 N-terminal tail interactions with Sir3p and Sir4p (26, 35) are consistent with this hypothesis. In addition, the histone H4 amino-terminal regions are indispensable for HM silencing (35, 58, 88). Overexpressed Sir3p has been shown by immunoprecipitation to physically spread in a histone H4-dependent manner as far as silencing extends both at the subtelomeric regions and at the HM loci (27). The extent of silencing can be correlated to the level of overexpression of Sir3p. Although Sir4p and Rap1p are required for that effect at telomeres (62, 78), their relative contributions to the postulated repressive structure at HML or whether they spread in association with Sir3p remains unclear.

Mutations of N-terminal tails of histone H3 alone have little effect on repression at the HM loci but appear to increase the severity of other mutations that affect silencing. These amino-terminal regions of the core histones are known to be sites for posttranslational modification by histone acetyltransferases, and nucleosomes of silent regions are hypoacetylated, similar to the histones in inactive chromatin from metazoan organisms (11, 91). SIR2, a protein involved in HM silencing, promotes deacetylation of histones, an activity which is characteristic of repressed chromatin (8). Other genes, such as NAT1, ARD1, SAS2, and SAS3, with predicted protein products bearing similarities to acetyltransferases also contribute to HM silencing (54, 60, 94). NAT1 and ARD1 are N-terminal acetylases with a different function from histone acetyltransferases. Their role in silencing is likely to be indirect; nearly 20% of yeast proteins have altered isoelectric points in a nat1 background, suggesting that many diverse effects could arise from mutations in these genes.

Derepression of the silent mating type loci leads to transcription of the a1 and a2 or 1 and 2 genes from HMRa or HML, respectively, as well as cleavage by HO endonuclease at its site located near the 3' end of the a1 and 1 coding sequences. Taken together, these studies suggest a correlation between a specific chromatin configuration and this transcriptional repression at HML and HMRa. The only previous study to address this suggestion at HML showed that the HO endonuclease recognition site of the HML locus was more accessible to DNase I cleavage in sir mutants than in wild type, as might have been expected from its differential accessibility to the HO endonuclease in the two genetic backgrounds (55). To directly examine the role of chromatin structure in silencing, we have performed a high-resolution analysis of the chromatin organization of ~4 kb of yeast chromosome III, which includes the silenced HML region. In addition, we compare this structure with that of the active MAT locus. Finally, we focus on modifications of chromatin structure at HML consequent to various null mutants of the SIR protein genes and an H4 amino-terminal-region deletion.

	MATERIALS AND METHODS

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S. cerevisiae strains, generously provided by J. E. Haber, were all derivatives of DBY745 (S288C): tNR (HML mat::LEU2 hmr::ADE1 lys5 leu2 ura3 trp1) ("HML only"); JKM115 (hml::ADE1 MAT hmr::ADE1 lys5 leu2 ura3 trp1) ("MAT only"); K30 (ho MAT leu2 trp1 his4 ura3); and 23-2 (ho mat 200 bp [a-like] leu2 trp1 his4 ura3). The mat deletion is between his mutations x109 x52 and was originally constructed by K. Tatchell (86) and by S. Y. Roth: PKY913 (MAT hhf1::HIS3 hhf2::LEU2, pUK613[hhf2 del(4-23)], lys2 leu2 ura3 trp1 ade2 arg4 his3 thr4 tyr) (35). The strains constructed during this study were YKW01 (tNR sir1::URA3), YKW03 (tNR sir3::URA3), and YKW04 (tNR sir4::URA3). They were propagated in yeast extract-peptone-dextrose (YEPD) complete medium. Mating ability was tested by mating tester strains, MATa or MAT ura2, with the strain to be assessed on YEPD plates for 16 h, and subsequently selecting for growth on minimal medium (SD) plates.

The SIR1, SIR3, and SIR4 genes of the HML-only strain (tNR) were replaced by standard methods (65) with the URA3 gene by transformation with the linearized disruption plasmids D1528 (77), pSR-sir4 (61), and pCTC73 (12), which were generously provided by D. Shore and S. Reimer. Transformation was assessed by uracil prototrophy. sir3 and sir4 mutants were screened for their ability to mate with a cells. The wild-type tNR cells mate with  cells. sir1 mutants were screened for their ability to mate with both cell types. The disruption of SIR1, SIR3, and SIR4 was verified by PCR analysis.

Yeast were grown in YEPD at 30°C to mid-log phase (optical density at 600 nm of ~1). Nuclei were isolated and then digested with micrococcal nuclease or DNase I (Worthington); DNA was purified as described previously (67, 85) with modifications as detailed by Weiss and Simpson (93). Protein-free DNA controls were obtained by either digesting purified, previously undigested DNA with a 50-fold-lower concentration of enzyme or by digesting a PCR product. Portions (4.4 kb) of the sequences including HML were amplified with oligonucleotides p108 and q152 (see below) as primers. PCR product (~100 ng) was digested with 1.0 U of MNase or 0.05 U of DNase I per ml at 37°C for 3 min in the presence of 36 µg of carrier DNA (calf thymus). After ethanol precipitation, DNA was resuspended in 50 µl of 0.1× TE buffer.

MNase and DNase I cleavage sites were located by primer extension assay with Taq polymerase as described previously (70) with minor modifications (93). Oligonucleotides used as primers included (coordinates are base pair positions in the published sequence of S. cerevisiae chromosome III (56): p108, 10830-10855; p111, 11107-11134; p129, 12874-12895; p134, 13362-13386; p136, 13654-13673; p140, 13984-14013; q152, 15213-15187; q140, 14043-14017; q134, 13386-13362; q123, 12306-12283; q120, 12030-12008; q113, 11388-11367; and q1999, 199946-199916.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Unique organized chromatin domain at HML. A high-resolution map of the chromatin structure of an ~4-kb domain spanning HML was established by using primer extension analysis of micrococcal nuclease digests of isolated nuclei. Nuclease cutting patterns of the silent HML locus were compared to the pattern of identical sequences of the transcribed MAT locus in regions where they overlap as well as to nuclease digests of protein-free DNA. Nucleosome positions are inferred from areas of nuclease protection extending about 150 bp which are flanked by nuclease-sensitive cleavage sites. Due to the sequence identity of portions of the three mating-type loci, strains with deletions of MAT and HMR (i.e., the "HML only" strain) or HML and HMR (i.e., the "MAT only" strain) were used to create unique primer extension sites at HML and MAT, respectively. The inferred chromatin structure of HML in a wild-type background is summarized in Fig. 1.

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the chromatin map of the entire HML locus. Map units correspond to base-pair positions of the published sequence of chromosome III (56). White boxes labeled E and I identify the silencer sequences; boxes labeled W, X, Y, Z1, and Z2 identify the mating-type-locus regions. Black arrowheads identify sites that are hypersensitive to micrococcal nuclease; tick marks correspond to regions generally sensitive to nuclease cleavage and detailed in other figures. The black rectangles indicate the Rap1p binding sites. Dark-shaded ellipses indicate precisely positioned nucleosomes. Light-gray ellipses indicate more loosely positioned nucleosomes, and dashed ellipses indicate less-defined chromatin structure of the W region. The 1 and 2 coding regions are identified by arrows.

View larger version (66K): [in this window] [in a new window]   FIG. 2.   HML chromatin near the I silencer. The chromatin structure of the Crick strand was mapped by primer extension analysis of micrococcal nuclease cleavage sites with primer p140. Wild-type (WT) and sir1 and sir3 mutant cells are as indicated. Extensions of undigested (0) and micrococcal-nuclease-digested chromatin are also presented. The D columns indicate protein-free DNA digests as a control for micrococcal-nuclease sequence specificity. The C column shows a dideoxycytosine-terminated sequencing reaction. Coordinates are positions in the published sequence of S. cerevisiae chromosome III. The silencer is represented by a shaded rectangle. Ellipses correspond to inferred positions of nucleosomes.

View larger version (58K): [in this window] [in a new window]   FIG. 3.   HML chromatin of the region between Z1 and the I silencer. The chromatin structure of the Crick strand was mapped by primer extension analysis of micrococcal nuclease cleavage sites with primer p136. Wild-type (WT) and sir1 and sir3 mutant cells are as indicated. Symbols are as detailed in the legend to Fig. 2. Rectangular boxes indicate the locations of mating-type-locus regions. The 3' end of the 1 gene is indicated by an arrow.

View larger version (89K): [in this window] [in a new window]   FIG. 4.   HML, wild type, sir3, and sir4, and MAT chromatin near the HO site and Y-Z1 border. The chromatin structure of the Crick strand was mapped by primer extension analysis of micrococcal nuclease cleavage sites with primer p134. Wild-type (WT) MAT and wild-type and sir3 and sir4 mutant HML cells are as indicated. Symbols are as detailed in the legend to Fig. 2. The coding region of the 1 gene is indicated by an arrow.

View larger version (69K): [in this window] [in a new window]   FIG. 5.   Chromatin structure of the 1 and 2 promoter region in HML and MAT. The chromatin structure was mapped by primer extension analysis of micrococcal nuclease cleavage sites with primer p129 (Crick strand) (A) and q134 (Watson strand) (B). Wild-type MAT and HML cells are as indicated. Symbols are as detailed in the legend to Fig. 2. The M column shows X174/HinfI-digested DNA fragments for size indication. Sequence numbers shown correspond to those of HML and differ by 185,899 from the corresponding nucleotide in MAT. Shaded rectangles locate sequences necessary for transcription initiation; black rectangles indicate TATA elements, and the white box shows the shared UAS. Tick marks indicate points of transcription initiation of the 1 and 2 genes, and arrows show their coding sequences (CDS).

View larger version (91K): [in this window] [in a new window]   FIG. 6.   Impact of the sir mutations on the chromatin structure of the 1 and 2 promoter region. The chromatin structure of the Crick strand was mapped by primer extension analysis of micrococcal nuclease cleavage sites with primer p129. Wild-type (WT) and sir3, sir4, and histone H4 N-terminal deletion (H4) mutant HML cells are as indicated. Symbols are as detailed in the legend to Fig. 5.

View larger version (49K): [in this window] [in a new window]   FIG. 7.   Chromatin structure of the W region in HML and MAT. The chromatin structure of the Watson strand was mapped by primer extension analysis of micrococcal nuclease cleavage sites with primer q123. HML and MAT are as indicated. Symbols are as detailed in the legend to Fig. 2. The dark box identifies the TATAA box, and the arrow shows the beginning of the BUD5 coding sequence. Sequence numbers shown correspond to those of HML, differ by 185,899 from the corresponding nucleotide in MAT, and are derived from the size of the X174/HinfI-digested DNA fragments.

Impact of sir mutations on HML chromatin organization. SIR1, SIR3, and SIR4 have been shown to be required for the maintenance of the repressed state of HML (45). Null mutants of these genes were created by replacing the promoter and part of the coding region with the URA3 gene (65). The HML-only strain used mates such as an a cell (79). In contrast, sir3 or sir4 mutants mate as  cells due to lack of silencing of HML, resulting in transcription of the 1 and 2 genes. The sir1 mutants mate with both a and  cells because the derepression of HML is only partial and the resulting mating phenotype is mixed. The Sir proteins are required for establishing and maintaining a repressive chromatin structure at HML (24, 55). Once the regions of HML where a particular chromatin organization characterizes the silent state of the locus were identified, the impact of the sir mutations on those structures was evaluated. Micrococcal nuclease cleavage sites of sir mutant HML were compared to the wild-type HML and MAT, as well as to HML in an H4 amino-terminal region (amino acids 4 to 23) deletion (H4) mutant. In the H4 mutant, HML is totally derepressed (26).

View larger version (85K): [in this window] [in a new window]   FIG. 8.   Chromatin structure near the E silencer. Chromatin structure was mapped by primer extension analysis of micrococcal nuclease cleavage sites with primer q120 (A) for the Watson strand between the W region and the silencer in the HML wild type (WT) and sir mutants and with p111 (B) for the Crick strand of the E silencer and adjacent region inside the HML in wild type and sir mutants. Wild-type MAT and wild-type and sir1, sir3, and sir4 mutant HML cells are as indicated. Symbols are as detailed in the legend to Fig. 2. Column G shows a dideoxyguanosine-terminated sequencing reaction. Column M shows X174/HinfI-digested DNA fragments for size indication. Sequence numbers shown correspond to those of HML and differ by 185,899 from the corresponding nucleotide in MAT. Shaded boxes identify the Rap1p binding site and the ACS of the E silencer in panel A. MAT sequences are different from HML outside the W region (downstream [*] in panel B).

View larger version (63K): [in this window] [in a new window]   FIG. 9.   Chromatin structure near the E silencer outside HML in wild type and the sir1 mutant. The chromatin structure of the Watson strand was mapped by primer extension analysis of micrococcal nuclease cleavage sites with primer q113. Wild-type (WT) and sir1 mutant HML are as indicated. Symbols are as detailed in the legend to Fig. 2. Column DI shows extensions of DNase I-digested chromatin. Shaded boxes identify the Rap1p binding site and the ACS of the E silencer.

Inactivation of transcription at MAT is not sufficient to establish the HML specific nucleosomal organization. We questioned whether the disorganized chromatin structure observed for most of the active MAT was the consequence of active nucleosome disruption caused by transcription. A strain constructed by combining two isolated XhoI linker mutations at MAT abolishing 1 and 2 transcription (86), thus creating an a-like strain, was used to compare the chromatin of the Z1-Z2-1 region of HML with the active and transcription-blocked MAT. HML- and MAT-specific primers lying immediately outside the Z2 region were used. Positioned nucleosomes L5 to L8 are clearly seen in the Z1 and Z2 region and extending into the 1 coding region, blocking the HO endonuclease site, at HML in the wild-type strains (Fig. 10). These nucleosomes are disrupted at the active MAT, with extensive nuclease cutting across the mapped region. At the mutated, nontranscribed MAT locus, identical disruption of the nucleosomes is observed (Fig. 10). The highly organized chromatin structure of the HML locus is not present at the MAT locus irrespective of whether it is being actively transcribed or not. Particular features of the silent locus are necessary for establishing nucleosome positioning in the distinctive, silenced chromatin structure.

View larger version (90K): [in this window] [in a new window]   FIG. 10.   Chromatin structure of the Z2-Z1-1 region in a nontranscribed MAT compared to HML and MAT. The chromatin structure of the Watson strand was mapped by primer extension analysis of micrococcal nuclease cleavage sites with primers q140 for HML and q1999 for MAT. Wild-type HML and MAT are as indicated. MATpro designates the strain that has a 200-bp deletion of the promoter sequences of the 1 and 2 genes at MAT. The location of the deletion is indicated by a bracket (pro). Symbols are as defined in the legend to Fig. 2. Sequence numbers shown correspond to that of HML and differ by 185,899 from the corresponding nucleotide at MAT.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

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 (19), PHO5 (83), and ADH2 (92), 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 Mat2p-Mcm1p binding site and the 3' end of the transcription unit for the a-specific genes (59, 90) 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, 88) 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 1 and 2 genes (Fig. 1).

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, 9, 30). 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 Mat2p 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, 17, 36, 41, 76). 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, 78). It has been shown that placing either HML or HML-E and/or HML-I near heterologous genes on chromosome III or on a plasmid alters the level of silencing (3, 49, 69) 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, 15, 16, 71); 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 Mat2p-mediated chromatin assembly suggested above, the precise architecture around promoter elements differs strikingly for the two situations. At a-cell-specific promoters for STE6 and BAR1, a positioned nucleosome places the TATA box near the pseudodyad of the nucleosome core (59, 66, 90); inaccessibility of this critical element to the transcription machinery has been proposed as one mechanism that could lead to repression (70, 73, 74). Surprisingly, at HML, much of the 200-bp intergenic region between the divergently transcribed 1 and 2 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. Hence, transcriptional repression at HML 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, 84), and sequences that would be involved in such interactions are sequestered in the positioned nucleosomes. At MAT, 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 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, 38), 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, 53), 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. 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, 57). 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 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 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 HML 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.