Long Range Communication Between The Silencers Of Hmr

MOLECULAR AND CELLULAR BIOLOGY, Mar. 2008, p. 1924–1935
Vol. 28, No. 6
0270-7306/08/$08.00 0
doi:10.1128/MCB.01647-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Long-Range Communication between the Silencers of HMR
Lourdes Valenzuela,1 Namrita Dhillon,1 Rudra N. Dubey,2
Marc R. Gartenberg,2 and Rohinton T. Kamakaka1*
Department of Pharmacology, 683 Hoes Lane, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854,2 and
Department of MCD Biology, Sinsheimer Labs, 1156 High Street, University of California, Santa Cruz, California 950641
Received 5 September 2007/Returned for modiﬁcation 2 October 2007/Accepted 29 December 2007
Gene regulation involves long-range communication between silencers, enhancers, and promoters. In Sac-
charomyces cerevisiae, silencers ﬂank transcriptionally repressed genes to mediate regional silencing. Silencers
recruit the Sir proteins, which then spread along chromatin to encompass the entire silenced domain. In this
report we have employed a boundary trap assay, an enhancer activity assay, chromatin immunoprecipitations,
and chromosome conformation capture analyses to demonstrate that the two HMR silencer elements are in
close proximity and functionally communicate with one another in vivo. We further show that silencing is
necessary for these long-range interactions, and we present models for Sir-mediated silencing based upon these
results.
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Gene activation and gene repression are central to the
Similarly, in yeast the promoters and terminators of genes are
proper development and differentiation of organisms. DNA
in close proximity to one another (3, 48) and tethered to the
elements such as promoters, enhancers, and silencers play a
nuclear pore (10, 52). The consequence of this spatial organi-
central role in eukaryotic gene regulation. These elements are
zation is that the DNA between these regulatory elements is
separated from each other by several kilobase pairs of DNA
looped out. It is thought that the formation of these nuclear
but are able to communicate with one another to regulate the
substructures aids in transcription activation.
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activation or repression of genes. The exact mechanism by
Silencers are negative regulatory elements composed of
which distally located elements communicate with one another
binding sites for various factors that act collectively in the
is not clear and is one of the key questions in gene regulation.
establishment and stable inheritance of a repressed state. Like
Long-range communication between distantly located ele-
enhancers, silencers repress promoters in a distance- and ori-
by on March 21, 2010
ments in chromosomes is thought to occur by one of two
entation-independent manner (35). Silencers ﬂank the silenced
principal mechanisms (8). One class of models postulate that a
HML and HMR mating-type loci in yeast, while at telomeres
signal emanating from a distal regulatory element spreads
the terminal repeated TG1-3 sequences serve as silencers.
along the DNA ﬁber until it encounters a proximal regulatory
These silencers recruit the Sir proteins, Sir2p, Sir3p, and Sir4p,
element. A second class of models postulate that distal and
which then spread across several kilobase pairs of DNA via
proximal regulatory elements interact with one another di-
interactions with histones. Thus, our current understanding of
rectly, with the intervening DNA forming a loop. Both mech-
Sir-mediated repression is that it is an example of long-range
anisms must function within the context of the global chromo-
effects mediated via transmission along the DNA ﬁber rather
some structure, which appears to be composed of large
than direct long-range interactions between the silencers (60).
chromosome loops that attach to a proteinaceous superstruc-
DNA elements that restrict the action of long-distance reg-
ture (11). The nucleus appears to be divided further into dis-
ulatory elements, such as silencers and enhancers, are generi-
tinct chromatin compartments, with heterochromatic domains
cally called insulators. Insulators located between an enhancer
being present in regions near the nuclear periphery while eu-
and a promoter (called enhancer blockers) disrupt enhancer-
chromatic domains are found mainly in the interior of the
promoter communication and prevent the enhancer from ac-
nucleus, although a signiﬁcant portion of euchromatin is lo-
tivating that promoter, while insulators located between a si-
cated near nuclear pores.
lencer and a promoter (called barriers) block the silencer from
It has been suggested that the functionally and structurally
repressing the promoter. Numerous models have been pro-
deﬁned chromatin domains may be coincident (36). Enhancers
posed to explain how insulators function to block long-range
and locus control regions (LCRs) are long-range regulatory
communication. Some models postulate that insulators act as
elements that activate promoters in a distance- and orienta-
decoys, forming nonproductive interactions with distal regula-
tion-independent manner, and recent studies indicate that en-
tory complexes, or sequester these complexes in speciﬁc re-
hancers and LCRs often cluster together in three-dimensional
gions of the nucleus, while other models suggest that insulators
space to form an “active chromatin hub” (23, 49, 58, 59).
function locally by disrupting the propagation of a speciﬁc
chromatin domain (60).
In this paper we present evidence demonstrating that while
* Corresponding author. Mailing address: Department of MCD Bi-
silencers function via recruitment and transmission of Sir pro-
ology, Sinsheimer Labs, 1156 High Street, University of California,
teins along the DNA, they also directly communicate with each
Santa Cruz, California 95064. Phone: (831) 459-3391. Fax: (831) 459-
other. Functional analyses of silencer-mediated repression sug-
3139. E-mail: rohinton@biology.ucsc.edu.
Published ahead of print on 14 January 2008.
gest that silencer elements communicate with one another in
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LONG-RANGE COMMUNICATION BETWEEN HMR SILENCERS
1925
mediating repression in the nucleus. Our studies also show that
Plasmids. Plasmids RO590 and RO635 contained the full-length SAS2 or
DNA fragments containing silencers (separated by several ki-
NUP2 coding regions fused in frame to the Gal4 DNA binding domain (GBD),
with transcription driven by the ADH1 promoter in the pGBK-RC-TRP1 base
lobase pairs of DNA) are in close spatial proximity in the
plasmid (pGBD) (47).
nucleus and likely form chromatin loops. Interestingly, this
Serial dilutions. Yeast cells were grown overnight at 30°C in 5 ml YPAD or
long-range communication was lost in mutants of the Sir pro-
Hartwell’s complete (HC)) medium without tryptophan to allow maintenance of
teins. Our results suggest that silenced domains are formed by
the plasmids. Cells were diluted to an A
of 1.0 unit/ml in HC-trp medium and
600
the spreading of repressor proteins from silencers that interact
serially diluted 5- or 10-fold. Using a cell spotter, approximately 3
l of each
serial dilution was placed onto properly supplemented HC plates to assay for
with one another enabling, compaction of the chromatin ﬁber
ADE2 and URA3 expression, or onto properly supplemented YMD plates pre-
and stable repression. These results are similar to the long-
viously spread with 1.0 A
unit of mating lawn (strain JRY19a) diluted in 300
600
range interactions between LCRs and promoters and suggest
l of YPD, for the mating assays. For the mating assays, selection for plasmids
conservation in the mechanism by which genes are activated
was maintained. The plates were incubated at 30°C and photographed. Cells
and repressed.
grown in limiting amounts of adenine were kept at 4°C for an additional 2 days
for development of the color prior to photography.
ChIP. Quantitative chromatin immunoprecipitation (ChIP) analysis was per-
formed as previously described (46), with minor modiﬁcations. The program for
MATERIALS AND METHODS
the PCR was as follows: 95°C for 3 min (1 cycle), and 95°C for 1 s, 52°C for 30 s,
and 72°C for 1 min (45 cycles). The fold enrichment was calculated using the
The genotypes of the strains, the oligonucleotides, and the exact sequences of
formula 2CT(IP)
CT(input) as described previously (40a) and was normalized to
the various integrations generated and used in this study will be provided upon
the telomeric probe. For Rap1p immunoprecipitation, polyclonal antibodies
request.
(Y300; Santa Cruz Biotechnology, Inc.) were used, while antibodies against
Yeast strains. Yeast genomic integrations were performed by homologous
H4K16Ac were purchased from Upstate.
recombination and gene replacement, using PCR products or DNA fragments
3C. The chromosome conformation capture (3C) analyses of yeast strains were
derived from plasmids. Yeast transformations used the lithium acetate method
performed exactly as described previously (18) with a few speciﬁc changes. Each
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(34). PCR ampliﬁcations were carried out with Expand high-ﬁdelity DNA poly-
strain in each experiment was cross-linked for 0, 5, 10, and 20 min, and each
merase, and integrations were conﬁrmed by PCR and sequencing analysis. SIR2,
sample was independently processed and analyzed. The restriction enzyme used
SIR3, PPR1, and ADE2 genes were deleted from the start to the stop codon and
for the digestion was Sau3A, and the digestion buffers were as recommended by
replaced with HIS3 or kanMX markers. Deletion of MAT
was obtained by
the manufacturer of the enzyme. All primers used were tested with un-cross-
replacing MAT 2 and MAT 1 sequences (SGD coordinates 199731 to 200964)
linked/ligated DNA, and only primers with equal ampliﬁcation efﬁciencies were
with the kanMX cassette.
used for the 3C analyses.
The ADE2 gene ﬂanked by Gal4p binding sites (Gbs), present at HML, in
Fluorescence analysis. Two diploid strains were constructed to visualize the
strain KIY54 (32) was PCR ampliﬁed with appropriate primers and integrated at
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relative positions of HMR and the nucleolus. The strains differ only by the
the HMR locus in a sir2
strain (JRY4576) or in an HMR I sir4
strain
absence of the tRNA barrier at HMR. The lac operator array is telomere prox-
(ROY926). Sequencing of the PCR products indicated that a single Gal4p
imal to HMR-I. Both strains contain Sik1p fused to red ﬂuorescent protein (RFP)
binding site, which is contrary to published results (32), ﬂanked ADE2. The PCR
(a nucleolar marker), express lac-green ﬂuorescent protein (lac-GFP), and con-
product was integrated in the HMRa2 coding region (SGD coordinates 293212 to
tain a galactose inducible R recombinase.
293410) with the ADE2 promoter close to the HMR-E silencer. Strains with the
by on March 21, 2010
To examine the relative positions of HMR with respect to the nucleolus, the
integrated ADE2 gene (ROY2729 [MAT
HMR::Gbs-ADE2-Gbs sir2 ] and
ROY2914 [MAT
HMR-E-Gbs-ADE2-Gbs-HMR I sir4 ]) were crossed with a
cells were ﬁrst grown in SC-trp medium containing dextrose. Cells were ﬁxed 2 h
W303 wild-type strain to obtain ROY2770 and ROY3001, respectively.
with paraformaldehyde and mounted on microscope slides containing agar plugs.
The HMRa1 coding region in ROY2729 and ROY2914 was replaced by the
Parallel Z stacks of cells were obtained using both rhodamine and GFP ﬁlters to
URA3 coding region by homologous recombination, and transformants were
visualize the nucleolus and HMR, respectively (17 sequential images separated by
crossed with an ade2 ::kanMX strain to obtain ROY3182 and ROY3194, re-
0.2
m). The two landmarks were considered colocalized if the corresponding
spectively.
ﬂuorescence signals fully overlapped within the same plane or in adjacent planes.
Plasmid pJR1270 contains an EcoRI-HindIII fragment with the HMR locus
The landmarks were considered to touch if contact (but not overlap) was seen
where the HMR-I silencer has been deleted. This fragment contains two SpeI
between them within a plane or adjacent planes. The landmarks were considered
sites. The plasmid was partially digested with SpeI, end ﬁlled, and religated to
to be fully separated if no contact was observed or if image planes lacking
obtain pRO698, which contains only one SpeI site 290 bp upstream of the ARS
ﬂuorescence separated ﬂuorescent foci in different planes. Multiple ﬁelds of cells
element present at HMR-E. A pair of oligonucleotides with four Gbs ﬂanked by
were examined for each of the two trials. Cell morphology was used to estimate
SpeI sites were annealed, digested, and cloned into the SpeI site in pRO698 to
the cell cycle stage of each cell examined. However, the same general trends were
produce pRO700. The EcoRI-BglII fragment from pRO700 was used to replace
observed in G , S, and G phases, so these data were pooled (G and S phase
1
2
1
the HMR region in strain ROY2585 (HMRa2::URA3 sir2 ), to give strain
cells were well represented, whereas there were considerably fewer G cells).
2
ROY3285 (Gbs-HMR sir2 ).
The EcoRI-HindIII fragment in pRO700 was used to replace the HMR region
in ROY2585 to produce ROY3283 (Gbs-HMR I sir2 ). The URA3 cassette was
RESULTS
integrated between HMR-I or HMR I and the tRNA gene (SGD coordinates
295070 to 295281) in strains JRY4566 (W303 sir2 ), ROY3285, ROY3550
Silencers ﬂank silenced genes at HML and HMR. At HML,
(HMR I sir2 ), and ROY3283, and the transformants were crossed with a ppr1
silencing initiates from both silencers, but at HMR silencing
::kanMX strain to obtain ROY3495 to -3489, -3683, -3699, -3680, and -3697.
ROY3495 and -3497 were crossed with a Gal4-TAP-tagged strain to obtain
initiates only at the HMR-E silencer. Previous work from our
ROY4371 and -4372, respectively.
lab showed that tethering proteins with barrier activity near
The tRNA gene was deleted and the URA3 gene integrated in strain JRY4566
HMR-E blocked the spread of silencing from the HMR-E si-
and ROY3285 by PCR-mediated gene replacement. Transformants were then
lencer if the HMR-I silencer was absent (21). However the
crossed with a ppr1
strain to obtain ROY3688, -3703, -3686, and -3701.
barrier could be bypassed if a second silencer (HMR-I) was
Plasmid pJR1571 contains an EcoRI-HindIII fragment comprising the HMR
locus. Oligonucleotides with three new Sau3A sites were used to PCR amplify
positioned downstream of the barrier (references 21 and 47
the XbaI-EcoNI fragment of the MATa2 gene. The PCR product was cloned into
and data not shown). One explanation for this phenomenon is
the XbaI-EcoNI fragment of HMRa2 in plasmid pJR1571. The EcoRI-HindIII
that silencing nucleates at HMR-I, as well as HMR-E. However,
fragment of the new construct (pOS154) was used to replace the HMR locus in
functional data clearly indicates that HMR-I only augments the
strain ROY2800 (HMRa2::URA3), and transformants were crossed with
mat
::kanMX, sir3 ::HIS3, and hml ::TRP1 strains to obtain ROY4064 and
activity of HMR-E and does not possess an autonomous silenc-
ROY4065.
ing activity (1, 7, 50).

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by on March 21, 2010
FIG. 1. Boundary trap assays at HMR and HML. (A) HMR-I does not recruit Sir3p. ChIP was used to map the presence of Sir3p in strains with
mutant silencers at HMR, and the immunoprecipitated samples were analyzed by PCR. The locations of the PCR probes are shown in the
schematic diagram. WT, wild type. (B) At HML, only Nup2 allows discontinuous silencing. Strains with a boundary trap construct at HML were
transformed with TRP1-containing plasmids constitutively expressing the chimeric protein Gbd-Sas2p (pRO590) or Gbd-Nup2p (pRO635) or the
vector. Cells were grown in liquid YM medium (HC-trp), and expression of the ADE2 and URA3 genes was monitored by serial dilutions on HC-trp
plates lacking or containing adenine, uracil, and 5-FOA as indicated. The plates were photographed after 2 days. The panel labeled in gray allows
differentiation between “true barrier” proteins and “desilencing” proteins. (C) At HMR, both Nup2 and Sas2p allow discontinuous silencing.
Strains with a boundary trap construct at HMR were transformed with TRP1-containing vector or with TRP1 plasmids constitutively expressing
Gbd-Nup2p or Gbd-Sas2p. Cells were grown overnight in liquid HC-trp, and serial dilutions were spotted on appropriate plates. Cells were spotted
on HC-trp plates lacking adenine or containing 30
g per ml of adenine and allowed to grow at 30°C prior to photography. To assay for stable
repression of URA3, cells were spotted onto HC-trp plates containing 5-FOA and lacking adenine or containing 30
g per ml of adenine and
allowed to grow at 30°C prior to photography. The panel labeled in gray allows differentiation between “true barrier” proteins and “desilencing”
proteins.
Since our analyses suggested that silencing at HMR might be
Deleting HMR-I did not affect Sir3p levels at the HMR-E
initiating at HMR-I, we reasoned that if HMR-I was a silencer,
silencer, but deleting HMR-E resulted in loss of Sir3p localiza-
then it should be able to recruit at least some Sir proteins in the
tion from the HMR locus but not the telomeres. In the absence
absence of the HMR-E silencer. To directly test this possibility,
of HMR-E, the levels of Sir3p at HMR were equivalent to those
we used ChIP to examine the binding of Sir3p near HMR-E
observed at the negative control, the TEL6R 7.5kb probe,
and HMR-I in a variety of HMR variants. As expected, Sir3p
where Sir3p has not been found previously. Therefore, these
localized to the two silencers in the wild-type strain (Fig. 1A).
results, at this level of sensitivity, demonstrate that HMR-I

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LONG-RANGE COMMUNICATION BETWEEN HMR SILENCERS
1927
does not recruit Sir proteins in the absence of HMR-E and are
silencer from the boundary trap strain at HMR and analyzed
consistent with previous functional results (1, 7, 50).
the ability of these strains to grow on medium lacking adenine
These results lead to an alternative explanation that HMR-E
but containing 5-FOA. Our results (Fig. 2A) showed that si-
“communicates” with HMR-I to facilitate silencing at a dis-
lencing of the URA3 gene in the dual reporter system at HMR
tance. To explore this paradoxical phenomenon further, we
required HMR-I. When this silencer was removed, no colonies
monitored silencing of a dual reporter system known as the
formed on the plates lacking adenine and containing 5-FOA.
boundary trap assay that was developed and used to investigate
This result demonstrates that the Sir proteins recruited at
insulator proteins at HML (33). In this assay, the silenced locus
HMR-E can transpose across an active domain only when a
was modiﬁed and the mating-type genes were replaced with
silencer is present on either side of this domain. These results
ADE2 and URA3 genes. Gal4 binding sites ﬂank the ADE2
with the dual reporter systems are concordant with our earlier
gene, whereas a second reporter, URA3, is not ﬂanked by these
studies of single tethered barrier proteins (47). Silencing adja-
sites and resides adjacent to the I silencer (Fig. 1B and C). The
cent to HMR-I requires the HMR-I silencer if a barrier blocks
assay monitors the ability of a protein tethered to the Gal4p
the action of HMR-E.
binding sites to insulate the ADE2 gene from repression but
To explore the discontinuous silencing phenomenon at a
not the neighboring URA3 gene.
molecular level, we mapped the distribution of Sir3p and
The dual reporter system was ﬁrst used at HML. Unlike at
H4AcK16 across the HMR domain in the presence and ab-
HMR, at HML both silencers are able to independently initiate
sence of Gal4-Sas2p. We chose to analyze these two proteins
silencing (Fig. 1B). We tested the behavior of Gal4-Nup2p and
since they are markers of active and inactive chromatin (46). In
Gal4-Sas2p. Nuclear pore proteins such as Nup2p were
the absence of Gal4-Sas2p, there is no acetylation at either
claimed to be “true barrier” proteins that can insulate the
silencer or the ADE2 gene (Fig. 2B). When Sas2p is recruited
ADE2 gene while maintaining the neighboring URA3 gene in a
to sites ﬂanking the ADE2 gene, there is no detectable acety-
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silenced state (33). We also tested Gal4-Sas2p, since acetyl-
lation at the HMR-E and HMR-I silencers but there is a sig-
transferases are believed to function by a “desilencing” mech-
niﬁcant increase in H4K16 acetylation at the ADE2 gene, con-
anism. We measured expression of ADE2 by growth on me-
sistent with the observation that ADE2 is active in these cells.
dium lacking adenine, and we measured expression of URA3
On the other hand, Sir3p was present at the two silencers, in
by growth on medium containing 5-ﬂuoroorotic acid (5-FOA).
both the presence and absence of Gal4-Sas2p, but was reduced
Cells expressing URA3 convert 5-FOA to a toxic metabolite
at the ADE2 gene when Sas2p was tethered at the Gal4p
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and die. We used these assays because they are far more
binding sites ﬂanking the ADE2 gene. Thus, tethered Gal4-
sensitive to changes in the expression levels than Northern
Sas2p does not block the normal function of the two silencers,
blots. Furthermore, these assays allow us to determine the
and the growth phenotypes observed in Fig. 1 are indeed due
mitotic stability of these epigenetic states. Our results (Fig. 1B)
to discontinuous silenced domains that initiate from HMR-E.
by on March 21, 2010
are consistent with previously published data (33). Gal4-
Interestingly, we consistently see increased levels of Sir3p at
Nup2p insulated ADE2 from repression while allowing URA3
both the HMR-E and HMR-I silencers compared with the
to be stably repressed in a small percentage of cells. On the
ADE2 gene, in the presence or absence of Gal4-Sas2p. Our
other hand, Gal4-Sas2p derepressed both ADE2 and URA3,
results demonstrate that HMR-I alone cannot recruit Sir pro-
presumably by disrupting silencing across the entire silenced
teins, but in the presence of HMR-E it is able to stably maintain
domain.
elevated levels of Sir proteins. While the elevated levels of
We next constructed a dual reporter system at HMR that was
Sir3p at HMR-E can be explained by the fact that HMR-E
similar to the system at HML, placing the ADE2 gene near
recruits the Sir proteins and initiates silencing, the reason for
HMR-E and the URA3 gene near HMR-I (Fig. 1C). The strain
the elevated levels at HMR-I were unexpected and not initially
was transformed with Gal4-Nup2p, Gal4-Sas2p, or vector
obvious. One possibility is that the increased levels of Sir pro-
alone. The cell growth assays in Fig. 1C clearly show that
teins at HMR-I may be due to the two silencers being in close
Gal4-Nup2 functions again as a true barrier, producing colo-
proximity to one another.
nies of cells in which ADE2 was active but URA3 was stably
Rap1p localizes to HMR-I. Our data indicate that HMR-E
repressed. However, unlike the situation at HML, Gal4-Sas2p
functionally communicates with HMR-I, resulting in a discon-
derepressed ADE2 expression while permitting URA3 repres-
tinuous silenced domain, but they do not specify how this
sion at HMR. Thus, Gal4-Sas2p also functions as a “true bar-
might occur. One possibility is that the two silencers reside in
rier” at HMR. Importantly, the ability of cells to form colonies
close proximity to one another.
on medium lacking adenine but containing 5-FOA indicates
To conﬁrm these long-range interactions, we asked whether
that the “discontinuous” silenced state that is established in
a DNA-bound protein at one end of the domain was in close
these cells is stably inherited for several generations, enabling
proximity to the other end of the domain, similar to the ex-
these cells to form colonies. A dual reporter system containing
periments used to show long-range interactions in Drosophila
ADE2 and MATa1 yielded similar results, suggesting that this
(5). There is a single binding site for Rap1p at the HMR-E
effect was not reporter speciﬁc (data not shown). Furthermore,
silencer, where the protein has been shown to bind (53). No
the fact that Sas2p, a bona ﬁde histone acetyltransferase which
Rap1p sites are known to exist at HMR-I. We used ChIP to
is expected to behave as a “desilencer,” can function as a “true
map the presence of Rap1p at HMR, as well as at loci on
barrier” protein indicates that the molecular underpinnings for
chromosome 6R (Fig. 3A). While we did not observe signiﬁ-
these deﬁnitions will need to be reconsidered.
cant binding of Rap1p to the telomere 6R 7.5-kb probe, the
We next determined whether the generation of the discon-
quantitative analyses showed that Rap1p was present immedi-
tinuous silenced state required HMR-I. We deleted the HMR-I
ately adjacent to telomere 6R and at HMR. At HMR we ob-

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by on March 21, 2010
FIG. 2. Discontinuous silencing at HMR. (A) HMR-I is necessary for discontinuous silencing. Strains with a boundary trap construct at HMR
but lacking the HMR-I silencer were transformed with TRP1-containing vector or with TRP1 plasmids constitutively expressing Gbd-Nup2p or
Gbd-Sas2p. The strains were assayed as described for Fig. 1C. (B) Mapping the distribution of acetylated histones and Sir3p in the boundary trap
constructs. Strains constitutively expressing Gbd-Sas2p or vector alone were grown in HC-trp medium, selecting for the plasmids (the medium was
supplemented with 90
g/ml adenine). ChIP with antibodies against H4K16Ac or Sir3p were performed exactly as previously described (46). The
graphs depict the enrichment of the immunoprecipitated sample over the input normalized to a telomeric probe. Enrichment and standard errors
were computed from at least two independent cross-linked samples and three independent immunoprecipitation experiments. Localization of the
PCR probes is depicted in the schematic.
served Rap1p binding to HMR-E, the silenced MATa1 gene at
on the assumption that when an upstream activation sequence
HMR and at HMR-I. This result was obtained with two differ-
(UAS) is brought in close spatial proximity to the promoter of
ent commercially available antibodies (data not shown), vali-
a repressed gene, it will activate that gene. We placed Gal4p
dating the presence of Rap1p at HMR silencers.
binding sites upstream of HMR-E and placed a repressed re-
We next determined if loss of Sir3p affected the distribution
porter gene (URA3) several kilobase pairs (4 kb) downstream
of Rap1p. Loss of Sir3p did not lead to any decrease in the
from the Gal4p binding sites on the distal side of HMR, be-
amount of Rap1p at HMR-E, but there was a complete loss of
tween HMR-I and the tRNA barrier. Transcription of the re-
Rap1p from HMR-I. These results are consistent with the ob-
porter was directed toward the Gal4p binding sites (Fig. 3B).
servation that HMR-E was in close proximity to HMR-I, al
At this location, the URA3 gene was subjected to repression by
though it is also possible that despite Rap1p being a sequence-
Sir-mediated silent chromatin, and in the absence of the Sir
speciﬁc DNA binding protein, it spread along the silenced
proteins, URA3 was active in both glucose and galactose (data
chromatin through interactions with the Sir proteins.
not shown). Interestingly, stable repression of URA3 located
“Enhancer” activity at HMR. To investigate the spatial lo-
downstream of HMR-I was dependent upon the HMR-I si-
calization of the silencers relative to each other, we decided to
lencer, because in the absence of this silencer, the reporter was
develop an “enhancer assay” (Fig. 3B). The assay is premised
no longer stably silenced (data not shown).

VOL. 28, 2008
LONG-RANGE COMMUNICATION BETWEEN HMR SILENCERS
1929
We next tested whether binding of Gal4p to its sites up-
stream of HMR-E could disrupt the silencing of the URA3 gene
located downstream of HMR-I. When cells were grown in glu-
cose, Gal4p was not activated and URA3 remained silenced in
strains that contained or lacked Gal4p binding sites (Fig. 3C,
top). When these cells were grown in medium containing ga-
lactose, however, URA3 was activated and cells were able to
grow in medium lacking uracil and not in medium containing
5-FOA (Fig. 3C, bottom). This result was observed only in
strains that contained Gal4p binding sites, indicating that Gal4
binding upstream of HMR mediated the galactose-dependent
URA3 induction.
One possibility is that Gal4p was disrupting silencer func-
tion. If Gal4p was disrupting silencing across the entire do-
main, then the MATa1 gene located in the silenced region
should also be activated. We therefore monitored expression
of the MATa1 gene located between the two silencers by per-
forming a mating assay and selecting for diploids. The appear-
ance of diploids (Fig. 3C) demonstrated that at this level of
sensitivity, MATa1 was silenced.
We also investigated whether Gal4p binding upstream of
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HMR was disrupting Sir3p binding at HMR by quantitative
ChIP. If this was the case, then in strains containing Gal4p
binding sites grown in galactose, one might expect to see a
reduction in the levels of Sir3p at HMR. We mapped the levels
of Sir3p by ChIP across the entire HMR domain in strains
grown in galactose with and without Gal4p binding sites. Our
mcb.asm.org
analyses showed only a slight change in the levels of Sir3p at
the two silencers, with no discernible change at the MATa1
gene located between the two silencers in the presence or
absence of Gal4p (data not shown). These results are consis-
by on March 21, 2010
tent with our mating assays (Fig. 3C) showing that silencing did
not signiﬁcantly change at HMR.
These results demonstrate “enhancer” function of a gene
across a silenced domain. They also demonstrate that a protein
bound to a UAS located several kilobase pairs from the pro-
moter of a gene could derepress that gene in yeast. This result
is highly unusual, since activation over such long distances has
not been observed in yeast (20). The simplest explanation for
these results is that the UAS was in close spatial proximity to
the promoter of the reporter gene in the nucleus, which then
alleviated silencing of the reporter. However one cannot rule
out other possibilities due to the inherent limitations of these
assays.
Loss of the barrier does not affect long-range communica-
tions. Our results suggested that the two silencers might be in
FIG. 3. Functional long-range communication at HMR. (A) Mapping
close proximity to one another; we were interested in deter-
Rap1p at HMR. Antibodies against the Rap1p C terminus were used to map
mining the DNA elements and factors that affected this local-
the distribution of Rap1p across the HMR locus in a wild-type (WT) strain
and a sir3 strain. Quantitative ChIPs were performed as described for Fig.
ization. In chicken cells the globin insulator helps tether the
2. The PCR probes used are shown in the schematic diagram. Error bars
globin domain to the nucleolus and aids in the formation of
indicate standard errors. (B) Schematic representation of the enhancer con-
chromatin loops (61, 62). In yeast, tRNA genes are dispersed
struct at HMR. The locations of Gal4p binding sites and the URA3 gene are
throughout the genome, but in situ hybridization demonstrated
shown. (C) “Enhancer” activity at HMR. Strains with URA3 located down-
that the genes are clustered adjacent to the nucleolus (30).
stream of HMR-I containing no Gal4 binding sites (-Gbs) or four Gbs up-
stream of HMR-E ( Gbs), were grown in 5 ml YPD overnight. Cells were
One of the HMR barriers is a tRNA gene (21), and it is
washed, and ﬁvefold serial dilutions were prepared. Properly supplemented
therefore possible that tethering of the HMR barriers to the
YM plates containing 2% galactose (YMG) or 2% glucose (YMD) as a
nucleolus might be the mechanism by which the two silencers
carbon source were used to induce or to repress expression of Gal4p, respec-
were brought in close proximity to one another. A prediction
tively. To assay for expression of URA3, cells were spotted onto YMG or
YMD plates lacking or containing uracil or 5-FOA and photographed. To
of this model would be that the HMR locus would reside
assay for expression of MATa1 at HMR, cells were spotted onto properly
adjacent to the nucleolus and deletion of the barrier would
supplemented YMG or YMD plates with mating lawns.
result in a concomitant loss of this localization.

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by on March 21, 2010
FIG. 4. Long-range communication and barrier function. (A) HMR does not colocalize with the nucleolus. Fluorescence analysis was performed
with strains expressing Sik1-RFP and lac-GFP. A lac operator array placed approximately 4 kb from HMR, adjacent to the promoter of the GIT1
gene, allowed us to map the localization of the silenced domain relative to the nucleolus. The GFP and RFP signals were monitored in strains
containing or lacking the tRNA barrier. Colocalization was deﬁned as full overlap of the green and red signals within the same or adjacent image
planes. Over 200 cells were examined in two independent trials. Colocalization was monitored when the HMR domain was present on a
chromosome as well as on an episome (following recombination). A representative picture of the cells is shown above the graphs. (B) Loss of the
tRNA barrier does not affect long-range activation at HMR. Strains containing or lacking the tRNA barrier without or with four Gal4p binding
sites upstream of HMR-E and with URA3 located downstream of HMR-I were grown overnight. Properly supplemented YM plates containing 2%
glucose or galactose as carbon source were used to induce expression of Gal4p. To assay for expression of URA3, cells were spotted onto YMG
plates lacking or containing uracil or 5-FOA and photographed. To assay for expression of MATa1 at HMR, cells were spotted onto properly
supplemented YMG plates with mating lawns.
To determine whether the tRNA barrier adjacent to HMR
Stacks of ﬂuorescent images along the Z axis using both GFP
associated with the nucleolus, we used a cytological approach.
and rhodamine (red) ﬁlters were collected to determine the
A lac operator array was incorporated adjacent to HMR (in-
relative positions of HMR and the nucleolus. Colocalization
serted approximately 4 kb from HMR) in a strain that ex-
was deﬁned as full overlap of the green and red signals within
pressed lac-GFP, as well as Sik1-RFP. lac-GFP binds the lac
the same or adjacent image planes. Over 200 cells were exam-
operator array to create a bright green spot of ﬂuorescence
ined in at least three independent trials. The data in Fig. 4A
(marking HMR), whereas Sik1p, a nucleolar protein, imparts
show that in over 90% of the cases, HMR and the nucleolus did
red ﬂuorescence to the perinuclear crescent-shaped nucleolus.
not contact one another. The low level of coincident colocal-

VOL. 28, 2008
LONG-RANGE COMMUNICATION BETWEEN HMR SILENCERS
1931
ization is similar to that found for other noninteracting chro-
within this region (in the MATa2 gene). Furthermore, since
matin landmarks (9, 12). Similar results were found when HMR
regions at HMR are homologous to regions at HML
and
and the adjacent barrier were liberated from the chromosome
MAT, we performed this analysis with a strain in which these
by site-speciﬁc recombination to form an extrachromosomal
two loci were deleted. All of the primers that we used in this
DNA circle. These results indicated that HMR and the associ-
analysis were oriented in the same direction. Therefore, a PCR
ated boundary did not reside at the nucleolus. Furthermore,
product can arise only after restriction fragments were digested
deleting the barrier did not alter the localization of HMR in the
and religated. This eliminated PCR products that might arise
nucleus (Fig. 4A). It is therefore unlikely that the mechanism
from incomplete digestion of the cross-linked samples.
by which silencers are brought in close proximity is via tether-
We initially analyzed the ligations with a ﬁxed oligonucleo-
ing to the nucleolus.
tide located in a Sau3A fragment containing the HMR-E si-
Tethering of insulators to nuclear superstructures has been
lencer (primer A) with restriction fragments that encompassed
proposed to be important for insulation. In Drosophila, the
the silenced domain and beyond to determine which fragments
Su(Hw) insulators cluster in the nucleus, forming insulator
were in close proximity to the reference fragment. Our analy-
bodies (28), while in yeast, nuclear pore proteins localize to the
ses showed that the HMR-E fragment ligated most frequently
silenced chromatin (10), and models suggest that tethering of
to a single Sau3A fragment (ampliﬁed with primer G) contain-
insulators to the pores, forming a chromatin loop, is the mech-
ing the HMR-I silencer (Fig. 5A).
anism by which chromatin domains are organized and main-
To ensure that the PCR ampliﬁcation efﬁciencies between
tained (32).
different primer pairs were comparable, we digested plasmid
Therefore it was still possible that the barrier insulator ele-
DNA containing the HMR locus with Sau3A in the absence of
ments at HMR were important for the observed long-range
cross-linking, followed by ligation under conditions that fa-
communication between the two silencers, albeit not by teth-
vored intermolecular ligation. PCR analyses indicated that all
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ering to the nucleolus. If barrier elements were necessary for
of the primer pairs were approximately equally efﬁcient in
organizing chromatin domains into loops, then loss of a barrier
amplifying the ligated products (Fig. 5A). We conﬁrmed the
should result in loss or diminution of long-range communica-
equivalent PCR ampliﬁcation efﬁciencies of the primers used
tions. Using the enhancer assay, we investigated the role of the
by twofold serial dilutions of the un-cross-linked (intermolec-
HMR tRNA barrier in this process. We generated two strains
ular) ligation reaction prior to PCR (data not shown). These
lacking the tRNA barrier and possessing URA3 immediately
data showed that the differences in ampliﬁcation observed
mcb.asm.org
downstream of HMR-I. One strain contained Gal4p binding
across the HMR domain, in the nucleus, were due to differ-
sites located upstream of HMR-E, while the second strain
ences in cross-linking/ligation of various fragments to the ref-
lacked these binding sites. Monitoring the expression of URA3
erence fragment (HMR-E) and not due to differences in PCR
in these two strains in galactose showed that loss of the tRNA
ampliﬁcation. As an additional control, we puriﬁed the PCR
by on March 21, 2010
barrier did not adversely affect the long-range communication
products obtained from the cross-linked nuclear samples and
between the two ends of the silenced chromatin domain
sequenced them to unambiguously determine the identity of
(Fig. 4B).
the ligated fragments (data not shown).
It has been suggested that chromatin loops are formed by
We next used the primer in the Sau3A fragment containing
the attachment of barrier elements to the nuclear pore via
HMR-I (primer G) as the reference primer and assayed the
Nup2p (32). Analyses of Nup2p mutants indicate that Nup2p is
proximity of this fragment to other fragments across HMR. Our
necessary for robust tRNA barrier function, but loss of Nup2p
results, shown in Fig. 5A, revealed that the HMR-I silencer-con-
did not affect the long-range communication between the two
taining fragment ligated most frequently to the HMR-E silencer-
silencers (G. Ruben and R. T. Kamakaka, unpublished data).
containing fragment (primer A) as well as to a fragment down-
HMR-E and HMR-I are in close spatial proximity. All of our
stream of HMR-E (primer C) that harbored the end of the
analyses described thus far suggested that the two silencers
MATa2 gene. From these data we inferred that HMR-I was in
were in close spatial proximity to one another. We therefore
close proximity to HMR-E and the 3 end of the MATa2 gene.
directly analyzed the spatial relationships at the native HMR
It is possible that HMR-I is in close proximity with both frag-
locus in the yeast nucleus by using the 3C method, which was
ments simultaneously or exchanges rapidly between these two
developed to investigate the three-dimensional relationships
fragments.
between DNA elements (15, 18). Cells were brieﬂy treated
The 3C method was initially used to demonstrate the prox-
with formaldehyde to cross-link DNA to proteins, followed by
imity between various centromeres in yeast (18). We also
cleavage with a speciﬁc restriction enzyme; we digested the
tested this interaction with our cross-linked samples. Consis-
DNA with Sau3A since this enzyme generated small fragments
tent with previously published data, we found that EcoRI frag-
(55). The fragments were then diluted and ligated, such that
ments at Cen IV (primer 14) ligated only to fragments at Cen
ligations were primarily between cross-linked DNA fragments.
III (primer 6) and not to other chromosome III EcoRI frag-
The fragments that ligated to one another were identiﬁed using
ments (primers 5 and 7) (data not shown).
PCR with speciﬁc pairs of primers. Using this method, the
Long-range interactions require silencing. Our results sug-
cross-linking frequency between two restriction fragments is
gest that long-range communication between the two HMR
expected to be roughly proportional to their proximity to one
silencers was not a fortuitous result of the clustering or long-
another in the nucleus.
range interactions between the barrier elements that ﬂank the
The DNA between the HMR-E and HMR-I silencers does
two silencers at HMR. Silencing at HMR utilizes the Sir pro-
not contain many Sau3A sites. To improve the resolution of
teins that interact with the chromatin to mediate silencing. We
our analyses, we introduced three additional Sau3A sites
therefore investigated whether the communication between

1932
VALENZUELA ET AL.
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by on March 21, 2010
FIG. 5. Spatial organization at HMR. (A) 3C analysis of HMR. Wild-type strains (lacking HML and MAT) were cross-linked, digested with
Sau3A, and ligated, and the ligation products were analyzed by PCR (labeled 3C). Reference primer A or G was used along with the other primers
across the domain. The location and orientation of the primers are shown schematically. The primer control panel involved intermolecular ligations
and PCR analyses of un-cross-linked DNA. (B) Loss of Sir3p affects long-range interactions. Strains with a deletion of the SIR3 gene were analyzed
with the 3C assay as described above. (C) Schematic representation of the HMR domain in the yeast nucleus.
the two silencers was a result of silencing. We determined
fragments was dramatically reduced (Fig. 5B), suggesting that
whether the long-range interaction between the two silencers
these long-range interactions required the Sir proteins.
was disrupted in a sir3
mutant by 3C analyses. Our results
Since HMR-E no longer ligated to HMR-I in a sir3
mutant,
showed that in the absence of this repressor, the extent of
to ensure that the cross-linking and ligation were normal in this
ligation between the HMR-E- and HMR-I-containing Sau3A
sample, we examined the ligation between the centromeric

VOL. 28, 2008
LONG-RANGE COMMUNICATION BETWEEN HMR SILENCERS
1933
fragments as controls, since Sir3p is not present at yeast cen-
proximity so that HMR-I shares the nucleation activity of the
tromeres and Sir3p mutations have not been shown to affect
more potent HMR-E silencer. In this case, silent chromatin
centromere function. Our analysis of centromeric chromatin
would nucleate at both silencers and spread from both until
demonstrated that indeed CEN III remained in close proximity
encountering synthetic (or natural) barriers. Thus, when a pair
to CEN IV in the absence of Sir3p (data not shown).
of barrier proteins is situated between HMR-E and HMR-I, a
Our results in their totality demonstrate that silencers sep-
domain of active chromatin will reside between domains of
arated over several kilobase pairs of DNA functionally and
silent chromatin.
structurally interact with one another. We have begun to iden-
An alternative possibility is that the insulated ADE2 con-
tify the determinants necessary for these interactions and show
struct counteracts silent chromatin that has spread from a sole
that the Sir proteins are necessary for this long-range commu-
nucleation point at HMR-E. In this scenario, ADE2 activation
nications between silencers.
would occur following a cell cycle event, such as DNA repli-
cation, that compromises silencing efﬁciency (4). Silent chro-
matin would persist on both sides of the activated domain
DISCUSSION
because the silencers stabilized the existing repressed state (2,
The control of eukaryotic gene expression involves commu-
13, 14, 42, 50). However, it is hard to visualize how this mech-
nication between regulatory elements that are often separated
anism lends itself to stable inheritance (which we observe in
by great distances. There are now numerous examples of distal
our assays). An alternative model that combines these two
enhancers and LCRs that contact the genes they activate (re-
scenarios is possible where binding of Sir proteins to HMR-E
viewed in reference 60). More recent studies have even found
facilitates interactions between HMR-E and HMR-I. Once
interactions between regulatory elements and genes that reside
HMR-I is brought in proximity to HMR-E, it can also nucleate
on entirely different chromosomes (39, 54). In this report, for
silencing, which then would spread from both silencers.
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the ﬁrst time, we show long-range interactions between the
Sir3p and long-range repression. The long-range communi-
silencers that ﬂank the HMR locus in yeast, and we identify the
cation between the two silencer fragments is dependent upon
determinants required for these interactions. Interestingly, we
Sir3p. Sir3p is a structural repressor protein that binds the
have shown that deletion of the tRNA insulator element or the
histones in nucleosomes to mediate repression. In vitro binding
nuclear pore protein Nup2p did not affect functional long-
studies with oligonucleosomes have shown that Sir3p oligo-
range communication between the two silencers but that loss
mers bind multiple chromatin fragments and “cross-links” nu-
mcb.asm.org
of Sir3p did result in diminution of these interactions, suggest-
cleosomal arrays (27, 29, 40). It is therefore possible that Sir3p
ing that silencing itself may be important for organizing this
binding to chromatin cross-links the silenced domain and the
chromatin domain.
resulting compaction brings distal sites together. The depen-
Silencers and the mechanisms for silencing. Numerous
dence on Sir3p is reminiscent of long-range repression in Dro-
by on March 21, 2010
studies have shown that HMR-E is sufﬁcient to nucleate silenc-
sophila, where Polycomb-mediated repression involves inter-
ing at HMR. HMR-I cooperates with HMR-E, but it cannot
actions between chromatin memory module elements and
initiate silencing on its own (42, 50). Therefore, HMR-I is
distally repressed promoters via the association of Polycomb
analogous to proto-silencers that have been found at telomeres
group proteins (17, 44). We note, however, that Sir3p is not
and HML (6, 13). At telomeres, proto-silencers and proteins
sufﬁcient to hold HMR loci on sister chromatids together (12).
with barrier activity are interspersed in the subtelomeric blocks
Instead, silent chromatin recruits cohesin, which mediates
to yield domains of discontinuous silencing (24, 25, 38), and
pairing of the twin silent chromatin domains. Additional fac-
these functional assays have led to models where the proto-
tors could similarly facilitate interactions between distal silent
silencer elements might interact with one another, but direct
chromatin segments within the same chromatin ﬁber.
long-range interactions at telomeric loci have not been dem-
Regulatory elements important for long-range communica-
onstrated using the 3C technique. Long-range interactions be-
tion. An interaction speciﬁcally between the two silencer-con-
tween the proto-silencers and terminal telomeric sequences,
taining DNA fragments raises the question of which DNA
which function as silencers, may indeed be occurring, similar to
elements, if any, are required. Our results with the boundary
what we observe at HMR. The distribution of terminally bound
trap system demonstrate that HMR-I is necessary. Preliminary
Rap1 and Ku at telomeres is consistent with telomere loop
data using the 3C technique also suggest that the silencer
formation (43, 56), and enhancer assays like the one we have
elements are necessary. ORC and Abf1p bind HMR-I and
employed in this study have also suggested looping within
might be involved in mediating these long-range interactions.
silent chromatin at telomeres (16). However, this is the ﬁrst
Rap1p might also aid long-range communication. When bound
report to unambiguously describe long-range interactions and
to two sites on naked DNA, the protein induces loop formation
the formation of chromatin loops at an internal silenced locus
(31). Further experiments will be necessary to dissect the roles
in yeast.
of these elements and proteins in long-range communication.
Placement of the dual reporter constructs of the boundary
In an alternative scenario, the two silencers could be brought
trap assay at the silent mating-type loci created discontinuous
in close proximity by insulator elements that ﬂank the silenced
silencing states. At HMR, Sir3p was found at both silencers but
domain. It has been suggested that chromatin loops are formed
was consistently reduced at the insulated reporter gene in
through association of insulator elements with the nuclear
between. In agreement, the H4K16Ac mark for active chro-
pores (32, 33, 52). It is therefore possible that the barrier
matin was found in a reciprocal pattern. How can a discontin-
elements ﬂanking HMR (21, 22) or the hypersensitive sites at
uous state be created when HMR-I does not function on its
this locus (45) associate with nuclear pores or active chromatin
own? One possibility is that both silencers are held in close
hubs and cluster in the nucleus, the consequence of which

1934
VALENZUELA ET AL.
MOL. CELL. BIOL.
would be to align HMR-E and HMR-I in close proximity. How-
ACKNOWLEDGMENTS
ever, we have shown that deleting the barrier or Nup2p, which
We thank Genevieve Fourel, David Donze, Grant Hartzog, Michael
interacts with the barrier, had little effect on long-range com-
Lichten, Orna Cohen-Fix, Masaya Oki, and Catherine Fox for com-
munication between the silencers. It therefore seems unlikely
ments on the manuscript; C. Fox and U. Laemmli for speciﬁc strains;
that chromatin barriers play a major role in the observed in-
and Riza Ysla for technical assistance.
This work was supported by grants from the NIH to R.T.K.
teractions between HMR-E and HMR-I.
(GM078068) and M.R.G. (GM51402).
Sir spreading, long-range interactions, and silencing. While
we have shown that the HMR silencers are in close proximity to
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