Long Range Communication Between The Silencers Of HMR Lourdes ...
MCB Accepts, published online ahead of print on 14 January 2008
Mol. Cell. Biol. doi:10.1128/MCB.01647-07
Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Long-range communication between the silencers of HMR
Lourdes Valenzuela1, Namrita Dhillon1, Rudra N. Dubey3, Marc. R Gartenberg3 and
Rohinton T. Kamakaka1,2
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2 Corresponding Author
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3 Department of Pharmacology
683 Hoes Lane
UMDNJ - Robert Wood Johnson Medical School
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Piscataway, NJ 08854
1 Department of MCD Biology
Sinsheimer Labs
1156 High Street
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University of California
Santa Cruz, CA 95064
e-mail: rohinton@biology.ucsc.edu
Abstract
Gene regulation involves long-range communication between silencers, enhancers
and promoters. In Saccharomyces 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
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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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2
Introduction
Gene activation and gene repression is central to the proper development and
differentiation of organisms. DNA elements such as promoters, enhancers and silencers
play a central role in eukaryotic gene regulation. These elements are separated from each
other by several kilobase pairs of DNA but are able to communicate with one another to
regulate the activation or repression of genes. The exact mechanism by which distally
located elements communicate with one another is not clear and is one of the key
questions in gene regulation. Long-range communication between distantly located
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elements in chromosomes is thought to occur by one of two principle mechanisms (8).
One class of models postulate that a signal emanating from a distal regulatory element
spreads along the DNA fiber until it encounters a proximal regulatory element. A second
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class of model postulates that distal and proximal regulatory elements interact with one
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another directly with the intervening DNA forming a loop. Both mechanisms must
function within the context of global chromosome structure, which appears to be
composed of large chromosome loops that attach to a proteinaceous superstructure (11).
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The nucleus appears to be divided further into distinct chromatin compartments with
heterochromatic domains being present in regions near the nuclear periphery while
euchromatic domains are mainly found in the interior of the nucleus, although a
significant portion of euchromatin is located near nuclear pores.
It has been suggested that the functionally and structurally defined chromatin
domains may be coincident (36). Enhancers and locus control regions (LCRs) are long-
range regulatory elements that activate promoters in a distance and orientation
independent manner and recent studies indicate that enhancers and LCRs often cluster
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together in three dimensional space to form an “active chromatin hub” (23, 49, 58, 59).
Similarly in yeast the promoters and terminators of genes are in close proximity to one
another (3, 46) and tethered to the nuclear pore (10, 52). The consequence of this spatial
organization is that the DNA between these regulatory elements is looped out. It is
thought that the formation of these nuclear sub-structures aids in transcription activation.
Silencers are negative regulatory elements composed of binding sites for various
factors that act collectively in the establishment and stable inheritance of a repressed
state. Like enhancers, silencers repress promoters in a distance and orientation
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independent manner (35). Silencers flank the silenced HML and HMR mating-type loci in
yeast while at telomeres the terminal repeated TG1-3 sequences serve as silencers. These
silencers recruit the Sir proteins, Sir2p, Sir3p and Sir4p, which then spread across several
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kilobase pairs of DNA via interactions with histones. Thus our current understanding of
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Sir-mediated repression is that it is an example of long-range effects mediated via
transmission along the DNA fiber rather than direct long-range interactions between the
silencers (60).
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DNA elements that restrict the action of long-distance regulatory elements, like
silencers and enhancers, are generically called insulators. Insulators located between an
enhancer and a promoter (called enhancer blockers) disrupt enhancer-promoter
communication and prevent the enhancer from activating that promoter while insulators
located between a silencer and a promoter (called barriers) block the silencer from
repressing the promoter. Numerous models have been proposed to explain how insulators
function to block long-range communication. Some models postulate that insulators act
as decoys, forming non-productive interactions with distal regulatory complexes or
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sequester these complexes in specific regions of the nucleus while other models suggest
that insulators function locally by disrupting the propagation of a specific chromatin
domain (60).
In this manuscript we present evidence demonstrating that while silencers function
via recruitment and transmission of Sir proteins along the DNA, they also directly
communicate with each other. Functional analyses of silencer-mediated repression
suggest that silencer elements communicate with one another in mediating repression in
the nucleus. Our studies also show that DNA fragments containing silencers (separated
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by several kilo-base pairs of DNA) are in close spatial proximity in the nucleus and likely
form chromatin loops. Interestingly, this long-range communication was lost in mutations
in the Sir proteins. Our results suggest that silenced domains are formed by the spreading
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of repressor proteins from silencers that interact with one another enabling compaction of
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the chromatin fiber and stable repression. These results are similar to the long-range
interactions between LCR’s and promoters and suggest conservation in the mechanism
by which genes are activated and repressed.
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5
Results
Silencers flank silenced genes at HML and HMR. At HML, silencing initiates from
both silencers but at HMR silencing only initiates at the HMR-E silencer. Previous work
from our lab showed that tethering proteins with barrier activity near HMR-E blocked the
spread of silencing from the HMR-E silencer if the HMR-I silencer was absent (21).
However the barrier could be bypassed, if a second silencer (HMR-I) was positioned
downstream of the barrier (supplementary figure 1) (21, 48). One explanation for this
phenomenon is that silencing nucleates at HMR-I, as well as HMR-E. However functional
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data clearly indicates that HMR-I only augments the activity of HMR-E and does not
possess an autonomous silencing activity (1, 7, 50).
Since our analyses suggested that silencing at HMR might be initiating at HMR-I, we
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reasoned that if HMR-I was a silencer, then it should be able to recruit at least some Sir
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proteins in the absence of the HMR-E silencer. To directly test this possibility, we used
chromatin immunoprecipitation to examine the binding of Sir3p near HMR-E and HMR-I
in a variety of HMR variants. As expected, Sir3p localized to the two silencers in the
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wild-type strain (Figure 1A). Deleting HMR-I did not affect Sir3p levels at the HMR-E
silencer, but deleting HMR-E resulted in loss of Sir3p localization from the HMR locus
but not the telomeres. In the absence of HMR-E, the levels of Sir3p at HMR were
equivalent to those observed at the negative control the TEL6R 7.5kb probe, where Sir3p
has not been found previously. Therefore these results, at this level of sensitivity,
demonstrate that HMR-I does not recruit Sir proteins in the absence of HMR-E and are
consistent with previous functional results (1, 7, 50).
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These results lead to an alternative explanation that HMR-E “communicates” with
HMR-I to facilitate silencing at a distance. To explore this paradoxical phenomenon
further, we monitored silencing of a dual reporter system known as the boundary trap
assay that was developed and used to investigate insulator proteins at HML (33). In this
assay, the silenced locus was modified and the mating type genes were replaced with
ADE2 and URA3 genes. Gal4 binding sites flank the ADE2 gene whereas a second
reporter, URA3 is not flanked by these sites and resides adjacent to the I silencer (see
figure 1B and 1C). The assay monitors the ability of a protein tethered to the Gal4p
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binding sites to insulate the ADE2 gene from repression but not the neighboring URA3
gene.
The dual reporter system was first used at HML. Unlike HMR, at HML both silencers
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are able to independently initiate silencing (Figure 1B). We tested the behavior of Gal4-
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Nup2p and Gal4-Sas2p. Nuclear pore proteins like Nup2p were claimed to be “true
barrier” proteins that can insulate the ADE2 gene while maintaining the neighboring
URA3 gene in a silenced state (33). We also tested Gal4-Sas2p since acetyltransferases
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are believed to function by a “desilencing” mechanism. We measured expression of
ADE2 by growth on medium lacking adenine and we measured expression of URA3 by
growth on medium containing 5-FOA. Cells expressing URA3 convert 5-FOA to a toxic
metabolite and die. We used these assays because they are far more sensitive to changes
in the expression levels than northern blots. Furthermore these assays allow us to
determine the mitotic stability of these epigenetic states. Our results shown in figure 1B
are consistent with previously published data (33). Gal4-Nup2p insulated ADE2 from
repression while allowing URA3 to be stably repressed in a small percentage of cells. On
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the other hand, Gal4-Sas2p derepressed both ADE2 and URA3, presumably by disrupting
silencing across the entire silenced domain.
We next constructed a dual reporter system at HMR similar to the system at HML,
placing the ADE2 gene near HMR-E and the URA3 gene near HMR-I (Figure 1C). The
strain was transformed with Gal4-Nup2p, Gal4-Sas2p or vector alone. The cell growth
assays in figure 1C clearly show that Gal4-Nup2 functions again as a true barrier,
producing colonies of cells in which ADE2 was active but URA3 was stably repressed
(see panels labeled –ADE +5-FOA). However unlike the situation at HML, Gal4-Sas2p
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de-repressed ADE2 expression while permitting URA3 repression at HMR. Thus, Gal4-
Sas2p also functions as a “true barrier” at HMR. Importantly, the ability of cells to form
colonies on media lacking adenine but containing 5-FOA indicates that the
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“discontinuous” silenced state that is established in these cells is stably inherited for
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several generations enabling these cells to form colonies. A dual reporter system
containing ADE2 and MATa1 yielded similar results, suggesting that this effect was not
reporter specific (supplementary figure 2). Furthermore, the fact that Sas2p, a bona fide
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histone acetyltransferase which is expected to behave as a “desilencer” can function as a
“true barrier” protein indicates that the molecular underpinnings for these definitions will
need to be reconsidered.
We next determined whether the generation of the discontinuous silenced state
required HMR-I. We deleted the HMR-I silencer from the boundary-trap strain at HMR
and analyzed the ability of these strains to grow on media lacking adenine but containing
5-FOA. Our results (Figure 2A) showed that silencing of the URA3 gene in the dual
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reporter system at HMR required HMR-I. When this silencer was removed, no colonies
formed on the plates lacking adenine and containing 5-FOA.
This result demonstrates that the Sir proteins recruited at HMR-E can only transpose
across an active domain when a silencer is present on either side of this domain. These
results with the dual reporter systems are concordant with our earlier studies of single
tethered barrier proteins (48). Silencing adjacent to HMR-I requires the HMR-I silencer if
a barrier blocks the action of HMR-E.
To explore the discontinuous silencing phenomenon at a molecular level, we mapped
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the distribution of Sir3p and H4AcK16 across the HMR domain in the presence and
absence of Gal4-Sas2p. We chose to analyze these two proteins since they are markers of
active and inactive chromatin (47). In the absence of Gal4-Sas2p, there is no acetylation
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at either silencer or the ADE2 gene (Figure 2B). When Sas2p is recruited to sites flanking
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the ADE2 gene there is no detectable acetylation at the HMR-E and HMR-I silencers but
there is a significant increase in H4K16 acetylation at the ADE2 gene consistent with the
observation that ADE2 is active in these cells.
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On the other hand, Sir3p was present at the two silencers, both in the presence and
absence of Gal4-Sas2p but was reduced at the ADE2 gene when Sas2p was tethered at
the Gal4p binding sites flanking the ADE2 gene. Thus, tethered Gal4-Sas2p does not
block the normal function of the two silencers and the growth phenotypes observed in
figure 1 are indeed due to discontinuous silenced domains that initiate from HMR-E.
Interestingly, we consistently see increased levels of Sir3p at both the HMR-E and
HMR-I silencers compared with the ADE2 gene, in the presence or absence of Gal4-
Sas2p. Our results demonstrate that HMR-I alone cannot recruit Sir proteins, but in the
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presence of HMR-E is able to stably maintain elevated levels of Sir proteins. While the
reason for the elevated levels of Sir3p at HMR-E can be explained by the fact that HMR-
E recruits the Sir proteins and initiates silencing, the reason for the elevated levels at
HMR-I were unexpected and not initially obvious. One possibility is that the increased
levels of Sir proteins at HMR-I may be due to the two silencers being in close proximity
to one another.
Rap1p localizes to HMR-I
Our data indicates that HMR-E functionally communicates with HMR-I resulting in a
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discontinuous silenced domain but it does not specify how this might occur. One
possibility is that the two silencers reside in close proximity to one another.
To confirm these long-range interactions we asked if a DNA bound protein at one end
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of the domain was in close proximity to the other end of the domain similar to the
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experiments used to show long-range interactions in Drosophila (5). There is a single
binding site for Rap1p at the HMR-E silencer where the protein has been shown to bind
(53). No Rap1p sites are known to exist at HMR-I. We used ChIP to map the presence of
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Rap1p at HMR, as well as at loci on chromosome 6R (Figure 3A). While we did not
observe significant binding of Rap1p to the telomere 6R 7.5 kb probe, the quantitative
analyses showed that Rap1p was present immediately adjacent to telomere 6R and at
HMR. At HMR we observed Rap1p binding to HMR-E, the silenced MATa1 gene at HMR
and at HMR-I. This result was obtained with two different commercially available
antibodies (data not shown) validating the presence of Rap1p at HMR silencers.
We next determined if loss of Sir3p affected the distribution of Rap1p. Loss of Sir3p
did not lead to any decrease in the amount of Rap1p at HMR-E but there was a complete
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loss of Rap1p from HMR-I. These results are consistent with the observation that HMR-E
was in close proximity to HMR-I, though it is also possible that despite Rap1p being a
sequence specific DNA binding protein, it spread along the silenced chromatin through
interactions with the Sir proteins.
“Enhancer” activity at HMR
To investigate the spatial localization of the silencers relative to each other we
decided to develop an “enhancer assay” (see figure 3B). The assay is premised on the
assumption that when an UAS is brought in close spatial proximity to the promoter of a
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repressed gene, it will activate that gene. We placed Gal4p binding sites upstream of
HMR-E, and placed a repressed reporter gene (URA3) several kilobase pairs (4 kb)
downstream from the Gal4p binding sites on the distal side of HMR, between HMR-I and
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the tRNA barrier. Transcription of the reporter was directed towards the Gal4p binding
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sites (Figure 3B). At this location, the URA3 gene was subjected to repression by Sir-
mediated silent chromatin and in the absence of the Sir proteins, URA3 was active in both
glucose and galactose (see supplementary figure 3). Interestingly, stable repression of
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URA3 located downstream of HMR-I was dependent upon the HMR-I silencer because in
the absence of this silencer, the reporter was not stably silenced anymore (see
supplementary figure 3).
We next tested whether binding of Gal4p to its sites upstream of HMR-E could
disrupt the silencing of the URA3 gene located downstream of HMR-I. When cells were
grown in glucose, Gal4p was not activated and URA3 remained silenced in strains that
contained or lacked Gal4p binding sites (Figure 3C top panel). When these cells were
grown in medium containing galactose, however, URA3 was activated and cells were able
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to grow on medium lacking uracil and not on medium containing 5-FOA (Figure 3C
bottom panel). This result was only observed 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 function. If Gal4p was disrupting
silencing across the entire domain, 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 performing a mating assay and selecting for diploids. The
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appearance of diploids (see mating assay panel in Figure 3C) demonstrated that at this
level of sensitivity, MATa1 was silenced.
We also investigated whether Gal4p binding upstream of HMR was disrupting Sir3p
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binding at HMR by quantitative ChIP. If this were the case then in strains containing
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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 analyses
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(Supplemental Figure 4) showed only slight change in the levels of Sir3p at the two
silencers with no discernible change at MATa1 gene located between the two silencers in
the presence or absence of Gal4p. These results are consistent with our mating assays in
figure 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 an UAS located several kilobase pairs from
the promoter 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
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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 communications
Our results suggested that the two silencers might be in close proximity to one
another; we were interested in determining the DNA elements and factors that affected
this localization. In chicken cells the globin insulator helps tether the globin domain to
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the nucleolus and aids in the formation of chromatin loops (61, 62). In yeast, tRNA genes
(tDNAs) are dispersed throughout the genome but in situ hybridization demonstrated that
the genes are clustered adjacent to the nucleolus (30). One of the HMR barriers is a tRNA
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gene (21) and it is therefore possible that tethering of the HMR barriers to the nucleolus
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might be the mechanism by which the two silencers were brought in close proximity to
one another. A prediction of this model would be that the HMR locus would reside
adjacent to the nucleolus and deletion of the barrier would result in a concomitant loss of
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this localization.
To determine whether the tRNA barrier adjacent to HMR associated with the
nucleolus, we used a cytological approach. A lac operator array (lacop array) was
incorporated adjacent to HMR (inserted approximately 4 kb from HMR) in a strain that
expressed lac-GFP, as well as Sik1-RFP. Lac-GFP binds the lacop array to create a bright
green spot of fluorescence (marking HMR) whereas Sik1p, a nucleolar protein, imparts
red fluorescence to the perinuclear crescent-shaped nucleolus. Stacks of fluorescent
images along the Z-axis using both GFP and rhodamine (red) filters were collected to
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determine the relative positions of HMR and the nucleolus. 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 at least three independent trials. The data in Figure 4A show
that in over 90% of the cases HMR and the nucleolus did not contact one another. The
low level of coincident colocalization is similar to that found for other non-interacting
chromatin landmarks (9, 12). Similar results were found when HMR and the adjacent
barrier were liberated from the chromosome by site-specific recombination to form an
extrachromosomal DNA circle. These results indicated that HMR and the associated
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boundary did not reside at the nucleolus. Furthermore, deleting the barrier (-tRNA) did
not alter the localization of HMR in the nucleus (Figure 4A). It is therefore unlikely that
the mechanism by which silencers are brought in close proximity is via tethering to the
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nucleolus.
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Tethering of insulators to nuclear super-structures has been proposed to be important
for insulation. In Drosophila, the Su(Hw) insulators cluster in the nucleus forming
insulator bodies (28) while in yeast, nuclear pore proteins localize to the silenced
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chromatin (10) and models suggest that tethering of insulators to the pores, forming a
chromatin loop, is the mechanism by which chromatin domains are organized and
maintained (32).
Therefore it was still possible that the barrier insulator elements at HMR were
important for the observed long-range communication between the two silencers albeit
not by tethering to the nucleolus. If barrier elements were necessary for organizing
chromatin domains into loops then loss of a barrier should result in loss or diminution of
long-range communications. Using the enhancer assay we investigated the role of the
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HMR tRNA barrier in this process. We generated two strains, lacking the tRNA barrier
and possessing URA3 immediately downstream of HMR-I. One strain contained Gal4p
binding sites located upstream of HMR-E while the second strain lacked these binding
sites. Monitoring the expression of URA3 in these two strains in galactose showed that
loss of the tRNA barrier did not adversely affect the long-range communication between
the two ends of the silenced chromatin domain (Figure 4B).
It has been suggested that chromatin loops are formed by the attachment of barrier
elements to the nuclear pore via Nup2p (32). Analyses of Nup2p mutants indicate that
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Nup2p is necessary for robust tRNA barrier function but loss of Nup2p did not affect the
long-range communication between the two silencers (G. Ruben and RTK persnl.
commun.).
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HMR-E and HMR-I are in close spatial proximity
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All of our analyses described thus far suggested that the two silencers were in close
spatial proximity to one another. We therefore directly analyzed the spatial relationships
at the native HMR locus in the yeast nucleus using the chromosome conformation capture
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(3C) method developed to investigate the three-dimensional relationships between DNA
elements (15, 18). Cells were briefly treated with formaldehyde to crosslink DNA to
proteins, followed by cleavage with a specific restriction enzyme- we digested the DNA
with Sau3A since this enzyme generated small fragments (55). The fragments were then
diluted and ligated, such that ligations were primarily between cross-linked DNA
fragments. The fragments that ligated to one another were identified using PCR with
specific pairs of primers. Using this method, the cross-linking frequency between two
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restriction fragments is expected to be roughly proportional to their proximity to one
another in the nucleus.
The DNA between the HMR-E and HMR-I silencers does not contain many Sau3A
sites. To improve the resolution of our analyses we introduced three additional Sau3A
sites within this region (in the MATa2 gene). Furthermore, since regions at HMR are
homologous to regions at HMLα and MAT, we performed this analysis in a strain where
these two loci were deleted. All of the primers we used in this analysis were oriented in
the same direction. Therefore a PCR product can only arise after restriction fragments
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were digested and religated. This eliminated PCR products that might arise from
incomplete digestion of the cross-linked samples.
We initially analyzed the ligations with a fixed oligonucleotide located in a Sau3A
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fragment containing the HMR-E silencer (Primer-A) with restriction fragments that
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encompassed the silenced domain and beyond to determine which fragments were in
close proximity to the reference fragment. Our analyses showed that the HMR-E
fragment ligated most frequently to a single Sau3A fragment (amplified with Primer-G)
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containing the HMR-I silencer (Figure 5A).
To ensure that the PCR amplification efficiency between different primer pairs was
comparable, we digested plasmid DNA containing the HMR locus with Sau3A in the
absence of cross-linking, followed by ligation under conditions that favored
intermolecular ligation. PCR analyses indicated that all of the primer pairs were
approximately equally efficient in amplifying the ligated products (see panel primer
control, Figure 5A). We confirmed the equivalent PCR amplification efficiencies of the
primers used by two-fold serial dilutions of the uncrosslinked (inter-molecular) ligation
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reaction prior to PCR (Supplementary figure 5). These data showed that the differences in
amplification observed across the HMR domain, in the nucleus, were due to differences
in cross-linking/ligation of various fragments to the reference fragment (HMR-E) and not
due to differences in PCR amplification. As an additional control, we purified the PCR
products obtained from the cross-linked nuclear samples and sequenced them to
unambiguously determine the identity of the ligated fragments (data not shown).
We next used the primer in the Sau3A fragment containing HMR-I (Primer-G) as the
reference primer and assayed the proximity of this fragment to other fragments across
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HMR. Our results, shown in figure 5A, revealed that the HMR-I silencer-containing
fragment ligated most frequently to the HMR-E silencer-containing fragment (Primer-A)
as well as to a fragment downstream of HMR-E (Primer-C) that harbored the end of the
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MATa2 gene. From these data we inferred that HMR-I was in close proximity to HMR-E
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and the 3’ end of the MATa2 gene. It is possible that HMR-I is in close proximity with
both fragments simultaneously or exchanges rapidly between these two fragments.
The 3C method was initially used to demonstrate the proximity between various
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centromeres in yeast (18). We also tested this interaction with our cross-linked samples.
Consistent with previously published data we found that EcoRI fragments at Cen IV
(Primer 14) ligated only to fragments at Cen III (Primer 6) but not to other chromosome
III EcoRI fragments (Primers 5 and 7) (Supplementary Figure 6).
Long-range interactions require silencing
Our results suggest that long-range communication between the two HMR silencers
was not a fortuitous result of the clustering or long-range interactions between the barrier
elements that flank the two silencers at HMR. Silencing at HMR utilizes the Sir proteins
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that interact with the chromatin to mediate silencing. We therefore investigated whether
the communication between the two silencers was a result of silencing. We determined
whether the long-range interaction between the two silencers was disrupted in a sir3∆
mutant by 3C analyses. Our results showed that in the absence of this repressor, the
extent of ligation between the HMR-E and HMR-I containing Sau3A fragments were
dramatically reduced (Figure 5B), suggesting that these long-range interactions required
the Sir proteins.
Since HMR-E no longer ligated to HMR-I in a sir3∆, to ensure the cross-linking and
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ligation were normal in this sample, we examined the ligation between the centromeric
fragments, as controls, since Sir3p is not present at yeast centromeres and mutants in
Sir3p have not been shown to affect centromere function. Our analysis of centromeric
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chromatin demonstrated that indeed CEN III remained in close proximity to CEN IV in
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the absence of Sir3p (data not shown).
Our results in their totality demonstrate that silencers separated over several kilo-base
pairs of DNA functionally and structurally interact with one another. We have begun to
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identify the determinants necessary for these interactions and show that the Sir proteins
are necessary for this long-range communications between silencers.
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Discussion
The control of eukaryotic gene expression involves communication between
regulatory elements that are often separated by great distances. There are now numerous
examples of distal enhancers and LCRs that contact the genes they activate (reviewed in
(60)). More recent studies have even found interactions between regulatory elements and
genes that reside on entirely different chromosomes (39, 54). In this report for the first
time we show long-range interactions between the silencers that flank the HMR locus in
yeast and we identify the determinants required for these interactions. Interestingly, we
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have shown that deletion of the tRNA insulator element or the nuclear pore protein
Nup2p did not affect functional long-range communication between the two silencers but
loss of Sir3p did result in diminution of these interactions suggesting that silencing itself
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may be important for organizing this chromatin domain.
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Silencers and the mechanisms for silencing
Numerous studies have shown that HMR-E is sufficient to nucleate silencing at HMR.
HMR-I cooperates with HMR-E but it cannot initiate silencing on its own (42, 50).
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Therefore, HMR-I is analogous to proto-silencers that have been found at telomeres and
HML (6, 13). At telomeres, proto-silencers and proteins with barrier activity are
interspersed in the subtelomeric blocks to yield domains of discontinuous silencing (24,
25, 38) and these functional assays have led to models where the proto-silencer elements
might interact with one another, but direct long-range interactions have not been
demonstrated at telomeric loci using the 3C technique. Long-range interactions between
the proto-silencers and terminal telomeric sequences, which function as silencers, may
indeed be occurring similar to what we observe at HMR. The distribution of terminally
19
bound Rap1 and Ku at telomeres is consistent with telomere loop formation (43, 56), and
enhancer assays like the one we have employed in this report have also suggested looping
within silent chromatin at telomeres (16). However this is the first report to
unambiguously describe long-range interactions and the formation of chromatin loops at
an internal locus in yeast.
Placement of the dual reporter constructs of the boundary trap assay at the silent
mating-type loci created discontinuous silencing states. At HMR, Sir3p was found at both
silencers but was consistently reduced at the insulated reporter gene in between. In
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agreement, the H4K16Ac mark for active chromatin was found in a reciprocal pattern.
How can a discontinuous state be created when HMR-I does not function on its own? One
possibility is that both silencers are held in close proximity so that HMR-I shares the
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nucleation activity of the more potent HMR-E silencer. In this case, silent chromatin
by on February 10, 2010
would nucleate at both silencers and spread from both until encountering synthetic (or
natural) barriers. Thus, when a pair of barrier proteins is situated between HMR-E and
HMR-I, a domain of active chromatin will reside between domains of silent chromatin.
ACCEPTED
An alternative possibility is that the insulated ADE2 construct counteracts silent
chromatin that has spread from a sole nucleation point at HMR-E. In this scenario, ADE2
activation would occur following a cell cycle event, such as DNA replication, that
compromises silencing efficiency (4). Silent chromatin would persist on both sides of the
activated domain because the silencers stabilized the existing repressed state (2, 13, 14,
42, 50). However it is hard to visualize how this mechanism lends itself to stable
inheritance (which we observe in our assays). An alternative model that combines these
two scenarios is possible where binding of Sir proteins to HMR-E facilitates interactions
20
between HMR-E and HMR-I. Once HMR-I is brought in proximity to HMR-E, it can also
nucleate silencing which then would spread from both silencers.
Sir3p and long-range repression
The long-range communication between the two silencer fragments is dependent
upon Sir3p. Sir3p is a structural repressor protein that binds the histones in nucleosomes
to mediate repression. In vitro binding studies with oligonucleosomes have shown that
Sir3p oligomers binds multiple chromatin fragments and “cross-links” nucleosomal
arrays (27, 29, 40). It is therefore possible that Sir3p binding to chromatin cross-links the
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silenced domain and the resulting compaction brings distal sites together. The
dependence on Sir3p is reminiscent of long-range repression in Drosophila where
Polycomb-mediated repression involves interactions between chromatin memory module
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elements and distally repressed promoters via the association of polycomb group proteins
by on February 10, 2010
(17, 44). We note, however, that Sir3p is not sufficient to hold HMR loci on sister
chromatids together (12). Instead, silent chromatin recruits cohesin, which mediates
pairing of the twin silent chromatin domains. Additional factors could similarly facilitate
ACCEPTED
interactions between distal silent chromatin segments within the same chromatin fiber.
Regulatory elements important for long-range communication
An interaction specifically between the two silencer containing DNA fragments raises
the question of which DNA element if any, are required. Our results with the boundary
trap system demonstrate that HMR-I is necessary. Preliminary data using the 3C
technique also suggest that the silencer elements are necessary. ORC and Abf1p bind
HMR-I and might be involved in mediating these long-range interactions. Rap1p might
also aid long-range communication. When bound to two sites on naked DNA, the protein
21
induces loop formation (31). Further experiments will be necessary to dissect the role of
these elements and proteins in long-range communication.
In an alternative scenario, the two silencers could be brought in close proximity by
insulator elements that flank the silenced domain. It has been suggested that chromatin
loops are formed through association of insulator elements with the nuclear pores (32, 33,
52). It is therefore possible that the barrier elements flanking HMR (21, 22) or the
hypersensitive sites at this locus (45) associate with nuclear pores or active chromatin
hubs and cluster in the nucleus, the consequence of which would be to align HMR-E and
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HMR-I in close proximity. However we have shown that deleting the barrier or Nup2p,
which interacts with the barrier, had little effect on long-range communication between
the silencers. It therefore seems unlikely that chromatin barriers play a major role in the
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observed interactions between HMR-E and HMR-I.
by on February 10, 2010
Sir spreading, long-range interactions and silencing
While we have shown that the HMR silencers are in close proximity to one another, it
is unclear if this long-range communication between silencers has any functional
ACCEPTED
significance at the native locus. It is possible that the two silencers are fortuitously
brought in close proximity to one another simply by the compaction of chromatin.
However data show that the HMR-I silencer is important for stabilizing the repressed
state (50). In its absence, the silent state is reduced and silencing is weakened in a
population of cells. Importantly, our data with the boundary trap assay showed that HMR-
I becomes an essential silencing element when propagation of silent chromatin from
HMR-E is blocked. One possibility, as described above, is that an interaction between the
silencers permits the robust nucleation activity of HMR-E to act locally via a spreading
22
mechanism as well as distally at HMR-I via long-range interactions. Another possibility,
that is not mutually exclusive, is that silencers associate with one another and with
telomeres to bring both ends of the silenced domain into a nuclear compartment that
favors silencing (Figure 5C). HMR resides at the nuclear periphery, and frequently
colocalizes with the highly concentrated foci of Sir proteins associated with telomeres
(26, 57). Consistent with this is the demonstration that telomere 3L is in proximity to the
silenced HML locus (37). The clustering of regulatory elements in the nucleus is a
recurring theme utilized by a variety of elements (60) to robustly activate and repress
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genes. It is thought to increase the local concentrations of proteins and thus improve the
probability that these elements will function efficiently (19).
The role of the Sir proteins in this scenario would be to stabilize the long-range
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interactions that arise due to tethering of the silencers. This would be consistent with in
by on February 10, 2010
vitro evidence showing that the Sir proteins preferentially bind two DNA fragments (27,
29) and our 3C data showing that the long-range interactions are lost in the absence of
Sir3p. This role for the Sir proteins is also consistent with the observation that loss of
ACCEPTED
silencing affects telomere-telomere interactions and telomeric foci formation in yeast.
Besides playing a role in tethering the HMR domain to telomeric foci, the role of the
silencers would also be to nucleate the spread of Sir proteins (41, 51). Our quantitative
analysis demonstrating increased concentrations of Sir proteins at silencers with reduced
levels of these proteins the further one traverses from the silencer would be consistent
with this role for the silencers thought other models are equally possible.
The mechanism by which long-range silencing occurs may be more complicated than
previously anticipated. Interactions between silencer elements mediated by the repressor
23
proteins may compete with interactions between enhancer and promoters to affect the
three-dimensional organization and functional status of the nucleus. Further studies
should help understand the significance of long-range interactions in chromatin domain
organization and gene regulation.
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by on February 10, 2010
ACCEPTED
24
Figure Legends
Figure 1
Boundary trap assays at HMR and HML
A) HMR-I does not recruit Sir3p
Chromatin immunoprecipitation 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.
B) At HML, only Nup2 allows discontinuous silencing.
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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 media (Hartwell
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complete (HC)- tryptophan) and expression of the ADE2 and URA3 genes was monitored
by on February 10, 2010
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 red allows
differentiation between “true barrier” proteins and “desilencing” proteins.
ACCEPTED
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 µg 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 ug per ml of adenine and allowed to grow at 30°C prior to photography.
25
The panel labeled in red allows differentiation between “true barrier” proteins and
“desilencing” proteins.
Figure 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 in Figure
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1C.
B) Mapping the distribution of acetylated histones and Sir3p in the boundary trap
constructs
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Strains constitutively expressing Gbd-Sas2p or vector alone were grown in HC-trp media
by on February 10, 2010
selecting for the plasmids (Media was supplemented with 90 ug/ml adenine). Chromatin
immunoprecipitation with antibodies against H4K16Ac or Sir3p were performed exactly
as previously described (47). The graphs depict the fold enrichment of the
ACCEPTED
immunoprecipitated sample over the input normalized to a telomeric probe. Fold
enrichment and SE values 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 figure.
Figure 3
Functional long-range communication at HMR
A) Mapping Rap1p at HMR
26
Antibodies against Rap1p C-terminus were used to map the distribution of Rap1p across
the HMR locus in a wild type and sir3∆ strain. Quantitative ChIPs were performed as
described in Figure 2. The PCR probes used are shown in the schematic diagram.
B) Schematic representation of the enhancer construct at HMR. The location of Gal4p
binding sites and the URA3 gene are shown.
C) “Enhancer” activity at HMR
Strains with URA3 located downstream of HMR-I containing no Gal4 binding sites (-
Gbs) or 4xGbs upstream of HMR-E (+Gbs), were grown in 5 ml YPD overnight. Cells
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were washed and 5-fold serial dilutions were prepared. Properly supplemented YM plates
containing 2% galactose (YMG) or 2% glucose (YMD), as carbon source, were used to
induce or to repress expression of Gal4p respectively. To assay for expression of URA3,
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cells were spotted onto YMG or YMD plates lacking or containing uracil or 5-FOA, and
by on February 10, 2010
photographed. To assay for expression of MATa1 at HMR, cells were spotted onto
properly supplemented YMG or YMD plates with mating lawns.
Figure 4
ACCEPTED
Long-range communication and barrier function
A) HMR does not colocalize with the nucleolus.
Fluorescence analysis was performed in strains expressing Sik1-RFP and lac-GFP. A
lacop 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 2 independent trials.
27
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 4 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
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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.
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Figure 5.
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Spatial organization at HMR
A) Chromosome conformation capture analysis of HMR
Wild type strains (lacking HML and MAT) were cross-linked, digested with Sau3A,
ACCEPTED
ligated and the ligation products were analyzed by PCR (labeled 3C). Reference primers
A or G were used along with the other primers across the domain. The location and
orientation of the primers is shown schematically. The primer control panel involved
inter-molecular ligations and PCR analyses of uncrosslinked DNA.
B) Loss of Sir3p affects long-range interactions.
Strains with a deletion of the SIR3 gene were analyzed by the chromosome conformation
capture assay as described above.
C) Schematic representation of the HMR domain in the yeast nucleus.
28
Materials and Methods
The genotypes of the strains used in this study are shown in supplementary table 1
The oligonucleotides used in this study are listed in supplementary table 2.
The exact sequences of the various integrations generated and used in this study are
listed in supplementary table 3.
Yeast strains
Yeast genomic integrations were performed by homologous recombination and gene
replacement, using PCR products or DNA fragments derived from plasmids. Yeast
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transformations used the lithium acetate method (34). PCR amplifications were carried
out with Expand High Fidelity DNA polymerase, and integrations were confirmed by
PCR and sequencing analysis. SIR2, SIR3, PPR1 and ADE2 genes were deleted from the
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start to the stop codon and replaced with HIS3 or kanMX markers. Deletion of MATα was
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obtained by replacing MATα2 and MATα1 sequences (SGD coordinates 199731 to
200964) with the kanMX cassette.
The ADE2 gene flanked by Gal4p binding sites (Gbs), present at HML, in strain
ACCEPTED
KIY54 (32), was PCR amplified with appropriate primers and integrated at the HMR
locus in a sir2∆ strain (JRY4576) or in a HMR∆I sir4∆ strain (ROY926). Sequencing of
the PCR products indicated that a single Gal4p binding site, which is contrary to
published results (32), flanked ADE2. The PCR product was integrated in the HMRa2
coding region (SGD coordinates 293212 to 293410) with the ADE2 promoter close to the
HMR-E silencer. Strains with the integrated ADE2 gene (ROY2729: MATα HMR::Gbs-
ADE2-Gbs sir2∆ and ROY2914: MATα HMR-E-Gbs-ADE2-Gbs-HMR∆I sir4∆) were
crossed with a W303 wild type strain to obtain ROY2770 and ROY3001 respectively.
31
The HMRa1 coding region in ROY2729 and ROY2914 was replaced by URA3
coding region by homologous recombination, and transformants were crossed with an
ade2∆::kanMX strain to obtain ROY3182 and ROY 3194 respectively.
Plasmid pJR1270 contains an EcoRI-HindIII fragment with the HMR locus where the
HMR-I silencer has been deleted. This fragment contains two SpeI sites. The plasmid was
partially digested with SpeI, end-filled and religated to obtain pRO698, which contains
only one SpeI site 290 bp upstream of the ARS element present at HMR-E. A pair of
oligonucleotides with 4xGbs flanked by SpeI sites were annealed, digested and cloned
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into the SpeI site in pRO698 to produce pRO700. The EcoRI-BglII fragment from
pRO700 was used to replace the HMR region in strain ROY2585 (HMRa2::URA3 sir2∆),
to give strain ROY3285 (Gbs-HMR sir2∆).
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The EcoRI-HindIII fragment in pRO700 was used to replace the HMR region in ROY
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2585 to produce ROY3283 (Gbs-HMR∆I sir2∆). The URA3 cassette was integrated
between HMR-I or HMR∆I and the tRNA gene (SGD coordinates 295070 to 295281) in
strains JRY4566 (W303 sir2∆) and ROY3285, ROY 3550 (HMR∆I sir2∆) and ROY
ACCEPTED
3283 and the transformants were crossed with a ppr1 ∆::kanMX strain to obtain
ROY3495- ROY3498, 3489, 3683, 3699, 3680 and 3697. ROY 3495 and 3497 were
crossed with a Gal4-TAP tagged strain to obtain ROY4371 and 4372 respectively.
The tRNA gene was deleted and the URA3 gene integrated in strain JRY4566 and
ROY 3285 by PCR mediated gene replacement. Transformants were then crossed with a
ppr1∆ strain to obtain ROY 3688, 3703, 3686 and 3701.
Plasmid pJR1571 contains an EcoRI-HindIII fragment comprising the HMR locus.
Oligonucleotides with three new Sau3A sites were used to PCR amplify the XbaI-EcoNI
32
fragment of the MATa2 gene. The PCR product was cloned into the XbaI-EcoNI
fragment of HMRa2 in plasmid pJR1571. The EcoRI-HindIII fragment of the new
construct (pOS154) was used to replace the HMR locus in strain ROY2800
(HMRa2::URA3), and transformants were crossed with matα∆::kanMX, sir3∆::HIS3 and
hml∆::TRP1 strains to obtain ROY4064 and ROY4065.
Plasmids
Plasmids RO590 and RO635 contained the full length SAS2 or NUP2 coding regions
fused in frame to the Gal4 DNA binding domain (GBD), with transcription driven by the
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ADH1 promoter in the pGBK-RC-TRP1 base plasmid (pGBD) (48).
Serial dilutions
Yeast cells were grown overnight at 30°C in 5 ml YPAD or HC (Hartwell’s
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complete) medium without tryptophan to allow maintenance of the plasmids. Cells were
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diluted to a concentration of 1.0 A600/ml in HC-trp medium and serially diluted five or
ten-fold. Using a cell spotter, approximately 3 µl of each serial dilution were placed onto
properly supplemented HC plates to assay for ADE2 and URA3 expression, or onto
ACCEPTED
properly supplemented YMD plates previously spread with 1.0 O.D. A600 of mating lawn
(strain JRY19a) diluted in 300 µl of YPD, for the mating assays. For the mating assays,
selection for plasmids was maintained. The plates were incubated at 30°C and
photographed. Cells grown in limiting amounts of adenine were kept at 4°C for two
additional days for development of the color, prior to photography.
Chromatin Immunoprecipitation
Quantitative ChIP analysis was performed as previously described (47), with minor
modifications. The program for the PCR was as follows: 95o C for 3 min (1 cycle), 95 o C
33
for 1 sec, 52oC for 30 sec, and 72oC for 1 min (45 cycles). The fold enrichment was
Ct(IP)-Ct(input)
calculated using the formula 2
as described in Litt et al., 2001 and was
normalized to the telomeric probe. For Rap1p immunoprecipitation, polyclonal
antibodies were used (Y300, Santa Cruz Biotechnology, Inc) while antibodies against
H4K16Ac were purchased from Upstate.
Chromosome conformation capture
The 3C analyses of yeast strains were performed exactly as described (18) with a few
specific changes. Each strain in each experiment was cross-linked for 0, 5, 10 and 20 min
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and each sample was independently processed and analyzed. The restriction enzyme used
for the digestion was Sau3A, and the digestion buffers were as recommended by the
manufacturer of the enzyme. All primers used were tested with uncrosslinked/ligated
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DNA and only primers with equal amplification efficiencies were used for the 3C
by on February 10, 2010
analyses.
Fluorescence analysis
Two diploid strains were constructed to visualize the relative positions of HMR and
ACCEPTED
the nucleolus. The strains differ only by the absence of the tRNA barrier at HMR. The lac
operator array is telomere proximal to HMR-I. Both strains contain Sik1p fused to RFP (a
nucleolar marker), express lac-GFP and contain a galactose inducible R recombinase.
To examine the relative positions of HMR to the nucleolus, the cells were first grown
in SC-trp media containing dextrose. Cells were fixed 2 hours with paraformaldehyde
and mounted on microscope slides containing agar plugs. Parallel Z-stacks were obtained
of cells using both rhodamine and GFP filters to visualize the nucleolus and HMR,
respectively (17 sequential images separated by 0.2 microns). The two landmarks were
34
considered colocalized if the corresponding fluorescence signals fully overlapped within
the same plane or in adjacent planes. The landmarks were considered to touch if contact
(but not overlap) was seen between them within a plane or adjacent planes. The
landmarks were considered to be fully separated if no contact was observed or if image
planes lacking fluorescence separated fluorescent foci in different planes. Multiple fields
of cells were examined for each of the two trials. Cell morphology was used to estimate
the cell cycle stage of each cell examined. However, the same general trends were
observed in G1, S and G2 phase so these data were pooled (G1 and S phase cells were
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well represented, whereas there were considerably fewer G2 cells).
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Acknowledgements
by on February 10, 2010
We would like to thank Genevieve Fourel, David Donze, Grant Hartzog, Michael
Lichten, Orna Cohen-Fix, Masaya Oki and Catherine Fox for comments on the
manuscript and to C. Fox and U. Laemmli for specific strains. This work was supported
ACCEPTED
by a grant from the NIH to RTK (GM078068).
35
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Figure 1A
mcb.asm.org
E
I
by on February 10, 2010
a1
6R 7.5
I
II
6R 0.5
III
Strain:
I II III IV V
α-SIR3
WT
Input
ACCEPTED
α-SIR3
HMR-I only
Input
α-SIR3
HMR-E only
Input
Downloaded from
mcb.asm.org
by on February 10, 2010
Figure 1B
GBS
GBS
HML-E
ADE2
URA3
HML-I
ACCEPTED
+ ADE
- ADE
+ ADE
-ADE
+ URA
+ URA
5-FOA
5-FOA
vector
GBD-NUP2
GBD-SAS2
Downloaded from
mcb.asm.org
Figure 1C
by on February 10, 2010
GBS
GBS
HMR-E
ADE2
URA3
HMR-I
ACCEPTED
+ ADE
- ADE
+ ADE
-ADE
+ URA
+ URA
5-FOA
5-FOA
vector
GBD-NUP2
GBD-SAS2
Downloaded from
mcb.asm.org
by on February 10, 2010
Figure 2A
GBS
GBS
HMR-E
ADE2
URA3
ACCEPTED
+ ADE
- ADE
+ ADE
-ADE
+ URA
+ URA
5-FOA
5-FOA
vector
GBD-NUP2
GBD-SAS2
Figure 2B
40
+ vector
+ pGBD-SAS2
ence 30
H4 AcK16
20
Fold Differ
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10
0
mcb.asm.org
I II III IV V
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200
+ vector
+ pGBD-SAS2
150
ence
Sir3p
ACCEPTED
100
Fold Differ
50
0
I II III IV V
Gbs
E
Gbs
I
Chr 6R
∆
ADE2
∆
I
II
III
IV
V
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Figure 3A
mcb.asm.org
Rap1p
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8
WT
6
sir3 ∆
ence
4
Fold Differ
2
ACCEPTED
0
I II III IV V
E
I
Chr 6R
a1
I
II
III
IV
V
ACS Rap1 Abf1
ACS Abf1
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mcb.asm.org
by on February 10, 2010
Figure 3B
GBS
a2
a1
URA3
HMR-E
HMR-I
?
ACCEPTED
GBS
HMR-E
a2
a1
URA3
HMR-I
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Figure 3C
mcb.asm.org
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∆
Gbs
a1
URA3
HMR-E
HMR-I
GLUCOSE
Mating assay
Growth control
- URA
+ 5-FOA
(-his)
- Gbs
ACCEPTED
+ Gbs
GALACTOSE
- Gbs
+ Gbs
Figure 4A
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No contact
mcb.asm.org
Contact
80
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60
40
ACCEPTED
20
HMR
HMR
HMR
HMR
-tRNA
-tRNA
+tRNA
+tRNA
Chr
Epi
Chr
Epi
RS
RS
E
HMR
I
GIT1
+/- tRNA
Figure 4B
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mcb.asm.org
barrier
∆
Gbs
a1
by on February 10, 2010
URA3
HMR-E
HMR-I
GLUCOSE
Mating assay
Growth control
- URA
+ 5-FOA
(-his)
_ Gbs
+ barrier + Gbs
_ Gbs
ACCEPTED
- barrier
+ Gbs
GALACTOSE
_ Gbs
+ barrier + Gbs
_ Gbs
- barrier
+ Gbs
Figur
Primer
e 5A
-A
+
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Primer-B
Primer-C
A
Primer-E
HMR-E
mcb.asm.org
Primer-F
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Primer-G
Primer-H
C
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Primer-J
3C
Primer
Contr
E
ol
Primer
F
-G +
HMR-I
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G
Primer-A
H
Primer-B
Primer-C
Primer-E
J
Primer-F
Primer-H
Primer-J
3C
Primer
Contr
ol
Figur
e 5B
Primer
Downloaded from
HMR
-A
+
mcb.asm.org
Primer-B
Primer-C
A
Primer-E
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HMR-E
B
Primer-F
Primer-G
C
Primer-H
Primer-J
sir3
∆
E
Primer
ACCEPTED
F
-G +
HMR-I
G
Primer-A
Primer-B
H
Primer-C
Primer-E
J
Primer-F
Primer-H
Primer-J
sir3
∆
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mcb.asm.org
tRNA
tRNA
Silencers
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Telomer e
HMR
ACCEPTED
