Global identication of yeast chromosome interactions using Genome

conformation capture
C.D.M. Rodley
a
, F. Bertels
a
, B. Jones
b
, J.M. OSullivan
a,
*
a
Institute of Molecular Biosciences, Massey University, Albany, New Zealand
b
Centre for Mathematical Biology, Massey University, Albany, New Zealand
a r t i c l e i n f o
Article history:
Received 25 May 2009
Accepted 16 July 2009
Available online 21 July 2009
Keywords:
Genome
Architecture
Organization
Conformation
Spatial arrangement
3C
GCC
Saccharomyces cerevisiae
Capture
a b s t r a c t
The association of chromosomes with each other and other nuclear components plays a critical role in
nuclear organization and genome function. Here, using a novel and generally applicable methodology
(Genome conformation capture [GCC]), we reveal the network of chromosome interactions for the yeast
Saccharomyces cerevisiae. Inter- and intra-chromosomal interactions are non-random and the number of
interactions per open reading frame depends upon the dispensability of the gene product. Chromosomal
interfaces are organized and provide evidence of folding within chromosomes. Interestingly, the genomic
connections also involve the 2 lm plasmid and the mitochondrial genome. Mitochondrial interaction
partners include genes of a-proteobacterial origin and the ribosomal DNA. Organization of the 2 lm plas-
mid aligns two inverted repeats (IR1 and IR2) and displays the stability locus on a prominent loop thus
making it available for plasmid clustering. Our results form the rst global map of chromosomal interac-
tions in a eukaryotic nucleus and demonstrate the highly connected nature of the yeast genome. These
results have signicant implications for understanding eukaryotic genome organization.
2009 Elsevier Inc. All rights reserved.
1. Introduction
Genomes are highly ordered yet dynamic entities in which
chromosomal positions, structures and interactions are controlled
in order to regulate nuclear processes (Dekker, 2008; Miele and
Dekker, 2008 and references therein). Recent research (Mitchell
and Fraser, 2008; Spilianakis et al., 2005) using microscopy (e.g.
Berger et al., 2008; Bolzer et al., 2005) and Chromosome Conforma-
tion Capture strategies (i.e. 3C (Dekker et al., 2002), 4C (Simonis
et al., 2006) and 5C (Dostie et al., 2006)) has begun to unravel
the structural (Guelen et al., 2008) and functional (Spilianakis
et al., 2005) signicance of chromosomal interactions at specic
loci including the b-Globin locus (Dostie et al., 2006; Simonis
et al., 2006), the Igf2/H19 imprinting control region (Ling et al.,
2006; Zhao et al., 2006), and the T
H
2 locus (Spilianakis et al., 2005).
The analysis of each of these loci and ne scale mapping of in-
ter- and intra-chromosomal interactions has required a priori
knowledge of postulated contact point(s). The need for such
knowledge clearly limits global insight into the network-like nat-
ure of chromosomal interactions. Instead, function must be dened
experimentally. Therefore, to accurately study DNADNA interac-
tions requires an approach that is free of any preconceived ideas
of the functions of the interacting sequences. Here, using a novel
and generally applicable strategy (Genome conformation capture
(GCC)), we describe the global chromosomal interaction map for
Saccharomyces cerevisiae.
2. Materials and methods
2.1. Genome conformation capture (GCC)
GCC involves sequencing a DNA fragment library generated by
the intra-molecular ligation of linked restriction fragments pro-
duced from reversibly cross-linked chromatin (Fig. 1). Chromatin
fragmentation is achieved using restriction enzymes (Fig. 1). Once
cleaved, the chromatin is greatly diluted to promote intra-molecu-
lar ligations within single restriction fragments or between multi-
ple fragments that are cross-linked together directly or through a
complex (Fig. 1). Following ligation, the sample is reverse cross-
linked, treated with proteinase K and spiked with pUC19. Finally
the DNA fragments are puried by phenol:chloroform and column
purication to produce a GCC library.
The GCC library is nebulized, prepared, and sequenced accord-
ing to the platform that is being used. Following sequencing, se-
quences are sorted based on the presence or absence of the
restriction site of interest (Fig. S2b). This step is performed if
paired-end sequences are being analyzed, to ensure that composite
1087-1845/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2009.07.006
* Corresponding author. Address: Institute of Natural Sciences, Massey Univer-
sity, Private Bag 102 904, NSMC, Auckland, New Zealand. Fax: +64 9 4418142.
E-mail address: j.m.osullivan@massey.ac.nz (J.M. OSullivan).
Fungal Genetics and Biology 46 (2009) 879886
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sequences are correctly positioned. Following sorting, the se-
quences (or more correctly, the restriction fragments that make
up the sequences) are positioned onto the reference genome
(including all episomes and the pUC19 control using the Topogra-
phy software and either the BLAST (Altschul et al., 1990) or SOAP
(Li et al., 2008) algorithms. Sequences with mismatches are
ignored to ensure positioning delity.
Accurate positioning of query sequences onto the genome
scaffold depends upon sequence length, genome size and com-
plexity. The minimum sequence length is determined using
pUC19. If the query sequence length is too short then pUC19 se-
quences are positioned onto the reference genome resulting in
the identication of pUC19-genomic interactions. Logically the
GCC methodology precludes the formation of any pUC19-genomic
ligation products since samples are spiked with pUC19 after the
ligation step (Fig. 1). In this instance the pUC19 sequence is used
as a bioinformatic control. In silico simulations determined that
15 bp is the minimum length to ensure pUC19 sequences anking
MspI restriction sites do not map onto the yeast genome, in par-
ticular a region on chromosome VII (42,48842,500). However,
GCC analysis identied 13 bp as the minimum yeast (S. cerevisiae)
restriction fragment sequence length that could be accurately
positioned in this analysis, because there were no false interac-
tions linking pUC19 to the yeast genome. Therefore, sequences
anking MspI restriction sites had to be P13 bp to be included
in this analysis.
Accurate positioning is enhanced by the segregation of interac-
tions involving unique and repetitive loci (e.g. sequences that are
positioned to more than one locus). Once positioned, the output
is analyzed according to user dened criteria. Low frequency or
random interactions constitute the background which is removed
during the statistical analysis.
2.2. GCC library preparation
Saccharomyces cerevisiae (BY4741 [Mata his3D1 leu2D0
met15D0 ura3D0]) was cultured (30 C, 160 rpm) to an A
600
= 0.6
in synthetic complete medium containing amino acid supplements
and glucose (2% w/v). Chromatin was prepared using a modied 3C
protocol (supplementary methods; Dekker et al., 2002; OSullivan
et al., 2009). The GCC library was prepared from 1.425 10
9
expo-
nentially growing yeast cells using the restriction enzyme MspI,
and a total of 5 lg DNA was sent for sequencing on the Illumina
Genome Analyser (Allan Wilson Centre, Massey University) to en-
sure the network was representative of the chromosomal interac-
tions within the population.
2.3. GCC network assembly
The GCC network was assembled from a total of 8,434,102 se-
quences of 36 bp in length. Sequences have been deposited in Gene
Expression Omnibus under accession number (GSE13648). Se-
quences were processed using the Flp_spin_man suite of programs
(supplementary information). 160,169 sequences (1.9%) contained
an MspI restriction site anked by restriction fragments of P13 bp
(Table S3).
Bioinformatic and statistical analyses (supplementary statistics)
were performed on the unique loci (except where indicated). Inter-
actions were analysed according to various criteria including: (1)
whether the restriction fragment was part of an essential or non-
essential ORF; (2) if the restriction fragment was contained solely
within an ORF or inter-genic region and (3) whether both partners
were on the same (intra) or different (inter) chromosomes. Con-
nections within the ribosomal DNA, 2 lm plasmid and mitochon-
drial genomes were considered as unique because they could be
positioned to a 1 MB region of Chromosome XII, the 6318 bp
2 lm plasmid or 85 kb mitochondrial genome, respectively. All
statistical analyses involving repeated sequences (e.g. 2 lm plas-
mid, mitochondrial, or rDNA sequences) included copy number
corrections.
Pseudo-replicates consisting of 10.9 million sequences were
randomly sampled from two solexa sequence sets, each containing
23 million sequences. Pair-wise comparisons were made between
the pseudo-replicates to determine the percentages of signicant
interactions that were conserved.
2.4. Conrmation of interactions
Chromosome conformation capture (3C) (Dekker et al., 2002;
OSullivan et al., 2009). Samples were prepared as for the GCC li-
brary preparation except that the column purication step was
omitted.
The gDNAgDNA, Mito-gDNA, Mito-rDNA, and Plas-gDNA inter-
actions were conrmed by 3C with primers designed, using Primer
3, to amplify across the ligation site (Tables S5 and S6). The gDNA
gDNA interaction primers are presented in Table S6. The gDNA
gDNA interaction product was present after one round of PCR how-
ever a second round of nested amplication improved the yield
and enabled purication for sequencing. First round PCR conditions
were 95 C for 2 min followed by 40 cycles of: 95 C for 30 s,
58.3 C for 30 s and 72 C for 1 min; and one cycle of 72 C for
3 min. First round PCR product was diluted 200-fold for use in
the second round. Second round PCR conditions were 95 C for
2 min followed by 40 cycles of 95 C for 30 s, 58.3 C for 30 s and
72 C for 30 s; and one cycle of 72 C for 3 min.
BHQ
TM
and BHQplus
TM
probes containing a 5
0
FAM label and Prim-
ers (Table S5) were designed using RealTimeDesign
TM
on-line soft-
ware and purchased from Biosearch Technologies Incorported
(CA, USA). Quantitative 3C assays were performed using an
Fig. 1. Schematic of the Genome conformation capture (GCC) methodology. An
interaction library was prepared using 3C methodology (Dekker et al., 2002).
Briey, the cells are cross-linked with formaldehyde; the chromatin digested (using
a restriction enzyme of choice [e.g. MspI]), diluted and ligated to generate the
interaction library. pUC19 is added to facilitate coverage calculations and to control
for purication efciency. The puried interaction library was nebulised into 150
180 bp fragments before sequencing.
880 C.D.M. Rodley et al. / Fungal Genetics and Biology 46 (2009) 879886
SDS7000. Reactions were performed in triplicate and contained:
Taqman

Gene Expression Master Mix (Applied Biosystems); 2 ll

of sample; 900 nM primers and 250 nM probes (Table S5) in a nal
reaction volume of 20 ll. A 3-stage program was used for Taqman
analyses (50 C, 2:00 min; 95 C, 10:00 min; 45 [95 C, 0:15 s;
60 C, 1:00 min]). Dedicated standards, which corresponded to
the interaction under investigation, (concentration from: 2 ng ll
1
to 2 10
15
g ll
1
) were prepared for each Taqman assay. Results
were expressed as the Interaction frequency compared to the glu-
cose sample (100%), once samples had been standardized for the
number of genomes contained in each sample (see below).
Genome copy number was calculated based on the concentra-
tion of the GAL1 locus within each sample (OSullivan et al.,
2009). Briey, quantitative PCR of the GAL1 locus within each sam-
ple was performed in triplicate in 15 ll reactions containing: the
GAL1 primer pair (Table S6; OSullivan et al., 2009), 2 ll of sample,
and Bio-RAD iTaq SYBR Green Supermix with ROX. Quantitative
PCR was performed on a SDS7000 using a ve stage program:
50 C, 2:00 min; 95 C, 2:00 min; 40 [95 C, 0:15 s; 59.5 C,
0:30 s; 72 C, 0:30 s]; 55 C, 1:00 min; followed by a dissociation
analysis. A cut and ligated genomic DNA sample (concentration
from: 3 ng ll
1
to 1.2 10
12
g ll
1
) was used as a control for all
Sybr-green assays. To facilitate inter-sample comparisons, the
GAL1 concentrations were used to standardize each sample to a
matched glucose sample according to the following formula:
GAL Ratio
test
GAL
test
=GAL
Glucose
% Interactions Taqman
test
GALRatio
test
=Taqman
Glucose

100=1
2.5. Negative interaction control
PCR positive controls for the theoretical ligation of Segments
10,330 Chr XIII [650,483654,199] and 6897 Chr X [144,850
146,958]) were constructed by the ligation of MspI digested ampli-
cons that spanned these segments. The amplicons were prepared
by the PCR amplication of each segment from outside of the MspI
restriction sites (using primer pairs: Sfh5(OverMspI6897)F Sfh5
(OverMspI6897)R; and Mrpl24(OverMspI10330)F Mrpl24(Over
MspI10330)R; Table S6). Standard PCR conditions were used ([1
95 C-2 min][30 95 C, 30 s; 58 C, 30 s; 72 C, 3 min][1 72 C,
7 min] 4 C, 1).
Primers (10330_3
0
MspI3C, 10330_5
0
MspI3C, 6897_3
0
MspI3C,
and 6897_5
0
MspI3C; Table S6) were designed, using Primer 3, to
amplify the products formed from the theoretical ligation of Seg-
ments 10,330 Chr XIII [650,483654,199] and 6897 Chr X
[144,850146,958]). Four independent 3C samples were screened
by PCR using combinations of primer 10330_3
0
MspI3C with
6897_3
0
MspI3C or 6897_5
0
MspI3C and combinations of primer
10330_5
0
MspI3C with 6897_3
0
MspI3C or 6897_5
0
MspI3C. 3C
samples were also screened by PCR using primer pairs (Table S6)
specic for three additional randomly chosen non-interacting frag-
ments: Chr XVI [579,477580,469, Ran1RPA135] Chr I [61,710
62,192, Ran2ERV46], Chr XII [115,597117,393, Ran1BPT1] Chr
IX [369,034369,502, Ran2PAN1], and Chr XIII [51,35552,627,
Ran1ZDS2] with Chr XIV [638,758642,168, Ran2VPS27]. Standard
PCR conditions were ([1 95 C, 2 min][30 (95 C, 30 s; 55.6 C,
30 s; 72 C, 30 s)][1 72 C, 3 min] 4 C, 1). PCR primer concentra-
tion (0.32.4 lM), template and temperature gradients were
performed.
3. Results
GCC involves sequencing a DNA fragment library that is formed
by the intra-molecular ligation of linked restriction fragments pro-
duced from chromatin that was reversibly cross-linked in vivo
(Fig. 1). As such, it is a modication of the 3C (Dekker et al.,
2002; OSullivan et al., 2009) technology which enables the identi-
cation of chromosomal interactions within cells. However, unlike
3C, GCC results in a global interaction map without any require-
ments for a priori assumptions about the function of a particular
sequence.
4. The network
The yeast GCC network (Fig. 2a) was assembled from a total of
160,169 sequences that contained the MspI restriction site (Table
S3) and represented 38,064 inter- and intra-chromosomal interac-
tions (Fig. S2a) between unique loci. Both fragments anking the
MspI restriction site had to be a minimum of 13 bp in length in or-
der for the MspI containing sequence to be included in this analysis
(see Section 2). A separate map was constructed for a further
22,220 interactions involving repeated loci. Adjacent interactions
(Fig. S2a) were not evenly distributed through-out the network.
This observation does not appear to link with the frequency of MspI
restriction sites or the length of the fragments. It is possible that
the frequency of the adjacent interactions provides information
about the level of proteins associated with the fragments of inter-
est. As such, it may represent a measure of the accessibility of the
restriction sites within the chromatin. In theory, inaccessible
restriction sites would impact on the detection of inter- and non-
adjacent intra-chromosomal interactions by inhibiting the genera-
tion of free ends for subsequent ligation at that site. While possible,
such a scenario is unlikely because both ends of the restriction
fragment would have to be simultaneously protected to inhibit
the generation of ligation competent free ends diagnostic for that
fragment.
Direct comparisons, of the proportion of rDNA, telomeric DNA
and a reference gene set within the short read sequences and the
genome (Table S1), demonstrated that the sequences were unbi-
ased. Similarly, the purication of circular and linear fragments
was also unbiased (Fig. S1). Moreover, 98% (13,560/13,786) of the
MspI fragments that were P13 bp in length were involved in at
least one interaction in the GCC network. Therefore, the GCC net-
work is representative of the chromosomal interactions within
the test population.
The unique GCC network (Fig. 2a) contains 1059 novel inter-
and non-adjacent intra-chromosomal interactions (Fig. S2a) that
occur at levels elevated above random (expected false positive
rate = 0.001; see Statistical methods). Published mitotic recombi-
nation rates are too low (between 5 10
4
and 3.5 10
10
per
generation) (Chen and Kolodner, 1999; Mieczkowski et al., 2006;
Szostak and Wu, 1980) to explain these interactions in terms of
the co-localization of sequences involved in recombination.
Estimates or measurements of the numbers of inter- and intra-
chromosomal interactions within the yeast genome do not exist.
To this end, pUC19 was added to the GCC library, following the
ligation step (Fig. 1). Extrapolation of the coverage level of the
pUC19 MspI sites (Table S2) determined the likelihood that we
are identifying a genomic chromosomal interaction to be 0.31
(condence interval 0.1030.61). Therefore, there are approxi-
mately 3000 signicant non-adjacent inter- and intra-chromo-
somal interactions within the genome of exponentially growing,
fermenting yeast cells.
5. Validation
FISH studies (Haeusler et al., 2008; Thompson et al., 2003;
Thomsen et al., 2003) clearly show an association between
tRNA genes and the ribosomal DNA (rDNA). Our data conrmed
C.D.M. Rodley et al. / Fungal Genetics and Biology 46 (2009) 879886 881
interactions between the tRNA and rDNA (Fig. 3a). These interac-
tions were not clustered within one rDNA region but spanned
the rDNA repeat (Fig. 3b). Telomeric clustering (Bystricky et al.,
2005; Gotta et al., 1996; Heun et al., 2001) cannot be conrmed
in the GCC map, as the reads which mapped to telomeric regions
were not long enough to be positioned to a single locus.
To conrm reproducibility, pseudo-replicates consisting of 10.9
million sequences were randomly sampled from two samples of 23
million sequences. Signicant adjacent, non-adjacent intra-chro-
mosomal, and inter-chromosomal interactions were highly con-
served between six pseudo-replicates (percentage of interactions
shared between a pair of replicates ranging from 96.3% to 98.7%,
77.5% to 84.2%, 67% to 77.5% respectively). In contrast, comparisons
between the pseudo-replicates and a separate experimental data-
set demonstrated that the adjacent interactions were highly
similar (range 81.7591.04%), but that the non-adjacent intra-
chromosomal (range 9.0419.2%) and inter-chromosomal interac-
tions (range 02.04%) were dependent upon the culture history
and environment.
As part of the validation, we randomly selected and conrmed
three sets of interactions by quantitative 3C (Dekker et al., 2002),
using separately prepared samples (Fig. 2b). The randomly selected
interactions were reproducible in different strains. Moreover,
studies demonstrated that the frequencies of the three interactions
alter, independently fromeachother, inthe different carbonsources
(i.e. glucose, glycerol-lactate, or galactose; Fig. 2b). Crucially, we
conrmed that randomly selected non-interacting loci, within the
GCC network (i.e. Chr XIII [650,483654,199] and Chr X [144,850
146,958], Fig. S4; Chr XVI [579,477580,469] and Chr I [61,710
62,192], Chr XII [115,597117,393] and Chr IX [369,034369,502],
Chr XIII [51,35552,627] and Chr XIV [638,758642,168]) do not
interact by 3C. Therefore, the observation that the interactions are
reproducible in the same and different strains, and are regulated,
independently of each other, further validates the assay.
6. Do all the chromosomes interact?
We looked for evidence of chromosome territories, chromo-
some pairing and interactions between particular loci, which
would be expected if yeast nuclei are structured. There was a linear
relationship between chromosome length and the number of sig-
nicant unique interactions in the GCC network (Fig. 2c). However,
not all possible chromosome pairings were observed. For example,
chromosomes I and VI interact with only 11 chromosomes while
no interactions were observed between chromosomes V and II
Fig. 2. Yeast chromosomes are linked by an intricate network of environmentally dependent inter- and intra-chromosomal interactions. (a) The GCC network map for unique
loci within exponentially growing yeast cells. The map is composed of interactions between unique loci that were represented by two or more sequences (false positive
rate = 0.001; supplementary statistics). Chromosomes are represented as circles, formed by the virtual fusion of the two telomeres, around the outside of the network. Circle
size is proportional to chromosome length. Chromosome XII appears small because the sequence used in the analysis only contained two rDNA repeats. Lines indicate
interactions between the loci they connect. The map as illustrated represents an overview of the entire system. Detail on individual interactions can be obtained by zooming
in on a particular locus. (b) Quantitative 3C analysis of three interactions in S. cerevisiae BY4741 cells grown on different carbon sources demonstrates the interaction
frequencies are independently environmentally dependent. gDNAgDNA, interaction between loci located on Chromosomes VII (bp 868,673873,686) and IX (bp 172,565
173,311); Mito-gDNA, interaction between loci located on the Mitochondrial genome (bp 24,87226,193) and Chromosome XVI (bp 365,496365,760); Plas-gDNA,
interaction between loci located on the 2 lm plasmid (bp 45865008) and Chromosome XI (bp 240,262240,547). The 3C interaction was quantied by real-time PCR
(methods) using BHQ
TM
and BHQplus
TM
probes (Table S5). Results were standardized according to the number of genomes present in each sample (Section 2) and are presented
as the interaction frequency compared to the glucose sample (100%). Results are the means of three replicates StDev. Standard 3C data for the gDNAgDNA interaction is
shown in Figure S3. (c) The number of inter-chromosomal interactions correlates with chromosome length. (d) Inter-chromosomal interactions between specic
chromosomes occur in clusters, indicating there are interaction faces.
882 C.D.M. Rodley et al. / Fungal Genetics and Biology 46 (2009) 879886
(576,869 and 813,178 bp, respectively). These observations may be
idiosyncratic to these particular chromosomes, the population
structure, cell cycle stage, or the coverage level.
The GCC network provides evidence for inter-chromosomal
interaction faces (Fig. 2a and d). Moreover, intra-chromosomal
loops form on average 28 14 (mean StDev, range 6 loops on
Chr164 loops on Chr4) times per chromosome. The number of in-
tra-chromosomal loops correlates (r
2
= 0.8594) with chromosome
size, in agreement with the observation of a linear relationship be-
tween chromosome length and the number of signicant unique
interactions (Fig. 2c). These loops are variously arranged as large
loops encompassing, and adjacent to, smaller loops and linker
DNA sections. There are also examples, within the GCC network,
where loops appear to be anchored by inter-chromosomal interac-
tions (Fig. S5), although the function(s) of the loops, if any, and how
that relates to the ORFs involved are unknown. Macro-domains
reminiscent of this organization has been observed in Escherichia
coli (Valens et al., 2004) and mammalian cells (Yokota et al.,
1995), where it is described in terms of the Giant loop random
arrangement for chromosome organization (Sachs et al., 1995). In
conclusion, chromosomes within exponentially growing yeast
nuclei show obvious signs of organization, despite the fact that
they are generally considered not to contain classical chromosome
territories (Bystricky et al., 2005; Haber and Leung, 1996).
7. Essential and non-essential genes behave differently
Inter- and intra-chromosomal interactions (Fig. S2a) were as-
sumed to occur with equal frequency at essential and non-essen-
tial ORFs. Loops formed within essential and non-essential ORFs
at similar frequencies (1% and 1.5%, respectively; Table S4). In con-
trast, essential genes are signicantly (p < 0.01, t-test) less con-
nected to non-adjacent loci than their non-essential counterparts
(Table S4). A similar difference is observed between intron-con-
taining verses non-interrupted ORFs (p < 0.01, t-test). ORFs are
designated as essential based on the function(s) of the encoded
product, rather than an inherent property of the DNA sequence.
Therefore, the reasons for the observed low connectivity of the
essential genes are not immediately obvious. We hypothesize that
essential genes are in isolated open sub-domains in order to pro-
mote their transcription and maintenance through uninhibited
access to non-DNA nuclear components.
8. Long distance inter- and intra-chromosomal interactions
occur in yeast
Our data identies long distance (non-adjacent) chromosomal
interactions in yeast. Interestingly, inter-genic fragments involved
in inter-chromosomal interactions were signicantly (p = 0.033,
Chi-squared test for independence) less connected to their adja-
cent sequences than non-interacting inter-genic regions. Compari-
sons of the relative positions of the interacting inter-genic
fragments demonstrated that they clustered approximately 400
600 bp upstream of the neighboring ORF (Fig. 4a). The interacting
partner sequences also clustered, within a 460 bp window located
300 bp downstream of the ATG (Fig. 4b). The interacting regions
overlap the start and stop sites for cryptic unstable transcripts
(CUTs), the presence of which is environmentally regulated (Neil
et al., 2009; Xu et al., 2009). Moreover, we have previously shown
that alterations to CUT levels alter the frequencies of certain chro-
mosomal interactions (OSullivan et al., 2009). Together these data
support a shift from the widely held view that yeast genes form
uninterrupted genomic segments (Dekker, 2008) to a new
hypothesis. Namely, that inter-chromosomal interactions form in
yeast to regulate pathways that are either redundant, didactically
opposite, or developmental. This type of reciprocal control has
been characterized at several mammalian loci (e.g. Dostie et al.,
2006; Qiu et al., 2008; Simonis et al., 2006; Zhao et al., 2006).
Fig. 3. Segments containing tRNA genes cluster with the ribosomal DNA. (a)
Interactions between tRNA containing segments and their partners were mapped
and a histogram plotted according to the segment (bins of 10 segments) with which
they interact. Major clusters occur at the rDNA repeats and within the mitochon-
drial genome. (b) The tRNA interaction pattern within the rDNA, limited to
statistically signicant interactions (i.e. allowing for rDNA copy number (statistical
methods)). The data plotted in (a) and (b) include data from the repeated
interactions.
Fig. 4. Non-adjacent interactions involving inter-genic regions occur predomi-
nantly within short windows in both partners of the interaction pair. (a) 122 inter-
genic restriction fragments involved in signicant non-adjacent interactions were
centered 400600 bp upstream of the nearest ORF A(+1)TG. Inter-genic fragments
were selected on the basis that the fragment did not overlap an ORF and comprised
<40% of the inter-genic region. Inter-genic fragments were classied according to
whether the surrounding genes were divergent, tandem on the Watson (tandem W)
or Crick (tandem C) strands. Interactions involving convergent inter-genic regions
were under-represented in our data due to a size bias (Figure S6). (b) The non-
adjacent interacting partner segment was typically intra-genic and was centred
between 300 and 760 bp downstream of the ORF A(+1)TG. The hatched boxes
illustrate the length of the regions involved in the interaction (mean + 1 StDev).
Frequency is a direct count of number of regions that are centred within the 200 bp
windows. The scale of the x axis is 100 bp.
C.D.M. Rodley et al. / Fungal Genetics and Biology 46 (2009) 879886 883
9. The 2 lm plasmid folds to align the inverted repeats and
expose the stability locus
S. cerevisiae harbours a 2 lm plasmid, which confers no partic-
ular advantage or burden on the host (Yeh and Bloom, 2006) and as
a result has been described as a model selsh DNA element (Mehta
et al., 2002). The 2 lm plasmid is relatively small (6318 bp) and en-
codes only four open reading frames (ORFs). However, it is main-
tained at 50100 copies per cell using partitioning and
amplication systems (Mehta et al., 2005). The partitioning mech-
anism requires that the 2 lm plasmids cluster about a stability lo-
cus (STB; Mehta et al., 2002; Scott-Drew et al., 2002). Therefore, we
predicted (Fig. 5a) and observed (Fig. 5b) an uneven distribution of
self-ligation products about the STB locus. The distribution of the
self-ligated products does not correlate with the length of the
restriction fragment. Rather, the distribution conrms the spatial
clustering of identical sequences, which appear as self-ligations
within the GCC network due to inter-repeat ligation (Fig. 5a).
Intriguingly, modeling of the plasmid structure (Fig. 5c) indicates
that the plasmid folds to present the STB locus on a prominent
loop, perhaps facilitating plasmid clustering about this locus.
The 2 lm plasmid contains two inverted repeats (IR1 and IR2)
which recombine as part of a copy number maintenance mecha-
nism (Futcher, 1986). GCC clearly identied spatial clustering of
these repeat sequences (Fig. 5c), which could help facilitate recom-
bination between these loci.
Partitioning of the 2 lm plasmid from mother to daughter cells
requires that the clustered plasmids interact with the yeast
chromosomes (Mehta et al., 2002). Such interactions were clearly
identied in the GCC network (Fig. 2a). However, the interacting
chromosomal regions did not share any obvious similarities, as
determined by gene ontology. Therefore, we propose that these
chromosomal loci act as the hitch-hiking sites for 2 lm plasmid
segregation.
10. Interactions occur between the mitochondria and yeast
chromosomes
Organelle genes have migrated from mitochondria to the nu-
cleus over the course of eukaryotic evolution (reviewed in Timmis
et al., 2004). Such transfer is central to endosymbiotic theory and is
a continual process as illustrated by the insertion of a near com-
plete chloroplast genome into the rice chromosome 10 (The,
2003) and the recent human insertions (Ricchetti et al., 2004). De-
spite the mechanism of transfer being unknown (Ricchetti et al.,
1999), studies have clearly demonstrated that mitochondrial
DNA is transferred to the yeast nucleus at rates of up to 2 10
5
per cell per generation (Thorsness and Fox, 1990). Similarly chloro-
plast transfers to the tobacco nucleus in 1:16,000 pollen grains
(Huang et al., 2003), or 1:5 10
6
leaf cells (Stegemann et al.,
2003). Moreover, mitochondrial sequences are used in the repair
of double strand breaks in haploid yeast (Ricchetti et al., 1999).
Transfer of mitochondrial genomic DNA to the nuclear genome
is frequent and on-going (Ricchetti et al., 1999, 2004; Timmis et al.,
2004). Thus, interactions between the mitochondrial genome and
the host chromosomes were expected. The 39 interacting host
chromosomal restriction fragments (expected false positive
rate = 0.05, Statistical Methods), contain parts of 51 nuclear ORFs.
The fact that 34% of these chromosomal ORFs had Paracoccus den-
itricans homologues is remarkable as it links the mitochondrial
genome with ORFs that were ancestrally of mitochondrial origin
(e.g. MAK5 and BNA4). Only two interactions mapped to regions
that are characterized as having mitochondrial sequences inserted
within the genome (NUMTs; Ricchetti et al., 1999) but neither
NUMT matched the interacting mitochondrial restriction fragment.
Characterized organelle insertions can be large (Martin, 2003;
Stegemann et al., 2003; The, 2003). It has been proposed that trans-
fer is due to the turn-over or degradation of organelles (reviewed
in Martin, 2003) or regulated by host encoded proteins (Shafer
et al., 1999). Alternatively, nuclear import of organelle genomes
may result from a combination these pathways. There is no direct
evidence to indicate whether the interactions between the mito-
chondrial and nuclear genomes we observed involve complete or
fragmented mitochondrial genomes.
Elevated interactions between a mitochondrial ORI sequence
(de Zamaroczy and Bernardi, 1986) and the yeast rDNA were ob-
served (Fig. 6). This interaction (Mitochondrial genome [50,520
50,804] and the rDNA (Chr XII [460,025460,609]) was conrmed
by 3C. This is remarkable because the level of mitochondrial DNA
affects the rate of replication of the host rDNA (Blank et al.,
2008) and thus yeast growth rate. Interestingly, mitochondrial es-
cape has been shown to occur at a higher rate in fermenting yeast
cells, where mitochondria only make up 3% of the total volume of
the cell (Shafer et al., 1999). This is in agreement with our observa-
tions that the frequency of the mitochondrial-genomic interaction
(i.e. Mitochondrial genome (bp 24,87226,193) and Chromosome
XVI (bp 365,496365,760)) was highest in fermenting yeast cells
(Fig. 2b). Therefore, it is possible that the interactions between
the mitochondrial and nuclear chromosomes are regulatory. In
effect the mitochondrion uses its own DNA to control host division
and expression of mitochondrial and related genes. This enables
population based control of single loci and ensures the mainte-
nance of a mutually benecial relationship. Such a role may
Fig. 5. The yeast 2 lm plasmid folds to maximize the interactions between the
inverted repeats, while the stability locus (STB) locus is on a prominent loop. (a)
Cartoon illustrating how Rep and STB mediated clustering of the 2 lm plasmid
results in self-ligated fragments due to inter-repeat ligation. The GCC method is as
shown in Fig. 1. (b) The distribution of self-ligated (circularized) restriction
fragments indicates functional clustering of the 2 lm plasmid about the STB locus
and the inverted repeats. Interaction frequencies were obtained from the combined
data le due to the repetitive nature of the inverted repeats and STB locus. Non-
circularizing fragments with similar sizes to the three circularizing fragments were
present (1032 bp [3] vs. 1100 bp [10], 950 bp [7] vs. 927 bp [0], and 720 bp [2] vs.
748 bp [0]; number of observed self-ligations in square brackets), within the 2 lm
plasmid indicating that size is not the sole criteria for self-ligation. The positions of
the circularizing fragments are shown relative to a cartoon of the 2 lm sequence,
below the graph. (c) Artists rendition of the 2 lm plasmid copied from a physical
model which integrated the non-adjacent intra-2 lm plasmid interactions. Intra-
molecular interactions conrm the co-localization of the inverted repeats (IR1 and
IR2) and demonstrate organization of the STB locus onto a prominent loop.
Restriction fragments are delineated by dividing lines. Interactions are denoted by
arrows annotated with the number of isolations of the interaction sequencing
product.
884 C.D.M. Rodley et al. / Fungal Genetics and Biology 46 (2009) 879886
explain why the loss of mitochondrial DNA causes alterations in
the expression of a diverse range of nuclear genes (Traven et al.,
2001) and similarly why NUMTs are not evenly distributed within
the nuclear genomes in which they are found (e.g. human (Ricch-
etti et al., 2004) and yeast (Ricchetti et al., 1999)).
11. Discussion
We have used GCC to demonstrate that the yeast genome is a
highly integrated network comprising nuclear and organelle chro-
mosomes, and episomes. Our results implicate chromosomal inter-
actions in the evolution of parasitic and mutualistic relationships,
perhaps through the modulation of intra-genomic conict. Previ-
ous observations indicate that chromosomal interactions, and
hence the structure of the genomic network, are controlled by epi-
genetic mechanisms (Espada and Esteller, 2007; Kim et al., 2009;
OSullivan et al., 2009; Osborne et al., 2007). As such, the genomic
network provides a mechanism by which genome function, be it
structural (Cavalier-Smith, 1978, 1982), regulatory (Dostie et al.,
2006; Ling et al., 2006; Martin et al., 2005; Osborne et al., 2004,
2007; Simonis et al., 2006; Zhao et al., 2006) or a combination of
both, can be integrated into the evolutionary and developmental
processes that regulate gene expression, nuclear and cellular size.
The GCC methodology is generally applicable to large genomes
(e.g. Human) and is designed to take advantage of future advances
in sequencing technology (i.e. longer reads and increasing capaci-
ties). Firstly, the algorithms (i.e. BLAST (Altschul et al., 1990)) and
SOAP (Li et al., 2008)), used by the Topography software, can posi-
tion longer sequences onto the reference genome. Secondly,
paired-end sequencing increases the useable sequences enabling
the identication of interactions on the basis that the paired se-
quences are derived from different genomic restriction fragments.
Thirdly, immune-precipitation, with inactivated restriction en-
zyme, enriches for useable sequences prior to sequencing. Finally,
renement of interaction zone resolution is achievable through
comparisons of GCC networks produced with different restriction
enzymes.
GCC produces a ne scale probabilistic map of the genomic
interactions occurring within a dened population of cells without
a priori constraints on the regions of interest. As such, GCC enables
an integrated empirical approach to global investigations of the
role that spatial organization has in genome function and
evolution. The next challenge will be to include a spatio-temporal
component to the global characterization of chromosomal interac-
tions to determine how genomic structures are assembled. More-
over, comparative GCC studies will identify regulatory
rearrangements involving inter-genic and genic regions alike.
These advances are crucial to improve our understanding of gene
expression, genome maintenance and evolution, given the increas-
ing evidence that the function(s) of the genome are determined by
multiple layers of regulatory control processes (Barabasi and Olt-
vai, 2004; Misteli, 2001).
Acknowledgments
The authors thank A. Rodrigo, A. Ganley, R McNab, P. Rainey,
and N. Proudfoot for critical discussions of this work and manu-
script. This work was funded by the Lottery Health (237394), Mau-
rice & Phyllis Paykel Trust and Massey University MURF fund. C.R.
is funded by an HRC Ph.D. scholarship (08/554).
Author contributions. C.R., prepared the GCC sample, was in-
volved in the bioinformatic analyses, and aided in the writing of
the manuscript. F.B., wrote the program suite and was involved
in the bioinformatic analyses. B.J. performed the statistical analy-
ses. J.O.S. designed the experiment and the software, performed
bioinformatic analyses, and wrote the paper.
The authors have no competing interests.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.fgb.2009.07.006.
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