Pan-S replication patterns and chromosomal domains
defined by genome-tiling arrays of ENCODE
Neerja Karnani,1Christopher Taylor,1,2Ankit Malhotra,1,2and Anindya Dutta1,3
1Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908, USA;
2Department of Computer Science, University of Virginia, Charlottesville, Virginia 22908, USA
In eukaryotes, accurate control of replication time is required for the efficient completion of S phase and
maintenance of genome stability. We present a high-resolution genome-tiling array-based profile of replication
timing for ∼1% of the human genome studied by The ENCODE Project Consortium. Twenty percent of the
investigated segments replicate asynchronously (pan-S). These areas are rich in genes and CpG islands, features they
share with early-replicating loci. Interphase FISH showed that pan-S replication is a consequence of interallelic
variation in replication time and is not an artifact derived from a specific cell cycle synchronization method or from
aneuploidy. The interallelic variation in replication time is likely due to interallelic variation in chromatin
environment, because while the early- or late-replicating areas were exclusively enriched in activating or repressing
histone modifications, respectively, the pan-S areas had both types of histone modification. The replication profile of
the chromosomes identified contiguous chromosomal segments of hundreds of kilobases separated by smaller
segments where the replication time underwent an acute transition. Close examination of one such segment
demonstrated that the delay of replication time was accompanied by a decrease in level of gene expression and
appearance of repressive chromatin marks, suggesting that the transition segments are boundary elements separating
chromosomal domains with different chromatin environments.
[Supplemental material is available online at www.genome.org.]
Although all the DNA in a eukaryotic cell replicates during the S
phase of cell cycle, there is a great variability in the actual point
in S phase when a given chromosomal segment replicates. Seg-
ments are known to reproducibly replicate early or late in S
phase, and it is generally believed that this is determined by the
time at which the origins in a segment fire. All origins of repli-
cation are licensed with MCM proteins by the time S phase be-
gins (Bell and Dutta 2002), and yet, once conditions in the cell
change to favor the firing of the origins, all origins do not fire at
the same time. In situ labeling techniques and other methods
have led to some general principles determining the time of rep-
lication of a segment in S phase (for review, see MacAlpine and
Bell 2005). Early-replicating segments are generally enriched in
euchromatin, while late-replicating segments are enriched in
heterochromatin. Some loci that are selectively expressed in spe-
cialized cells (e.g., immunoglobulin, beta-globin, or neural-
associated genes) show a change in time of replication from late-S
phase in undifferentiated, nonexpressing cells to early-S phase
after differentiation (Simon et al. 2001; Zhou et al. 2002; Perry et
al. 2004). The correspondence between the activation of chro-
matin at differentiation-induced genes with the advancement in
replication time also suggests that the chromatin environment
dictates time of replication (Bickmore and Carothers 1995; Roun-
tree et al. 2000; Demeret et al. 2001).
The completion of many genomic sequences and the advent
of genome-tiling microarrays provided an opportunity to corre-
late gene expression or chromatin structure with time of replica-
tion at a much finer resolution. DNA replicated at specific inter-
vals in S phase were hybridized to genome-tiling microarrays to
determine the exact time in S phase when specific genes repli-
cate. Early experiments in model organisms like Saccharomyces
cerevisiae and Drosophila melanogaster confirmed many of the
principles outlined above (Raghuraman et al. 2001; Schubeler et
al. 2002; MacAlpine et al. 2004).
Extending this method of analysis to human cells, specifi-
cally to Chromosomes 21 and 22, we confirmed that similar prin-
ciples dictate time of replication in human chromosomes (Jeon et
al. 2005). We made the surprising observation that almost 60% of
the chromosomal probes studied gave a replication signal at mul-
tiple times in S phase, described as a pan-S-phase pattern of rep-
lication. While asynchrony of replication between alleles of a
given gene would give rise to a pan-S-phase pattern of replica-
tion, it seemed highly unlikely to us that 60% of the human
chromosomes would show such asynchrony. In addition, it was
unclear whether the pan-S-phase replication was an artifact of
cells losing their synchrony of progression through the cell cycle,
of the thymidine-aphidicolin method of cell cycle synchroniza-
tion, or of the aneuploidy inherent in HeLa cells.
The ENCODE region encompasses 44 segments covering
∼1% of the human genome on which multiple groups are apply-
ing different techniques to find the best methods to annotate the
human genome (The ENCODE Project Consortium 2004). We
measured the replication time for this region and used the data to
improve our method of computing the replication profile of
chromosomal segments. The improvements in our algorithm de-
creased the pan-S replication pattern to ∼20% of the segments
interrogated. We confirmed the prediction of pan-S replication
E-mail email@example.com; fax (434) 924-5069.
Article is online at http://www.genome.org/cgi/doi/10.1101/gr.5427007.
Freely available online through the Genome Research Open Access option.
17:865–876 ©2007 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/07; www.genome.org
by an independent method of assessing replication time: inter-
phase FISH. The results demonstrate that pan-S-phase replication
is a real pattern of replication that cannot be explained by arti-
facts derived from microarray platform, methods of cell cycle
synchronization, or aneuploidy of cells. Instead, pan-S-phase
replication is a reflection of asynchrony of replication between
alleles in a given cell, suggesting that differences in the chroma-
tin environment of two alleles can be seen in up to 20% of the
human genome in some cells.
Finally, using the high-definition temporal profile of repli-
cation over the ENCODE areas, we identified adjoining chromo-
somal segments of a few hundred kilobases each with differing
times of replication. Hypothesizing that these areas are “replica-
tion domains,” we demonstrate for one such region that the ad-
joining domains have different levels of gene expression and
activating and repressing marks on histones. We believe that the
replication domains correspond to chromosomal domains sepa-
rated by boundary elements.
Replication timing analyses of 1% genome using synchronized
HeLa cells were synchronized at G1/S by thymidine-aphidicolin
block. After release from the block, cells were pulsed with bro-
modeoxyuridine (BrdU) at every 2-h interval of S-phase and ge-
nomic DNA isolated. In all, five time intervals (0–2, 2–4, 4–6, 6–8,
and 8–10 h) representing 10 h of the entire S phase were col-
lected. The BrdU-incorporated heavy/light (H/L) DNA was puri-
fied using a CsCl density gradient as described earlier (Jeon et al.
2005). Purified DNA from each time interval was hybridized to
the high-density genome-tiling Affymetrix array comprising
unique 25-mer oligonucleotides in the ENCODE-selected chro-
mosomal loci covering 1% of the human genome (∼30 Mb) (see
Methods for details of the ENCODE regions).
Segregation of chromosomal regions into temporally specific
and pan-S replicating segments
Probes that replicated in a discrete interval in S phase were called
temporally specific, while probes that replicated at multiple in-
tervals in S phase were called temporally nonspecific. The Meth-
ods and Supplemental Table 1 contain examples of the specificity
classification. For the Affymetrix ENCODE array, 26.115% of the
probes were temporally nonspecific.
In order to classify chromosomal segments as temporally
specific or asynchronously replicating (pan-S), a 10-kb sliding
window was passed along the chromosome and each window
defined as replicating in a pan-S manner if >60% of the probes in
that window are temporally nonspecific (see Methods for de-
tails). Thus, by ensuring that the majority of contiguous probes
in a given segment replicate in a temporally nonspecific manner,
we eliminate artifacts from cross-hybridization or from poor
probe hybridization. Since the estimated average speed of a rep-
lication fork is ∼1 kb/min, isolated segments <10 kb (<10 min)
that appeared to replicate in a nonspecific manner were signifi-
cantly below the resolution of the 2-h sampling method. Such
segments (<0.2% of the ENCODE region) were therefore elimi-
nated from our calculations. After these corrections, ∼20% of the
ENCODE area replicated in a pan-S-phase pattern as determined
by a base-pair count (Fig. 1A), while the remaining 80% shows a
temporally distinct profile. Individual chromosomal segments
showing these patterns are presented below.
Continuous TR50 profile along the length of a chromosomal
The time at which a temporally specific probe replicates to 50%
(TR50) is calculated by summing the replication signal over the
five time points and linearly interpolating the time when 50% of
the total signal was reached. Supplemental Table 1 gives ex-
amples of TR50 calculation for several probes. Plotting the TR50
values for specific probes against the linear coordinate of the
probe on the chromosome gives a view of the replication profile
of the chromosome. Because the raw TR50 data are noisy, Lowess
smoothing for all temporally specific probes in a 60-kb window
was performed to ascertain the trends in the replication pattern
along the length of the chromosome. Figure 1B shows the raw
TR50 data and a smoothed TR50 curve for a 1.9-Mb segment of
Chromosome 7 (ENm001). By averaging over relatively long seg-
ments of DNA, the smoothed curve corrects for scatter created by
(A) Temporally specific versus pan-S distribution of replication for 1% of
the human genome investigated in this study. (B) Raw TR50 data with a
smoothed TR50 curve overlaid from the 1.9-Mb region on Chromosome
7. According to the ENCODE Consortium nomenclature, this chromo-
somal segment is referred to as ENm001 (http://hgwdev.cse.ucsc.edu/
ENCODE/encode.hg17.html). (C) Smoothed TR50 data from the 1-Mb
beta-globin locus (ENm009) on Chromosome 11. The lowest point in
each valley indicates a site that is replicated before its adjoining segments
and thus is likely to contain origins of replication. The gaps in the TR50
plots indicate the presence of repeats. In order to minimize cross-
hybridization of oligonucleotides, repeat regions of the genome are not
spotted on the tiling arrays. The triangle on the X-axis indicates the
position of the known beta-globin origin.
Temporal profile of replication of chromosomal segments.
Karnani et al.
differences in probe hybridization effi-
ciency or from cross-hybridization of a
few errant probes and is very useful for
comparing the time of replication of ad-
joining segments of DNA. Figure 1C
shows another example of a smoothed
TR50 plot from a 1-Mb region of Chro-
mosome 11 containing the beta-globin
locus. The late replication of this seg-
ment in HeLa cells agrees with previ-
ous findings that the beta-globin locus
replicates late in S phase in nonery-
throid cells (Epner et al. 1988; Dhar et al.
1989). The TR50 profiles for all the 43
chromosomal loci can be viewed using
the UVa DNA Rep TR50 track at the
encode.hg17.html site. Temporal pro-
files from 12 of these regions are shown
in Supplemental Figure 1.
Local minima of the TR50 curve
show areas that replicate earlier than the
flanking regions and thus are likely to
contain origins of replication, as has
been shown previously in S. cerevisiae
(Raghuraman et al. 2001). Only one pre-
viously validated origin of replication
lies in the ENCODE area near the large
stretch of repeat sequences (chr11:
5124929–5193780) within the beta-
globin locus (Kitsberg et al. 1993; Alad-
jem et al. 1998; Wang et al. 2004). The
repeat sequences near the beta-globin
gene were not represented on the micro-
array, causing a gap in the TR50 profile
(Fig. 1C). However, the TR50 profile of
the regions immediately adjoining these
repeats clearly suggests that a minimum
in the TR50 profile is located somewhere
at or near these repeats, indicating the
presence of an origin of replication at
this site. Thus, the hundreds of minima
in the TR50 profile are likely to be at or
near origins of replication.
Segregation of temporally specific regions into early-, mid-,
and late-S replicating regions
The smoothed TR50 profile suffers from a compression of the
Y-axis values due to the smoothing operation; thus we do not get
an accurate estimate of the time of replication of a given segment
from the profile. We therefore processed the TR50 data to define
discrete segments with early-, mid-, and late-S-phase replication
in addition to the pan-S-phase replication patterns described
above. A temporally specific region is classified into early, mid, or
late replication based on the average TR50 of the temporally spe-
cific probes within a 10-kb window. TR50 cutoffs of 3.4 h (for
early- to mid-S transition) and 3.9 h (for mid- to late-S transition)
The top panel of Figure 2A shows the segregation of
ENm001 after these analyses. Tracks representing segments that
replicate in early-, mid-, late-, or pan-S-phase, respectively, are
indicated. Since ENm001 is an early-replicating region, only a
very small region shows up in the late-replication track. The gen-
eral trend of the right portion of the region replicating later can
be seen in the transition from a solid early-replication track into
mid-replicating regions as we move left to right. The tracks are
nonoverlapping at the base-pair level, and the apparent overlap
in certain places is due to the low resolution of the UCSC Browser
snapshot required to fit the whole region into a figure.
The second and third panels of Figure 2A show similar seg-
mentation of 500-kb chromosomal regions from Chromosomes
16 (ENr313) and 13 (ENr132), respectively. ENr313 replicates
late, while ENr132 shows a pan-S pattern of replication. TR50
segmentation profiles for 12 regions are shown in Supplemental
Figure 1. Profiles for all the 43 regions can be viewed in the UVa
DNA Rep Seg track at http://hgwdev.cse.ucsc.edu/ENCODE/
encode.hg17.html site. Eighty percent of the ENCODE area rep-
licates in a temporally specific interval (Fig. 1A). Within the spe-
cific pattern of replication. (A) On the basis of TR50, temporally specific regions are further segregated
and displayed as three tracks (UCSC Genome Browser), early-, mid-, or late-replicating, while chro-
mosomal regions undergoing temporally nonspecific replication are highlighted under the pan-S track.
The three panels in this figure show segregation of replication timing for three chromosomal segments.
(Top panel) The 1.9-Mb region (ENm001) of Chromosome 7; (middle and bottom panels) examples of
two 500-kb chromosomal segments from Chromosomes 16 (ENr313) and 13 (ENr132) that under-
went late and pan-S replication, respectively. The FISH track in all the three panels refers to the
chromosomal positions of BAC clones selected for the interphase FISH experiment shown in Figures 3
and 4. (B) Percent of temporally specific chromosomal segments replicating in early-, middle-, or late-S
Segregation of chromosomal segments with temporally specific and temporally nonspe-
Replication profile of 1% of human genome
cific regions, 31% segregates into early-, 34% into mid-, and 35%
into late-S-phase replicating patterns (Fig. 2B).
Validation of replication time by interphase FISH
To check the temporal profile of replication generated by the
microarray data, we used interphase FISH as an independent
method for determining replication time. Although labor-
intensive, this method has the additional advantage in that the
large sizes of the probes reduce errors from poor signal strength
and cross-hybridization. Ten BAC clones of 48–187 kb each (de-
tails in Supplemental Table 2) were selected to validate the mi-
croarray data for 10 segments from nine ENCODE areas: three
each with early and pan-S-phase and four with late-S-phase pat-
terns of replication. The positions of BAC clones used in Figure 3
and Figure 4 are highlighted in Figure 2A. HeLa cells were syn-
chronized and harvested at 2-h intervals during S phase, and BAC
clones were labeled and hybridized to denatured interphase nu-
clei. A single hybridization signal (visible as a dot under the mi-
croscope) indicates one copy of the targeted DNA. ENm001
showed 2 dots/cell in G1(0 h) and 4 dots/cell in G2, while the
remaining eight regions had 3 dots/cell in G1and 6 dots/cell in
G2because of the aneuploidy of HeLa cells. The percent replica-
tion of a probed segment in each time interval in S phase was
determined by counting the increments in dots/cell during that
interval, where 100% replication means that the number of dots/
cell is twice the G1value.
The RP11-51M22 probe shows that this region of ENm001
replicates early with the complete doubling of all signal in the
first 2 h of S phase (Fig. 3A). For a late-replicating region, RP11-
3I14 from ENr313, the increase in dot number was maximum in
the last 4 h of S phase (Fig. 3B). RP11-88E10 from the ENr132
region indicated that significant replication occurred in multiple
time intervals (Fig. 3C), consistent with the pan-S replication
detected in the microarrays (Fig. 2A). All the 10 segments tested
by FISH reproduced the microarray data for time of replication
(see Supplemental Table 2 for details).
Pan-S replication is due to interallelic variation in time
To ascertain whether the pan-S-phase pattern of replication was
due to intercellular or interallelic variation in replication time,
we calculated the percent nuclei in mid-replication. The Mid-
Score for a time point is defined as the percentage of cells in
mid-replication, having replicated one, but not all alleles, for a
given probe. Thus cells in mid-replication will have 3 dots/cell for
ENm001, and 4 or 5 dots/cell for ENr313 or ENr132. Segments
that replicate synchronously in a narrow interval of S phase are
expected to have a very narrow temporal window with a high
Mid-Score (a more detailed explanation is in Supplemental Fig.
2). ENm001 (early replicating) had no time point with a high
Mid-Score, while ENr313 (late replicating) had only two time
points with a Mid-Score of 5.6% and 7.9%, indicating that all the
alleles replicated in a narrow time window (Fig. 3D).
If pan-S-phase replication is due to intercellular variation in
time of replication of the chromosomal segment, the two alleles
in a cell will still replicate simultaneously so that the window of
time when a cell is caught in mid-replication will remain short.
Mid-Scores would be low or elevated for only a tightly restricted
time interval (Supplemental Fig. 2). However, the ENr132 region
(pan-S-phase replication) showed four time points with high
Mid-Scores (i.e., 12.1, 11.4, 30.7, and 24.5) (Fig. 3D), suggesting
that there was significant asynchrony in the time of replication
of the alleles in a given cell. Thus the asynchrony in replication
seen in the pan-S-phase pattern of replication is due to interal-
lelic variation in replication time.
Pan-S-phase pattern of replication is not due
to thymidine-aphidicolin block
We next investigated whether pan-S-phase replication was
caused by the prolonged arrest in S phase that is inherent to the
thymidine-aphidicolin double-block method of synchronization
of cells in the cell cycle. HeLa cells were synchronized in mitosis
using nocodazole and released. The time of replication was de-
termined by interphase FISH for five regions (Fig. 4B): three that
were temporally monophasic and two that had a pan-S-phase
(A–C) Synchronously progressing HeLa cells were hybridized to fluores-
cence-labeled probes of BAC clone DNA RP11-51M22, RP11-3I14 (for
early- and late-replicating areas, respectively) and RP11-88E10 (for pan-S
pattern of replication). The chromosomal locations of these BACs are
highlighted in Figure 2A. The percent replication at each interval of S
phase is plotted against time in S phase. (D) The interallelic variation in
replication for FISH data observed for each of the BAC clones mentioned
above was determined by calculating the Mid-Score (detailed in Results
and Supplemental Material).
Interphase FISH for validating replication timing in HeLa.
Karnani et al.
868 Genome Research
pattern of replication. The temporally specific segments still
replicated in the expected time frames despite the different
method of synchronization (Fig. 4B; Supplemental Table 2).
Most important, both the pan-S-phase regions continued to
replicate at multiple times in S phase (Fig. 4B; Supplemental
Table 2), suggesting that pan-S-phase replication was not an ar-
tifact of the synchronization method. The observed asynchrony
in replication was due to interallelic variation as determined by
the wide time interval when the cells displayed high Mid-Scores
Pan-S pattern of replication is not restricted to aneuploid
We wanted to rule out the possibility that the pan-S-phase pat-
tern of replication is seen only in aneuploid cancer cells like
HeLa. To address this, we repeated the interphase FISH experi-
ments with MCF10A, a breast epithelial cell line derived from
fibrocystic breast disease that is near diploid and nonmalignant
(Fig. 4D). The area covered by probe RP11-88E10 (a region with
pan-S-phase replication in HeLa cells) replicated at two time in-
tervals (Fig. 4E). The first peak at 4 h corresponded with the time
interval during which the Mid-Score increased (Fig. 4F). The Mid-
Score remained high until the 10-h time interval, when the sec-
ond peak of replication was observed, indicating a significant
time lapse in the replication of two alleles. Therefore, pan-S-
phase replication is also seen in MCF10A cells and is not unique
to HeLa cells. Replication of RP11-51M22 (early) and RP11-3I14
(late) was also consistent with that seen in HeLa cells. FISH analy-
ses for two more regions in MCF10A are detailed in Supplemental
Correlation of TR50 profile with genome sequence features
The replication timing for the 43 ENCODE regions were corre-
lated against genome sequence features such as AT content, CpG
islands, and gene density. AT content was computed using a 10-
kb sliding window and plotted against the smoothed TR50 curve.
A transition from low to high AT content is evident for early- to
late-replicating regions (Fig. 5A). The Spearman rank correlation
coefficient calculated from the plot was 0.257, suggesting a mod-
erate positive correlation. The Pearson correlation coefficient was
0.252, also indicating a moderate positive correlation. Computa-
tion of AT content at a window size of 1 kb gave a lower corre-
lation coefficient (0.19).
DNA methylation is an important epigenetic marker (Jones
and Takai 2001), with differential DNA methylation between al-
leles leading to monoallelic gene expression, interallelic differ-
ences in the chromatin, and asynchronous replication (Simon et
al. 1999; Rountree et al. 2001; Fournier et al. 2002; Jiang et al.
2004; Fuks 2005). Since the Mid-Score calculations above sug-
gested that the pan-S areas demonstrated interallelic differences
in replication, we wondered whether the pan-S replicating seg-
ments were enriched in CpG islands and thus potentially suscep-
released from mitosis followed by FACS for DNA content. (B,C) Interphase FISH was performed with HeLa cells synchronized with nocodazole and
released. The X-axis represents time in S phase such that 0 h = 12 h post-release from the nocodazole block. The rest is as in Figure 3. (D) MCF10A cells
released from a G1/S block with thymidine/aphidicolin followed by FACS for DNA content. (E,F) Interphase FISH with MCF10A cells synchronously
progressing through S phase to determine the replication profile and Mid-Score with the chromosomal segments mentioned in Figure 3.
Pan-S replication pattern is independent of cell synchronization method and aneuploidy. (A) HeLa cells blocked (by nocodazole) and
Replication profile of 1% of human genome
tible to regulation by differential DNA methylation. Indeed, the
pan-S-phase regions showed the maximum enrichment (1.86) of
CpG islands (Fig. 5B).
We next compared the replication time of a segment with its
gene density. In the chromosomal areas where replication was
temporally specific, a threefold higher enrichment of genes was
found in regions replicating early compared to those replicating
late (Fig. 5C). Interestingly, the pan-S-phase regions had gene
content (enrichment = 1.42) (Fig. 5C), comparable to early-
replicating chromosomal segments (enrichment = 1.41), consis-
tent with the idea that these regions could have replicated early
if not for interallelic variation in chromatin structure that re-
sulted in a subset of the alleles replicating late and producing a
pan-S-phase pattern of replication.
Early-replicating regions are highly transcribed
Active transcription of genes is associated with euchromatin
and may be expected to correlate with early replication. Total
RNA was prepared from logarithmically growing HeLa cells
and hybridized to an Affymetrix HG-U133 Plus 2.0 array to mea-
sure the level of expression of genes in different chromosomal
segments. Early-replicating segments have 5.34-fold higher
transcription over the late-replicating regions (Fig. 5D). The
pan-S regions had an intermediate level of gene expression, con-
sistent with the idea that all alleles of the genes in these segments
are not in favorable chromatin and are not uniformly well ex-
TR50 profile on one chromosomal segment defines
The global correlations described above are consistent with the
hypothesis that early-replicating regions are usually gene dense
and contain actively transcribed genes. The fine resolution of
replication profile possible with the genome-tiling arrays allowed
us to closely examine how such correlations hold up across con-
tiguous stretches of chromosomes. Intriguingly, the TR50 profile
of some regions revealed the presence of neighboring chromo-
somal segments with acute transitions in replication time. For
example, in ENm005, an ∼366-kb (Chr21: 33119705–33486048)
late-replicating stretch was bracketed by two early-replicating ar-
eas (Fig. 6A). Dual color interphase FISH was performed to con-
firm the transition in replication time from early to late in two
neighboring segments of ENm005 (Fig. 6B). BAC clones (sepa-
rated by ∼355 kb) from the early (RP11-54F16) and late (RP11-
79D9) replicating areas confirmed that the two DNA seg-
ments, indeed, replicated in two different intervals of S phase
The replication dissimilarities between the adjoining do-
mains correlated with dissimilarities in gene expression and gene
density (Fig. 6C). The late-replicating island was both gene-poor
and transcriptionally less active compared to the adjoining early-
replicating chromosomal segments.
These observations suggested the existence of two chro-
matin environments in a contiguous stretch of a chromo-
some separated by some type of boundary element. Since histone
modifications distinguish euchromatin from heterochroma-
tin, we decided to confirm the existence of two chromatin
environments in this locus in HeLa cells by performing a
chromatin immunoprecipitation (ChIP) assay for the active
and inactive chromatin marks. H3 Lys4 methylation is spe-
cific for active chromatin at active promoters (Bernstein
et al. 2005). We therefore selected nine genes, two in the late-
replicating region (OLIG1 and OLIG2) and seven in the adjoining
early-replicating chromosomal segments (C21orf119, SYNJ1,
C21orf66, IFNAR1, GART, ITSN1, and ATP5O) and designed prim-
ers to amplify unique 100–300-bp fragments from the
2-kb sequences upstream of the genes (see details for primers
in Supplemental Table 3). Chromatin immunoprecipitation
(ChIP) and amplification of these promoters revealed that
all seven genes in the early-replicating segments were positive
for H3 lysine 4 (H3K4) methylation, while the two embedded in
the late-replicating environment (005HM4 and 005HM5) lacked
this modification (Fig. 6D). Conversely, ChIP for markers of re-
pressed chromatin, H3 lysine 9 (H3K9) dimethylation and asso-
ciation of HP1?, showed that the two promoters in the late-
replicating domain were in repressed chromatin. Five out of the
(A) Plot of smoothed TR50 against AT content in a 10-kb sliding window.
Lowess smoothed curve done at f = 0.3 (fraction of the data included in
the running local fit) is overlaid in black to show the general trend. (B–D)
Histograms showing distribution of (B) CpG islands, (C) gene density, and
(D) transcripts (HeLa cells) against temporal segregation of replication.
Correlation between replication time and genomic features.
Karnani et al.
870 Genome Research
seven promoters in the early-replicating segments were negative
for markers of repressed chromatin, while the other two were
positive (Fig. 6E,F).
Therefore, the island of late-replicating DNA represents a
specific chromosomal domain with all the features of hetero-
chromatin: low gene density, low gene expression, lack of acti-
vating chromatin marks, and presence of repressive chromatin
marks. The rapid transition of the features of heterochromatin in
this late-replicating island to those of euchromatin in the flank-
ing areas suggests that the chromosome may be divided into
discrete domains with different chromatin features. In addition,
the existence of such discrete adjoining domains with different
chromatin structure suggests the presence of boundary elements
that prevent the spread of euchromatin from the neighboring
areas to this island of heterochromatin.
Pan-S segments contain markers for both active and repressed
The interallelic variation in replication time observed in pan-S
replicating segments predicts that one allele will be in active
chromatin and another in repressed chromatin, leading us to test
whether pan-S replicating segments are enriched in both types of
marks. ENr132 contained extensive stretches with the pan-S rep-
lication pattern with a few interspersed segments that were ex-
clusively late replicating. The two promoters in the pan-S repli-
(ENm005). This Browser picture highlights four tracks (I–IV): (I) FISH: chromosomal location of BAC clones (RP11-54F16 and RP11-79D9, from left to
right) selected for the interphase FISH experiment shown in B; (II) Primers: chromosomal locations of the primers (005HM1–9, left to right) selected for
ChIP assay to ascertain the histone modifications and HP1?-binding sites shown in D–F; (III) RefSeq: positions of all the genes in this chromosomal
segment; and (IV) the contiguous TR50 profile. (B) Dual color FISH was performed with HeLa cells synchronously progressing through S phase.
RP11-54F16 (from early-replicating area on left) was labeled with spectrum red dUTP, while RP11-79D9 (from late-replicating area) was labeled with
spectrum green dUTP. Dual color FISH with these two BAC clones ascertained the replication time of the two regions of Chromosome 21 set 355 kb
apart. (C) Plot of smoothed TR50 (Y-axis on left, gray) against level of transcription of genes (Y-axis on right, black). The two asterisk marks represent
transcripts whose transcription levels exceeded the Y-axis limit (i.e., 2346 and 10,010 for left and right asterisks, respectively). (D–F) ChIP-PCR assay
across ENm005 region (see Supplemental Table 3 for primers). PCR was performed on DNA chromatin immunoprecipitated with antibodies against
methylated histones (H3 Lys4 and H3 Lys9) and HP1?. (Input) DNA control before immunoprecipitation; (IgG) ChIP with rabbit IgG was negative control
for nonspecific precipitation. Forty cycles of PCR were performed for H3 Lys4 and HP1? and 30 cycles for H3 K9 di-Me. The asterisks refer to primer pairs
that gave positive ChIP signal for the indicated antibodies relative to the IgG negative control. 005HM4 and 005HM5 were from the late-replicating
island in ENm005.
Replication profile demarcates chromosomal domains. (A) UCSC Genome Browser display of a 1.7-Mb region from Chromosome 21
Replication profile of 1% of human genome
cating area, 132HM1 and 132HM2, were positive by ChIP for
both the activating histone modification (H3K4 methylation)
and repressive histone modification (H3K9 dimethylation) and a
marker for heterochromatin (HP1) (Fig. 7). In contrast, 132HM3,
from a late-S replicating segment only carried the repressed chro-
matin marks and not the activating histone modification. There-
fore, combining the data in Figure 6 and Figure 7, three out of
three late-replicating promoters were exclusively in repressed
chromatin, and five out of seven early-replicating promoters
were exclusively in activated chromatin. In contrast, the pro-
moter from the pan-S replicating segment carried marks of both
active and repressed chromatin, consistent with the pan-S repli-
cation pattern arising from interallelic variation in the chroma-
Since the ENCODE project specifically selected the target 1% of
the genome to be broadly representative of the whole genome
based on criteria like gene density and sequence conservation, we
expect that the lessons learned from these high-resolution repli-
cation time profiles can be extended to the entire genome. The
pan-S-phase pattern of replication; the correlation of replication
time with chromatin modifications, gene expression, and AT
content; and the significance of chromosomal domains and
boundary elements revealed by our studies are discussed here.
We still identify regions that replicate in multiple times in S
phase in mammalian cell lines (pan-S replication pattern). Since
the genome-based studies of replication in S. cerevisiae were ex-
ecuted only in haploid strains, they were not expected to identify
regions with interallelic difference in time of replication
(Raghuraman et al. 2001). Genome-based studies of replication in
diploid organisms were also unsuitable to identify this pattern
because of the study design (MacAlpine et al. 2004; Woodfine et
al. 2004). In those studies, the time of replication was assessed by
determining the ratio of DNA content for a locus in late-S (or G2)
cells compared to G1cells. In such experiments, segments show-
ing replication in both early- and late-S phase would appear to
replicate in mid-S phase, and the pan-S pattern would be missed.
In contrast, the sampling of cells in multiple intervals in S phase
and the use of a more sensitive method of detecting replication
dependent on a positive selection for BrdU-labeled DNA enabled
us to identify chromosomal segments that replicate in multiple
intervals in S phase.
In this study, 20% of the studied genome appeared to rep-
licate asynchronously, a value that is one-third that of our pre-
vious analysis on Chromosomes 21 and 22 (Jeon et al. 2005). This
difference is due to an important refinement in the method of
analysis in the present study. In the pre-
vious work, the hybridization data from
genome-tiling arrays was analyzed by
the standard Affymetrix GTRANS soft-
ware to generate a track that showed
when the replication signal from a given
time point was significantly enriched
over signal obtained from DNA repli-
cated for the entire duration of S phase.
Although this method provided an in-
tuitive belief for replication timing, not
surprisingly, replication signal was not
only seen in the time period when the
locus replicated but lower levels of signal
were seen in adjoining time intervals.
The presence of a signal in multiple time
tracks led us to overestimate that nearly
60% of sequences showed a pan-S repli-
cation pattern (Supplemental Figure 3,
ENm001). In contrast, in this study, we
segregate probes into those that are tem-
porally synchronous versus temporally
asynchronous by quantitative criteria
that take into account the spillage of
replication signal into adjoining time
points. In addition, only large contigu-
ous DNA segments (?10 kb) containing
>60% of probes with asynchronous rep-
lication signals are classified as pan-S.
This prevents mis-calling as pan-S short
stretches where low signal strength or
cross-hybridization from isolated probes
give an apparent replication signal in
multiple intervals in S phase. As is evi-
dent from the comparison of the two
methods in one segment (Supplemental
Fig. 3), the present method gives a more
conservative estimate of segments that
Genome Browser display of a 500-kb region from Chromosome 13 (ENr132). This Browser picture
highlights three tracks: (I) Primers: ChIP-PCR primers (132HM1–3, left to right) to study histone modi-
fications and HP1?-binding sites; (II) RefSeq: positions of all the genes in this chromosomal segment;
and (III) the temporal segregation of replication data. (B,C) ChIP-PCR assay across ENr132 region (see
Supplemental Table 3 for primers) against methylated histones (H3 Lys4 and H3 Lys9) and HP1? (as
Both active and repressive chromatin marks are present in a pan-S segment. (A) UCSC
Karnani et al.
872 Genome Research
replicate at multiple times in S phase. Because microarray-based
profiling of replication is a relatively new approach, we also vali-
dated the time and pattern of replication for some of the seg-
ments by a completely independent method, interphase FISH.
The confirmation of all three pan-S regions as replicating asyn-
chronously adds to the confidence that ∼20% of the chromo-
somal segments in HeLa cells, indeed, show this unexpected pat-
tern of replication.
All 10 regions tested by interphase FISH (including the tem-
porally specific regions) reproduced the time of replication esti-
mated by the microarray-based replication profile. In addition,
the time of replication for five of five tested chromosomal re-
gions remained unaltered when a different cell cycle block
method was used in HeLa cells. Interphase FISH allowed us to
check the time of replication of the same five regions in another
cell line, MCF10A, where we found replication times of 3/5 chro-
mosomal segments to match that of HeLa. The differences at the
other two loci are likely due to differences in the chromatin en-
vironment of these loci in the two cell lines. Since MCF10A cells
are near-diploid and untransformed, the detection of pan-S-
phase replication in these cells indicates that pan-S replication is
not an artifact arising exclusively from the aneuploidy or the
transformed state of HeLa cells. It is, of course, entirely possible
that aneuploidy or cell transformation increases the fraction of
the genome that shows pan-S replication.
Since FISH-based methods analyze replication in the context
of individual nuclei, the Mid-Scores showed that the asynchrony
in replication time was due to interallelic difference in replica-
tion. Homologous alleles usually replicate synchronously in S
phase, but there are some notable exceptions to this general rule.
In humans, examples of such exceptions include monoallelically
expressed genes such as those imprinted depending on parent of
origin (Simon et al. 1999), genes encoding olfactory receptors
(Chess et al. 1994), genes on the female X-chromosome (Avner
and Heard 2001; Boumil and Lee 2001), and immunoglobulin
and T-cell receptor genes (Mostoslavsky et al. 2001). We will test
in the future whether all the pan-S segments express all their
genes monoallelically. The interallelic asynchrony in replication
in the pan-S segments suggests that one allele is in euchromatin
and the other in heterochromatin. Consistent with this, pan-S
areas are unique in being enriched in both activating and repres-
sive marks (Fig. 7), with the different marks residing presumably
in the two different alleles.
Since the HeLa cell line is of female origin (XX), the inacti-
vation of one of the X-chromosomes predicts that segments from
the X-chromosome should replicate in a pan-S manner, unless
the long passage and aneuploidy of these cells have disrupted
such inactivation. There are two regions from the X-chromosome
included under ENCODE (Supplemental Fig. 4). The 1.2-Mb
ENm006 region had three areas of pan-S replication (126 kb, 62
kb, and 10 kb), one of which contained the Glucose-6-phosphate
dehydrogenase (G6PD) gene, which is known to be transcription-
ally repressed on the inactivated X-chromosome and delayed in
replication compared to its active counterpart (Hansen et al.
1996). The second region, ENr324 (ChrX: 122,507,850–
123,007,849), contained no pan-S replicating segments. Thus the
survey of the X-chromosome fragments for pan-S replication
gave mixed results. The lack of pan-S replication over the entire
stretch of X-chromosome in HeLa cells could not only be due to
transformation and long-term culture affecting inactivation, but
also because ENm006 and ENr324 contain blocks of genes that
normally escape X-chromosome inactivation, similar to many
reported X-linked genes (Chang et al. 1990; Disteche 1995; Miller
et al. 1995; Carrel et al. 1996; Vermeesch et al. 1997).
Correlation of gene expression with time of replication in
eukaryotes has produced contradictory results. In S. cerevisiae, the
expression of genes did not correlate with their time of replica-
tion in S phase. In contrast, in cultured Drosophila cells, there was
a positive correlation between early replication and gene tran-
scription (MacAlpine et al. 2004). In mammalian cells, house-
keeping genes like Hprt, histones, beta-tubulin, actin, and rDNA
are ubiquitously expressed and replicated in the first half of S
phase. On the other hand, tissue-specific genes such as those
coding for Factor IX, fibronectin, and myosin heavy chain rep-
licate late in the cell lines not expressing them (Holmquist et al.
1982; Iqbal et al. 1984; Goldman 1988). The previous study from
our laboratory on human Chromosomes 21 and 22 also showed
a positive correlation between early replication and gene expres-
sion, but the results could have been improperly skewed because
of atypical features of the two small chromosomes. The positive
correlation between early replication and gene expression in this
study is likely to be generally true throughout the genome, be-
cause it was obtained with a distributed set of segments that
together are representative of the entire genome.
The association of early replication with gene expression
suggests that there are consistent differences in chromatin envi-
ronment between the early- and late-replicating segments. Cyto-
logical studies have shown spatial differences in nuclear staining
for both activating and silencing histone modification marks,
and these spatial differences in histone modification are corre-
lated with differences in replication time (Wu et al. 2005). ChIP
for histone modification marks reported here strengthens the
correlation at a finer resolution: early replication and gene ex-
pression correlate with euchromatin, and conversely, late repli-
cation correlates with low gene expression and heterochromatin.
These results are confirmed in a wider study that correlates our
replication time data with ChIP-on-chip data for histone modi-
fications done by the ENCODE Consortium (The ENCODE
Project Consortium 2007).
Interestingly, the finer resolution offered by genome-tiling
microarrays identified chromosomal segments with acute transi-
tions in replication timing. For one particular segment (ENm005)
(Fig. 6), the replication time transition was confirmed by inter-
phase FISH and appeared to correlate with transitions in both
gene expression and chromatin modifications: a late-replicating
island of 355 kb had repressive chromatin marks and low gene
expression. The genes OLIG1 and OLIG2 in this island are known
to be expressed during development of oligodendrocyte (OL) lin-
eage (Jakovcevski and Zecevic 2005), and thus the island is ex-
pected to become early replicating in oligodendrocytes.
Identification of transition zones separating chromosomal
domains is an interesting outcome from the replication profiles.
Thirty-one genes of biomedical significance (including 10 onco-
genes/tumor suppressor genes on 11q and 21q) reside in or near
replication-timing transition regions (Watanabe et al. 2002). The
mechanism by which a boundary is maintained between euchro-
matin and heterochromatin around these transition zones is not
understood. At the major histocompatibility complex (MHC),
loci replication timing switches precisely where there is a transi-
tion in the GC% content and is associated with nuclear scaffold
attachment regions (Tenzen et al. 1997). A similar transition in
GC content in the neurofibromatosis 1 (NF1) gene demarcates
early replicating from late-replicating chromatin, and a stalled
replication fork was observed in this transition region (Schmeg-
Replication profile of 1% of human genome
ner et al. 2005). The sites of replication time switch identified by
our method will likely lead to the identification of more such
transition zones, and we are interested in determining in the
future whether such zones cause replication forks to slow down
or stall, whether they contain nuclear scaffold attachment re-
gions, and whether they act as boundary elements responsible
for keeping adjoining chromatin domains separate from each
In humans, the R and G chromosomal bands have been
linked to both gene density and AT/GC content. G bands are
AT-rich, while the R bands are more GC-rich. GC-rich regions are
not only enriched in genes but specifically in expressed genes
(Saccone et al. 1993; Caron et al. 2001; Lander et al. 2001; Ver-
steeg et al. 2003). The moderately positive correlation between
AT content and TR50 (0.26 at 10-kb, 0.19 at 1-kb resolution)
suggests a trend favoring an increase in AT content as we move
from early- to late-replicating chromosomal segments. This ob-
servation is also in concordance with our previous study on
Chromosomes 21 and 22 (Jeon et al. 2005). The correlation in-
creases as the computation is done at larger scales, suggesting
that the influence of AT content on TR50 occurs at scales greater
than tens of kilobases. Consistent with this, replication-timing
studies done at 1-Mb resolution show an even stronger positive
correlation with AT content (Woodfine et al. 2004).
Finally, the smoothed TR50 profile suggests locations of ori-
gins of replication at local minima and positions of replication
fork termination at local maxima. Replication speed can also be
estimated based on the slope of the smoothed TR50 profile at a
given locus. These possibilities will be explored in our future
Cell culture, synchronization, and FACS analysis
HeLa and MCF10A cells were cultured as per standard growth
conditions. For synchronous progression through S phase, HeLa
and MCF10A cell lines were arrested by thymidine-aphidicolin
block and released as described earlier (Jeon et al. 2005). For
nocodazole block, HeLa cells at 60% confluency were treated
with 40 ng/mL nocodazole for 10 h. This was followed by selec-
tion of cells blocked in mitosis by mitotic shake-off. These cells
were washed three times with PBS and released into fresh
medium for 12 h to reach the 0-h point when they enter S
phase. Cells harvested at indicated time points of S phase were
either used for FISH or fixed in 70% ethanol and stained with
propidium iodide (PI) for FACS by standard methods.
Newly replicated DNA (H/L DNA) purification
Synchronously released cells were labeled with 100 µM BrdU for
the indicated time interval; 10 ∼ 30 150-mm plates of cells were
used to purify H/L DNA from each time point as described earlier
(Jeon et al. 2005).
To generate replication time profiles, ENCODE01-Forward (P/N
900543; Affymetrix) tiling arrays were used. These arrays are de-
signed to study the pilot ENCODE regions of DNA, comprised of
30 Mb of DNA, or ∼1% of the human genome. These pilot regions
were selected by a committee of the National Human Genome
Research Institute (NHGRI). Half of the content on the
ENCODE01 Array was manually selected by the NHGRI commit-
tee, while the remaining 50% was randomly selected (The
ENCODE Project Consortium 2004). A total of 14.82 Mb of se-
quence constituted the manually selected regions and included
14 targets ranging in size from 500 kb to 2 Mb. The randomly
selected content includes 30 500-kb regions selected based on
gene density and level of nonexonic conservation.
Nonrepetitive, 25-mer oligonucleotide probe pairs (Perfect
Match, PM; Mis-Match control, MM) spaced at intervals of ∼22
bp as measured from the central nucleotide were spotted on ar-
rays. Heavy/light DNA from each time point was fragmented to
50–100 bp by DNase I digestion, end-labeled with biotin-ddATP
using terminal transferase, and hybridized to the arrays as per the
manufacturer’s protocol (Affymetrix). Each microarray was
scanned and analyzed for signal intensities using a GeneChIP
Scanner 3000 and GeneChIP Operating Software (GCOS; Af-
fymetrix). Two biological and one technical replicates were hy-
bridized to ascertain the reproducibility of array hybridizations.
The primary data in the form of .cel files can be accessed at
http://www.cs.virginia.edu/∼cmt5n/Rawtimepoints/. All the pri-
mary and processed data have been generated using the hg17
(NCBI Build 35, May 2004) build of the Human genome assem-
The replication signal for each probe was calculated as
PM ? MM. If the difference was negative, then the signal was
assigned a value of 0. For a given probe on the array, we have five
replication signals, one from each time point. Each probe is clas-
sified to be replicating either in a temporally specific or nonspe-
cific (asynchronous) manner as follows. Probes were temporally
specific if the signal in any one time point was at least twice the
signal of each of the other four time points. To accommodate the
possibility that a temporally specific replication signal could
span two adjacent time points, probes were also called specific if
the sum of any two adjacent time points was at least three times
the signal of each of the other three time points. Probes that do
not satisfy either of the criteria above are designated as tempo-
rally nonspecific. Supplemental Table 1 gives some examples of
the specificity classification. For the Affymetrix ENCODE array,
we classified 26.115% of the probes as temporally nonspecific in
their pattern of replication.
For studying gene expression, RNA was extracted from loga-
rithmically growing HeLa cells by using an RNeasy Kit (QIAGEN)
and hybridized to the Human HG-U133 Plus 2.0 array (contain-
ing ∼38,500 genes) as described in the Affymetrix GeneChIP pro-
tocol (Affymetrix). Each microarray was scanned, visualized, and
analyzed for the level of each individual transcript using a Ge-
neChIP Scanner 3000 and GeneChIP Operating Software (GCOS;
Affymetrix). Transcript signal was mapped against the chromo-
some coordinates (probe-by-probe basis) using the HG-U133A_2
Annotations, CSV provided by the manufacturer (Affymetrix).
Segregation of temporally specific and pan-S replicating
To segregate broad regions of replication, a sliding window of 10
kb was passed along each chromosomal segment, calculating the
percentage of temporally nonspecific probes within the window.
A pan-S region is begun when the percentage exceeds 60% and
continues until it drops below the 60% threshold minus a given
tolerance (e.g., 10% for our analysis). The tolerance is introduced
in order to avoid thrashing between nonspecific and specific re-
gions. Once the percentage drops below “threshold tolerance”
(e.g., 50% for our settings of threshold and tolerance), the current
pan-S region ends and a temporally specific region is started. The
temporally specific region is continued until the percentage
again rises above the threshold. In this manner, moving along
the chromosome, broad regions of replication are segregated into
temporally specific or pan-S classes.
Karnani et al.
The tolerance parameter, which helps us avoid thrashing
between the two classes, introduces a directional bias into the
segregation algorithm. As we move from lower chromosomal po-
sitions to higher chromosomal positions, the percentage must
exceed 60% in order to begin a pan-S region. But the pan-S region
does not end until the percentage drops below 50%. In order to
correct for this directional bias, we perform two passes of the
algorithm. One pass moves the window toward higher chromo-
somal positions, while the other pass moves the window toward
lower chromosomal positions. Then we merge the two passes
into a single segregation, which no longer suffers from a direc-
Interphase fluorescence in situ hybridization
Cells in S phase were harvested at indicated time points and
incubated in pre-warmed 75 mM KCl solution for 15 min at 37°C
to prepare nuclei. These nuclei were fixed in 3:1 (v/v) methanol/
glacial acetic acid and mounted on a slide. A nick translation kit
and SpectrumGreen dUTP/Spectrum Red dUTP (Vysis Inc.) were
used for labeling the probe. Hybridization was carried out in a
humidified chamber for 16 h at 37°C as described in the Vysis
FISH protocol (Vysis Inc.). Slides were washed with 0.4? SSC/
0.3% NP-40 for 2 min at 73°C and again with 2? SSC/0.1%
NP-40 solution for 1 min at room temperature. Chromosomal
DNA was counterstained with DAPI (VECTASHIELD Mounting
Medium; Vector Labratories) and visualized with a Nikon Micro-
phot.SA fluorescent microscope equipped with a DAPI filter,
FITC, and a TRITC cube set (for Spectrum Green and Spectrum
red fluors, respectively). Images were digitally obtained with a
Nikon UFX-DX camera and Spot version 3.5.4 software. All the
BAC clones were purchased from Children’s Hospital Oakland
The number of dots was visually counted in ∼100 cells at
each time interval, and the number of dots/cell was calculated;
100% replication (in G2cells) was when the increase in the num-
ber of dots/cell equaled the number of dots/cell observed in G1.
After determining the dots/cell at 0, 2, 4, 6, 8, and 10 h of S phase,
for each interval (e.g., 0–2 h, 2–4 h, etc.), we calculated the in-
crease in dots/cell during that interval and converted it to the
percent of replication.
Correlation of TR50 with genome features
CpG island annotations were obtained from the UCSC Ge-
nome Browser Web site (http://genome.ucsc.edu/cgi-bin/
hgTrackUi?hgsid=64765488&c=chr16&g=cpgIsland). The den-
sity of CpG islands was calculated for all the chromosomal re-
gions in each of the replication segments, that is, early-, mid-,
late-, and pan-S. For the CpG islands that overlapped two tem-
poral segments, the number of bases in the CpG island were
counted and a 60% cutoff was used to assign it a specific temporal
For determining gene density, we used the annotated
genes under the Refseq database from the UCSC Genome
Enrichment in each of the replication segments (early-,
mid-, late-, and pan-S) within a given data set (CpG islands, gene
density, and transcripts) was calculated as follows. The number
of elements from the data set whose majority base count fell into
early segments was calculated. This was divided by the total num-
ber of elements in the data set to get a ratio of early-replicating
elements. This ratio was divided by the ratio of early segments to
all segments to give the enrichment ratio. Hence, a value of 1.0
would indicate that the data set was distributed in early segments
as was expected by chance, while a value of 2.0 would indicate
twice as many as expected by chance. Enrichment of the mid-,
late-, and pan-S replicating regions was calculated similarly.
A chromatin immunoprecipitation assay was performed as per
the protocol described at http://www.upstate.com with a varia-
tion in the sonication step. Samples were sonicated using a Bran-
son microtip (3.2 mm) and Fisher Model 500 Sonic Dismembra-
tor. Eight cycles of 15-sec pulse at 50% amplitude and 45 sec of
cooling on ice were done to disrupt the cells. The antibodies used
for ChIP were for identifying sites of histone 3 Lys 4 mono-, di-,
and trimethylation (H3K4 Me), histone 3 Lys9 dimethylation
(H3K9 di-Me), and HP1?. These antibodies were purchased from
Upstate (Anti-H3K4 Me; 05-791 and H3K9 Anti-Me; 07-441) and
Abcam (Anti- HP1?; ab9057). To determine the ChIP signal for
H3K9 di-Me, 4 µL of ChIP DNA were first amplified in a linear
range (14 cycles) using the WGA2 kit from Sigma-Aldrich and
cleaned up by the QIAGEN PCR clean-up kit. Two microliters of
this purified DNA was used as template for ChIP assay with prim-
ers. To rule out any amplification bias, three independent am-
plifications were performed and PCR with primers repeated with
each of these template preparations. As a negative control, ChIP
DNA from an IgG sample was amplified in a similar way. The
details on primers used for ENm005 and ENr132 regions are pro-
vided in Supplemental Table 3.
This work was supported by National Institutes of Health Grant
HG003157 (to A.D.). We thank members of the Dutta laboratory
for helpful discussions.
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Received April 21, 2006; accepted in revised form October 30, 2006.
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