Developmental control of gene copy number
by repression of replication initiation
and fork progression
Noa Sher,1George W. Bell,1Sharon Li,1Jared Nordman,1Thomas Eng,1
Matthew L. Eaton,2David M. MacAlpine,2and Terry L. Orr-Weaver1,3
1Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA;
2Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on
a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be
repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are
transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of
the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically
reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. In-
hibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce
copy number. Although repressive histone marks are removed whenSUUR is mutated and copy number restored, neither
transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block
replication, however. These results establish that developmental control of DNA replication, at both the initiation and
elongation stages, is a mechanism to change gene copy number during differentiation.
[Supplemental material is available for this article.]
Precise DNA replication is crucial for genome maintenance, and
most cancer cells contain regions of increased gene copy number
(Kallioniemi 2008). The mechanisms controlling metazoan repli-
cation origin activation and fork progression have been difficult to
investigate not only because few origins have been delineated but
also because initiation and elongation are hard to capture and
mechanistically distinguish in vivo. Recent genomic approaches
defined sites and timing of replication initiation in cell culture
et al. 2008; Schwaiger et al. 2009; Sequeira-Mendes et al. 2009;
et al. 2011). These studies have established an association between
replication initiation, active transcription, and, in the case of Dro-
sophila cells, Origin Recognition Complex (ORC) binding. Com-
parison of different cell culture lines suggests that there is plasticity
in origin timing activation (Schwaiger et al. 2009; Hiratani et al.
2010; Mesner et al. 2011; for reviews, see Gilbert 2010; Mechali
2010). To understand the relationship between DNA replication
and differentiation, however, it is crucial to investigate replication
in vivo in tissues undergoing differentiation. For example, ORC
binding has yet to be mapped in a differentiated metazoan tissue.
Developmental modification of the parameters of DNA repli-
cation can provide powerful models both to identify metazoan
replication origins and to elucidate their regulation. Increased DNA
content at a genomic level is common throughout the plant and
animal kingdoms, resulting in polyploid cells such as mammalian
megakaryocytes or polytene cells such as rodent placental tropho-
blasts (Edgar and Orr-Weaver 2001; Lee et al. 2009; Ullah et al.
2009). Polyploid and polytene cells differ in that in the latter the
replicated sister chromatids remain physically aligned. In many
polyploid or polytene cells, the genome doubling is not integral,
and there is differential replication, i.e., genomic regions whose
copynumberisamplifiedabovethe overallploidyofthe genome or
under-replicated with reduced copy number at certain genomic re-
gions. Because this differential replication occurs in response to
can be analyzed at its time of occurrence and regulatory mecha-
nisms delineated. For example, analysis of developmentally regu-
lated gene amplification in Drosophila ovarian follicle cells revealed
that there are multiple mechanisms by which origins can be acti-
vated (Park et al. 2007; Xie and Orr-Weaver 2008; Kim et al. 2011).
Heterochromatin is known to be under-replicated in Drosoph-
ila polytene tissues (Spradling and Orr-Weaver 1987). Constitutive
heterochromatic regions, localized in large blocks surrounding the
centromeres, make up about a third of the Drosophila genome, but
because these regions are composed of highly repetitive DNA, they
are not molecularly tractable. Belyaeva and coworkers defined in-
tercalary heterochromatin in the larval salivary gland as regions
dispersed on the chromosome arms that appear condensed and
constricted, and therefore likely to be under-replicated (Belyaeva
et al. 2008). They identified a fascinating chromatin protein, SUUR
(Suppressor of Under-Replication), whose function is required for
under-replication of the centric and intercalary heterochromatin
(Belyaeva et al. 1998). By using microarrays of Drosophila EST se-
quences, genomic regions whose copy number is affected by SUUR
were identified by array–based comparative genomic hybridization
(aCGH), comparing copy number in strains with overexpressed
SUUR relative to a SuUR mutant (Belyakin et al. 2005). These studies
uncovered 52 SUUR-dependent under-replicated regions, and it was
shown that the protein localizes broadly to the intervals (Pindyurin
Article published online before print. Article, supplemental material, and pub-
lication date are at http://www.genome.org/cgi/doi/10.1101/gr.126003.111.
Freely available online through the Genome Research Open Access option.
22:64–75 ? 2012 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/12; www.genome.org
et al. 2007). Although these regions contain transcription units,
they have heterochromatic characteristics of late replication in dip-
loid cell culture (Belyakin et al. 2005; Pindyurin et al. 2007).
A recent analysis of transcription factor and chromatin pro-
tein binding, measured by DamID in transfected Drosophila Kc167
cell culture, defined five chromatin states (Filion et al. 2010). This
study showed that chromatin can exist in a state associated with
repression of transcription and these regions have reduced gene
density, but this is distinct from heterochromatin. Almost 50% of
the genome is in such a ‘‘BLACK’’ chromatin state, bound by the
proteins SUUR, histone H1, D1, and IAL, with Lamin, the Su(Hw)
insulator, and the Effete protein frequently also associated. BLACK
chromatin is likely to be repressive for replication, as these do-
mains undergo late replication and have reduced levels of ORC
binding in cell culture (MacAlpine et al. 2010). Whether the ge-
nomic localization of BLACK chromatin is conserved in differen-
tiated cells in vivo remains to be determined.
Here we use tiled genomic microarrays to identify all eu-
chromatic regions of differential replication in Drosophila salivary
glands and high-throughput RNA sequencing to quantify tran-
script levels across the genome. We have identified 34 under-rep-
licated regions that correspond to a subset of BLACK chromatin
and are repressed for transcription. We investigated the mecha-
nism by which replication is repressed in these domains by ana-
lyzing the localization of ORC in wild-type and SuUR mutants. We
find that ORC is absent from these domains and that loss of SUUR
function results in replication across the domains, apparently by
increased replication fork progression from flanking regions.
Euchromatic genomic regions are under-replicated throughout
salivary gland development
We used tiled microarrays spanning the entire Drosophila eu-
chromatin to compare the copy number from isolated third-instar
larval salivary glands with that of early-stage diploid embryos, in
wild-type strains in which replication was not perturbed. The
criteria for calling a sequence probe on these arrays euchromatic
is that it is not present in pericentric or telomeric heterochro-
matin blocks, corresponds to chromosomal arm regions visible
on polytene chromosomes, and is a nonrepetitive sequence. We
identified 34 domains ranging from 100–450 kb that were up to
10-fold under-represented in copy number (Fig. 1A; Supplemental
Fig. S1; Supplemental Table 1). In addition to these euchromatic
regions, there were five regions with pericentric heterochromatin
probes present on the arrays that were under-replicated. We de-
fined aregion asbeingunder-replicated ifthe copy number wasat
least twofold reduced in regions of $10 kb in two independent
aCGH experiments. The MA2C peak calling analysis software
(Song et al. 2007) also identified these domains as significantly
under-replicated, as well as 19 others that are less than twofold
under-replicated. We focused on the 34 domains in euchromatin
that were at least twofold under-replicated for our studies.
Quantitative PCR was used as an independent means to confirm
that the copy number was indeed reduced (Supplemental Fig.
S2). Twenty-five of the under-replicated regions identified in
this study were also found to be under-replicated in the study
by Belyakin et al. (2005), in which SUUR was overexpressed. Of
the nine unique regions, three were not probed by the micro-
arrays used in the previous study, as these contained solely EST
To ask when during salivary gland development this under-
replication begins, we dissected salivary glands from embryos and
from each larval instar, and measured copy number at four under-
replicated loci relative to a fully replicated control region by qPCR.
For dissection from embryos and first-instar larvae, salivary glands
were GFP-labeled with a Forkhead GAL4 driver to activate a UAS-
GFP reporter. From the earliest endocycles, the under-replicated
domainsarealreadyestablishedandbeginto be under-represented
(Fig. 1B; for model of replication forks at under-replicated regions,
see Fig. 1C).
Drosophila Oregon-R (OrR) third-instar salivary glands indicates the presence of euchromatic under-replicated regions. The genome profile shows the log2
ratio of copy number in the third-instar salivary gland genome relative to that of diploid embryonic DNA. Under-replicated loci are indicated by vertical
light green bars. OrR flies are the wild-type strain used throughout this study. For this and other figures, UCSC Genome Browser (build dm3) was used
(http://genome.ucsc.edu) (Rhead et al. 2010; Fujita et al. 2011). (B) Under-replication begins with the first endocycle in the embryonic salivary gland.
Levels of DNA at four under-replicated loci relative to a fully replicated control locus were measured by qPCR in embryonic and first-, second-, and third-
sample t-test, the embryonic salivary gland sample is significantly different from the diploid embryo sample, P = 0.012. (C) Model of replication fork
structure at under-replicated region.
Euchromatic regions are repressed for replication early in development. (A) aCGH of the proximal euchromatic half of chromosome 2L of
Developmental control of origins and fork movement
Transcription is repressed in under-replicated domains
Given the positive relationship previously observed between rep-
lication and active promoters in metazoan cells, we speculated
that genes within the under-replicated domains would show re-
duced transcript levels and that under-replication might be a
mechanism to reduce gene expression in polytene cells (Schubeler
et al. 2002; MacAlpine et al. 2004, 2010; Dutta et al. 2008; Karnani
et al. 2010). Using Illumina-based RNA sequencing (RNA-seq) of
RNA isolated from third-instar larval salivary glands, we per-
formed a genome-wide analysis of transcription levels. We also
measured the presence of RNA polymerase II (Pol II) by ChIP-chip
(chromatin immunoprecipitation from dissected salivary glands
followed by hybridization of the labeled precipitated DNA to
We found that genes not only at the bottom of under-repre-
sented (UR) troughs but also at their sides are silent, with the ex-
ception of six in the center of the UR troughs and nine more at the
edges of the troughs (Fig. 2B; Supplemental Fig. S1). These regions
lack RNA Pol II (Fig. 2C), and thus the absence of transcripts is due
to absence of transcription, as opposed to transcript instability.
Genome-wide, the RNA Pol II signal is highly predictive of the
presence of transcripts (Spearman’s rank correlation coefficient =
0.7, P < 2 3 10?16). Therefore, the under-replicated regions in the
salivary gland are transcriptionally repressed (Supplemental Fig.
S3). For a list of the GO terms of repressed transcripts, see Sup-
plemental Table 2.
Histone H3 trimethylated at lysine 27 (H3K27me3) is a mark
associated with transcriptional repression of euchromatic regions
(Schwartz and Pirrotta 2007) and has recently been shown to be
the only repressive histone modification tested whose binding
pattern parallels chromatin accessibility (Bell et al. 2010). There-
fore, we checked, by ChIP-chip of third-instar salivary glands,
whether the chromatin configuration in the under-replicated re-
gions was likely in a repressed, inaccessible state and found that all
under-replicated regions show the repressive H3K27me3 mark to
varying extents (Fig. 2D, see Fig 6B; Supplemental Fig. S4). The
under-replicated regions, however, were not the genomic intervals
most intensely marked by the presence of H3K27me3.
The ORC is absent in under-replicated regions
The reduction in copy number in the under-replicated domains
could be due to a lack of origin firing within under-replicated re-
gions, a block to replication fork progression through these re-
gions, or a combination of the two. Therefore, we wanted to as-
certain whether initiation complexes, marking potential origins of
replication, were present in the under-replicated regions. We per-
formed immunoprecipitations with anti-ORC2 antibody from dis-
sected third-instar larval salivary glands and analyzed the samples
either by ChIP-chip or by Illumina sequencing (ChIP-seq). Strik-
ingly, both ChIP-seq (Fig. 2E) and ChIP-chip (Supplemental Fig.
kb in under-replicated regions was greatly diminished compared
with that in the remainder of the fully replicated genome (see Fig.
6A). Drosophila origins tend to be spaced ;40 kb apart (Spradling
proximal half of chromosome 2L, as described in Figure 1A. (B–E) RNA-seq, RNA Pol II ChIP-chip, H3K27me3 ChIP-chip, and ORC2 ChIP-seq peaks in
Oregon-R (OrR) third-instar salivary glands, respectively. Total Illumina RNA sequencing reads are plotted; RNA Pol II ChIP was normalized to IgG control
ChIP, and H3K27me3 ChIP data to input DNA. ORC2 ChIP-seq peaks were called by SPP compared with input sequencing lane; peaks for these data are
organized downward by the UCSC Genome Browser.
Under-replicated regions are restrictive for ORC2 and RNA Pol II binding and are marked by weak binding of H3K27me3. (A) aCGH of
Sher et al.
harbor extremely sparse ORC binding sites or are devoid of ORC
binding sites and therefore likely do not initiate replication by
origin activation from a pre-RC complex.
Park and Asano (2008) argued that in the salivary gland cells,
ORC is dispensable for DNA replication. This conclusion implies
that there would be no consequence of an absence of ORC in the
under-replicated regions. Thus we examined the properties of ge-
nomic replication in the salivary glands of orc1- and orc2-null
mutants (Landis et al. 1997; Park and Asano 2008). We find by
microdensitometry that overall ploidy is reduced fourfold in orc2
and twofold in orc1 mutants (Fig. 3A for orc2; Supplemental Fig. S6
for orc1). Park and Asano (2008) also observed a twofold reduction
in ploidy in orc1 mutants. Our aCGH analyses showed a markedly
altered pattern of differential DNA replication in these mutants.
We found dramatic changes in the copy number associated with
a loss of ORC function. All but the most pronounced under-repli-
cated regions became fully replicated, and there were interspersed
regions of experimentally reproducible, but disorganized, ampli-
fication and under-replication (Fig. 3B; Supplemental Fig. S6).
Thus, ORC is indeed functioning in polytene replication and af-
fects the distribution of replication along the genome. The
remaining replication in the mutants may have been allowed by
maternal loading or residual activity in ORC complexes missing
the mutated subunit. In addition, ORC-independent mechanisms
cation. (A) Ploidy levels of orc2 mutants and heterozygous sibling controls were quantified using DAPI microdensitometry. The intensity of DAPI staining
was measured relative to diploid cells to calculate their ploidy. Each symbol represents the DAPI signal intensity from a single nucleus in two experimental
replicates, and the bar is the mean of the measured nuclear values. (B) aCGH was performed comparing DNA of orc2k43/Df(3R)Exel6288 and orc2k43/
Df(3R)Exel6171 third-instar salivary glands with that of diploid embryo. Oregon-R (OrR) aCGH is shown for comparison. The baseline of each aCGH is
based on the average of all points on the array, so that the baseline of the orc mutant aCGHs is fourfold below that of the OrR aCGH shown. Plotted is the
mean probe intensity. The asterisk marks examples of disordered but experimentally consistent overreplication, whereas the ^ symbol indicates disor-
ganized but reproducible under-replication.
orc2 mutants have reduced ploidy levels and show increased copy number of most under-replicated regions, as well as disorganized repli-
Developmental control of origins and fork movement
of DNA replication may have resulted in the altered patterns of
differential DNA replication in the orc mutants. Additional studies
will be required to ascertain this point.
Comparison of ORC binding in the salivary gland
and cell culture
As our study is the first in which ORC binding has been mapped in
a primary tissue, we compared the sites of ORC binding across the
with data sets from studies previously performed on several Dro-
sophila cell lines (Fig. 4A; Eaton et al. 2011). Whereas 1618 sites are
utilized by all cell types, there is a cell specificity to ORC binding,
with the salivary gland tissue differing most in its ORC binding
sites relative to the cell lines, perhapsdue to its more differentiated
state. Of the 5253 ORC binding sites in the salivary gland, 1501
cell lines (Fig. 4A).
In cell lines, ORC binding is associated with transcription start
sites (TSSs) of actively transcribed genes (MacAlpine et al. 2010).
Sixty-fourpercent ofORCsitesare withina kilobase ofan active TSS
(MacAlpine et al. 2010). We examined the relationship between
association and to determine whether differences in transcription
could account for the tissue-specific ORC binding sites (Fig. 4B). In
the salivary gland, 3819 ORC binding sites are within a kilobase of
a TSS (73%), showing the same principle of ORC localization in this
controlled by these promoters are not uniquely expressed in the
salivary gland (Fig. 4B).Thusthetissuespecificity of ORC binding is
not explained by active TSS sites.
We compared the pattern of ORC binding in the salivary
gland and Kc cell culture in the regions that become under-repli-
cated in the salivary gland (Fig. 4C). In Kc cells, these 34 regions
the levels are notably higher than in the salivary gland.
SuUR mutants restore replication, without reestablishing
transcription or ORC binding
regions, we turned to flies with mutated SUUR, a protein already
compared between the different cell types (cultured cell lines or salivary gland) indicated on the left. (Red boxes) Origins unique to a single cell type;
(orange) origins shared by two cell types; (green) origins shared by three cell types; (purple) origins shared by all four cell types studied. Bars in blue above
the boxes show the number of origins shared by the cell types indicated by the color-coded boxes below each bar. Less than 1 kb proximity of ChIP peaks
was required for binding site conservation. The number of ORC binding sites differs slightly from that reported by Eaton et al. (2011) because our data
merge some ORC binding sites into broader peaks. (B) Rectangular Venn diagram comparing the relationship between ORC binding and TSSs in salivary
glands and Kc cell culture, with rectangles drawn to scale. After identifying the TSS nearest to each ORC site, the percentile rank of the corresponding
transcript (in RPKM) was determined from the salivary gland and Kc cell RNA-seq data. From the difference in percentile ranks (DPR) between the salivary
gland and Kc cell, each transcript was classified as salivary gland specific (SG >> Kc, for DPR > 40, black), higher in salivary gland or no difference (for DPR
ORC binding across the under-replicated regions in the salivary gland and the corresponding regions in Kc cells. Each of the 34 domains is displayed
vertically, in the same order for ease of comparison. Each domain that is under-replicated in the salivary gland was divided into 100 windows, and each
site in the window.
Comparison of ORC binding sites in salivary gland cells (SG) to those in Kc, S2, and Bg3 Drosophila cell lines. (A) Origin binding sites were
Sher et al.
68 Genome Research
1998). We measured the copy number in SuUR mutants and found
all the under-replicated domains to be fully replicated by aCGH,
with slight partial under-replication only at cytological location
36D (2L: 16923976–17352361), the under-replicated domain with
the lowest copy number and spanning the largest region in the
entire Drosophila genome (Fig. 5B; for qPCR verification, see Sup-
plemental Fig. S7; for other chromosome arms, see Supplemental
We next utilized the SuUR mutant as a tool to dissect mech-
anistically how SUUR affects differential replication using the pa-
and the presence of the H3K27me3 repressive chromatin mark. We
first looked at ORC localization by ChIP-seq and ChIP-chip from
the salivary glands dissected from SuUR mutant larvae. Strikingly,
we found that loss of functional SUUR protein allowed a restora-
tion of copy number without a concomitant restoration of ORC
binding within the previously under-replicated domains (Fig.
5C,D; Supplemental Fig. S8,9). Figure 6A quantitatively illustrates
in the SuUR mutant.
Given that replication in SuUR mutants proceeds through
previously under-replicated regions, we asked whether the chro-
matin accessibility of these regions was altered, looking both at
transcription levels by RNA-seq and at the repressive H3K27me3
mark, which in wild-type flies marked all under-replicated do-
mains, by ChIP-chip. In SuUR mutants, transcription was still re-
pressed within the regions normally under-replicated, although
copy number was restored (Figs. 5E, 6C). As it was possible that
replication restored RNA Pol II binding but downstream factors
were lacking to fully restore transcription, we performed a ChIP-
chip with anti-RNA Pol II antibody and found that it did not bind
the under-replicated regions in SuUR mutants (Fig. 5F). Whereas
neither RNA Pol II binding nor transcription was restored in the
salivary glands of SuUR mutants, the H3K27me3 mark was lost
(Fig. 5G) and was restored to levels equal to those in the remainder
of the genome (Fig. 6B). The exceptions are several genes within
under-replicated regions that retain the H3K27me3 modification
in SuUR mutants and an under-replicated region at 89E on chro-
mosome 3R (12482529–12793969) that retains high levels of this
mark in the mutant.The loss of the H3K27me3mark in contrastto
the continued transcriptional repression and absence of ORC
binding in the SuUR mutant indicates that this histone modifica-
tion is not causal for these effects.
repressive H3K27me3 mark. (A,B) aCGH of proximal half of chromosome 2L in OrR (A) and SuUR mutant (B) third-instar salivary glands, as described in
Figure 1A. (C) ChIP-seq peaks of ORC in SuUR mutant third-instar salivary glands, called as described in Figure 2. (D) The ChIP-seq peaks of ORC from
wild type for comparison. (E) Number of reads from Illumina RNA sequencing of SuUR mutant third-instar salivary glands. (F ) RNA Pol II ChIP of SuUR
mutantthird-instarsalivaryglands, normalized to IgG controlChIP.(G)H3K27me3 ChIP-chip inSuURmutantthird-instarsalivary glands, normalized to
Loss of SuUR function restores genome replication without restoring ORC binding or transcription, but does lead to depletion of the
Developmental control of origins and fork movement
SuUR mutants show enhanced rates of replication
Two mechanisms could explain how under-replicated regions be-
come fully replicated in an SuUR mutant salivary gland. Either
there is ORC-independent initiation in under-replicated regions
normally repressed by SUUR, or ORC-initiated replication forks
from outside these regions travel farther in the absence of SUUR.
Although it is difficult to test for ORC-independent initiation, we
tested the second hypothesis by devising an assay to determine
whether SUUR affects replication fork progression.
To test the rate of replication fork progression in the SuUR
mutants, we exploited another model system of metazoan replica-
tion: gene amplification in the follicle cells of the Drosophila ovary.
Developmentally regulated gene amplification occurs in these
cells, producing 100-kb regions in which the DNA copy number
is increased in a symmetrical gradient. From the peak of each
amplicon,the copynumberdecreasesoutto ;50 kboneitherside.
Mutants that enhance replication fork movement cause gradients
extending to 200 kb (Park et al. 2007), and the gradients have been
shown to reflect the rate of replication fork movement (Claycomb
et al. 2002). We measured the copy number in the amplicons in
wild-type and SuUR mutant follicle cells. In comparison with the
wild type, SuUR mutant amplicons are broader but not taller (Fig.
7A), with forks traveling farther in the same developmental time
window (Fig. 7B; Supplemental Fig. S10). These results indicate
that absence of SUUR leads to increased fork movement but does
not affect the number of rounds of initiation. Either SUUR may act
at the replication fork or it could control chromatin across the
domain to make a barrier to fork movement.
SUUR protein tethered to a single site is not sufficient
to repress replication
We wished to ascertain whether a single SUUR protein tethered to
onesite couldblock replication. To thisend, wemadea GAL4DNA
binding domain fusion to SUUR (GAL4DBD-SUUR) (Fig. 8A) and
created heat-shock–inducible transgenic lines with this construct.
Complementation tests of our construct in SuUR mutant lines dem-
under-replicated regions and the remainder of the genome. Note that in the SuUR mutant these regions are no longer under-replicated, but we refer to
them as UR. (A) Comparison of number of ORC binding sites per 100 kb of genome, as assayed by ChIP-seq, shows that in both OrR and SuUR mutants the
under-replicated regions have many fewer ORC binding sites than the remainder of the genome. (B) Boxplots showing quantile-normalized H3K27me3
ChIP-chip data, comparing level of H3K27me3 in under-replicated regions with the rest of the genome, in OrR and SuUR mutant salivary glands. OrR UR
was significantly enriched for H3K27me3 over the SuUR mutant UR with a P-value < 1 3 10?15(unpaired Wilcoxon test). (C) Boxplots showing quantile-
normalized RNA sequencing data (figure from one replicate; second is indistinguishable) comparing transcriptional levels in under-replicated regions with
the rest of the genome in OrR and SuUR mutant salivary glands. Mutation of SuUR does not restore transcription in UR regions. In both strains, the
transcription levels in the under-replicated regions were significantly lower than the remainder of the genome (P < 1 3 10?15, Wilcoxon test). The
difference between the under-replicated regions in the two strains was not statistically significant (P = 0.3234, Wilcoxon test).
Quantitative comparisons of ORC binding, H3K27me3 enrichment, and RNA expression levels between Oregon-R (OrR) and SuUR mutant in
Sher et al.
onstrated that the fusion protein was indeed functional (Fig. 8B), as
it was able to rescue the SuUR mutant phenotype and restore un-
We used the functional GAL4DBD-SUUR lines to tether SUUR
SUUR to a single site was sufficient to induce under-replication in
the salivary gland at an ectopic locus. Figure 8C illustrates that
tethering of SUUR to cytological location 58D8 is not sufficient to
induce under-replication, despite the fact that ChIP-PCR demon-
strates the presence of the SUUR protein at that locus. Similarly,
tethering of SUUR to a UAS site adjacent to the origin of the
no decrease in amplification levels of that genomic locus in the
follicle cells (Fig. 8D). Together, these results show that whereas
SUUR marks under-replicated regions in the salivary gland, it ei-
ther is in itself not sufficient to inhibit replication or needs to be
present across a broad domain and not only at a discrete site to
exert its repressive function on replication.
Our study supports a model in which lack of ORC binding across
broad regions combined with inhibition of progression of repli-
cation forks initiated from flanking ori-
gins can lead to chromosomal domains
that are not replicated and thus under-
represented in polytene cells. In the SuUR
mutant, these regions are fully replicated,
but ORC binding is not altered. Thus, the
SUUR protein appears necessary to re-
press DNA replication by impeding rep-
lication fork progression.
The finding that under-replication
begins from the onset of polytenization
during embryogenesis has implications
for the mechanism of replication re-
pression and supports the model of in-
hibition of fork movement as well as ini-
tiation. The salivary glands undergo nine
to 10 endocycles, rounds of alternating S
and gap phases, to reach their ploidy of
1024C–2048C. The under-replicated re-
gions are maximally 10-fold under-rep-
the onset of under-replication in embryo-
genesis, eitherthese domains arefully rep-
licated in some endocycles, there is varia-
tion in replication between cells in each
salivary gland, or there is variation be-
tween DNA duplexes in the increasingly
polytene cells. A parsimonious explana-
tion is that SUUR is not able to block
replication fork progression with 100%
efficiency, and in some cells or on some
DNA helices, the flanking forks replicate
across the under-replicated domain. By
measuring just a single pulse of CldU in-
corporation in the SuUR mutant, it was
suggested that at the histone locus, nu-
not increased (Kolesnikova et al. 2009).
Because only a subset of DNA duplexes are under-replicated each S
phase, DNA combing studies are problematic for measuring fork
rate, as they cannot distinguish replication forks being affected by
SUUR and giving rise to under-replication.
A key conclusion is that although it is necessary for under-
replication,the SUUR proteinisnot sufficient. The under-replicated
domains identified in this comprehensive aCGH study are only
a subset of the chromosomal regions to which SUUR was shown to
bind on polytene salivary gland chromosomes (Zhimulev et al.
2003). Moreover, a comparison of the under-replicated regions in
larval fat body, the midgut, and the salivary gland demonstrated
that there is tissue-specific control of under-replication (Nordman
et al. 2011). Some under-replicated regions are shared between the
three tissues, but others are under-replicated in only one or two of
the tissues. Despite the tissue specificity of the location of under-
mutated (Nordman et al. 2011), demonstrating that SUUR is nec-
essary but not sufficient to block replication. Tethering of SUUR to
a specific site in either the salivary gland or the follicle cells did not
block replication or impede fork movement. Additional evidence
that SUUR is not sufficient to repress replication comes from the
analysis of ORC distribution. There are 22 euchromatic regions
of $200 kb that lack ORC binding but are not significantly under-
replicated. From the salivary gland immunofluorescence localiza-
of Oregon-R (OrR) and SuUR mutant FACS sorted 16C follicle cell nuclei at cytological locations
encompassing Drosophila amplicon in follicle cells DAFC-66D and DAFC-7F. (B) SuUR mutant forks travel
fartherin the same developmental timewindow at DAFC-66D. SuUR mutant replication fork progression
occurs between stage 11 and 13 in egg chamber development and is not due to alterations in egg
chamber development timing. Quantitative PCR with primers 20 kb apart along the DAFC-66D
amplicon was performed on hand-sorted egg chambers, relative to an unamplified control locus. The
distances are from the peak of amplification (0 point). The experiment was performed in triplicate.
SuUR mutantfolliclecellamplicons exhibitenhancedreplicationforkprogression. (A)aCGH
Developmental control of origins and fork movement
tion, it appears that at least nine of these are bound by SUUR
(Makunin et al. 2002). Thus despite the absence of ORC and the
presence of SUUR, replication occurred.
SUUR could act directly on replication proteins or indirectly
by affecting chromatin configuration. Although it is conceivable
that the SUUR protein acts directly at the replication fork to
movement in a follicle cell amplicon. (A)Insertionconstructedfortetheringexperiments,SUURfusedinframetotheDNAbindingdomain(DBD)ofGAL4,
with anhsp70 promoter. (B)Complementationassaysshowing that GAL4DBD-SUUR is capableoffunctioning as wild-type SUUR protein, restoring under-
replication in SuUR mutant larvae. Copy number is relative to a control fully replicated locus. (C) Tethering of GAL4DBD-SUUR at a fully replicated locus is
not sufficient to induce under-replication. (Inset) GAL4DBD-SUUR was bound to the UAS as analyzed by ChIP analysis relative to a negative control locus
(pol a, where GAL4 is not bound). (D) Tethering of GAL4DBD-SUUR to the DAFC-62D amplicon in follicle cells does not affect amplification levels. The left
panel shows the DNA copy number across the amplicon with GAL4DBD-SUUR tethered at the position designated by the asterisk. The right panel shows
UAS, as determined by ChIP analysis.
Tethering experiments indicate that tethering of a single SUUR is not sufficient to induce under-replication in salivary glands or to impede fork
Sher et al.
72 Genome Research
control processivity or rate of movement, we think it most likely
impedes replication fork progression. The necessity but in-
sufficiency of SUUR raises the possibility that it acts with other
chromatin proteins to repress replication. In DamID studies in
cell culture, the SUUR protein localizes across the domains of
BLACK chromatin (Pindyurin et al. 2007; Filion et al. 2010).
Immunostaining of SUUR on polytene larval salivary gland chro-
mosomes also indicates that SUUR is bound across large regions
(Makunin et al. 2002). Interaction between SUUR and specific
chromatin proteins may establish a configuration inhibitory to
This study provides the first map of ORC binding in differ-
entiated, primary metazoan cells. As suggested by cell culture
studies, ORC indeed displays different binding sites in different
cell types. The positioning of ORC in polytene salivary glands is
considerably more distinct than the differences observed be-
tween diploid Drosophila cell lines, as 28% of the ORC binding
sites observed in the salivary glandare uniqueto that tissue,nearly
triple the number of cell-type–specific origins seen in cell culture.
The position of ORC binding sites in salivary glands supports the
emerging paradigm that ORC localization frequently is linked
to active TSSs (MacAlpine et al. 2004, 2010). Active transcrip-
tion, however, does not appear responsible for directing ORC
binding, and other factors must contribute. Thus ORC binding
may require open chromatin configurations that show tissue
What could be the developmental function of under-repli-
cation? The domains are repressed for transcription, yet the ef-
fects on transcription and replication are separable by SuUR mu-
tation. Thus under-replication is not required for silencing of
transcription, and restoration of transcription is not needed for
replication. The properties in cell culture of these regions that
become under-replicated in the salivary gland and in some cases
in the larval fat body and midgut suggest genomic hardwiring.
There is less exon density across these domains, and in cell cul-
ture, transcription and ORC binding are reduced in these regions
compared with flanking sequences. They also are late replicating.
During salivary gland differentiation, this blueprint becomes
more exaggerated because transcription is inhibited and ORC bind-
ing lost. The tissue specificity seen between the three polytene
tissues, however, reveals that these under-replicated domains are
not solely the passive consequence of late replicating regions
established to control replication timing in diploid cells. In ad-
dition to tissue specificity with regard to the extent of replication,
in the fat body many genes with the under-replicated regions are
transcribed (Nordman et al. 2011). The genome structure appears
transcription, but this can be increased or overcome in response
to differentiation. It is interesting from an evolutionary per-
spective to ask what selective pressures drive the establishment of
domains repressed for replication and the maintenance of the
number during the differentiation of polyploid cells. Strikingly,
theyoperate by developmental control ofboth initiation andactive
control of fork movement. The recognition that SUUR controls fork
movement and that it is present in diploid cells suggests that in
diploid cells, proper regulation of DNA replication occurs not only
at initiation but also via active regulation of replication elongation.
Such regulation may be critical in diploid cells to maintain copy
number, preventing changes associated with cancer.
Comparative genome hybridization
Seventy third-instar salivary glands from either Oregon-R (OrR) or
Ringer’s (EBR) solution (Ephrussi and Beadle 1936). DNA from
salivary glands or 0- to 2-h OrR embryos was phenol-chloroform–
aCGH labeling kit. Slides were hybridized to a custom Agilent tiling
array spanning the entire Drosophila euchromatin and were washed
as per Agilent’s recommendations.
Real-time qPCR was performed with primers against under-repli-
cated or amplified regions and normalized to DNA levels at a fully
replicated control locus at cytological location 93F4 (primer se-
Instrument according to the manufacturer’s instructions.
Salivary glands were dissected from wandering third-instarlarvae in
EBR for no longer than 30 min in the presence of Roche Complete
Protease Inhibitor Cocktail, fixed, and processed according to the
method described by Lee et al. (2006). Each ChIP was performed
2010) according to the method described by Austin et al. (1999).
Immunoprecipitation was performed as described by Lee et al.
(2006) with 2 mL (1:200 dilution) of anti-DmORC2 serum (kindly
provided by Stephen Bell, Massachusetts Institute of Technology,
Cambridge), anti-RNA Pol II (Upstate CTD4H8), anti-H3K27me3
(Abcam 6002), or normal IgG (Millipore 12-370 and 12-371). Un-
amplified DNA was processed for hybridization as described for
aCGH, omitting the BioPrime restriction enzyme digestion step.
Array data analysis
Array intensities were median normalized across channels and
smoothedby genomic windowsof 1 kb using the Ringopackagein
R (Toedling et al. 2007). For ChIP experiments, enriched peaks
were identified where at least three adjacent probes showed en-
richment above the threshold determined by the upper bound of
the null distribution, using the Ringo package in R.
Following ChIP, sequencing libraries were prepared with the Illumina
and were sequenced as described. High-quality samples were con-
distribution between 150 and 300 bp on an Agilent Bioanalyzer.
After subsequent qPCR library quantification, 2–7 pM of linker-li-
gated DNA was applied to a flow cell using the Illumina Cluster
Station fluidics device. Thirty-six-base sequencing was performed
on an Illumina Genome Analyzer II (GAII) sequencer, according to
Illumina’s standard protocols. Images acquired were processed by
the bundled Illumina image extraction pipeline version 1.6 and
ChIP peak calling
The ChIP-seq reads were mapped back to the Drosophila mela-
nogaster genome (FlyBase version 5.2) using MAQ (version 0.7.1)
Developmental control of origins and fork movement
(Li et al. 2008) with a maximum of three mismatches per read. For
et al. 2008) to remove anomalous reads and detect peaks using the
as determined by the SPP package’s MSER function. For the Kc,
BG3, and S2 cells, we used Peakseq3(Rozowsky et al. 2009) to de-
tect peaksin paired replicates with a maximumP-value of 0.05 and
a minimum FDR of 0.05. The replicates were then combined by
taking theirintersectionand throwingout any peaksthat were not
present in both samples. Additional information is available with
associated metadata in the GEO repository (GSE20889, GSE20888,
Third-instar salivary glands dissected in EBR were immediately
transferred into TRIzol (Invitrogen) and were RNA-extracted ac-
cording to the manufacturer’s protocol. Ten micrograms of RNA
was processed with Illumina mRNA Sample Preparation kit ac-
cording to the manufacturer’s protocol.
Developmental time course
Salivary glands from embryonic and larval stages were dissected
from homozygous UAS-GFP; fkhIII-GAL4 lines (fkhIII-GAL4 flies
kindly provided by Deborah Andrew, Johns Hopkins Medical
School, Baltimore, MD; UAS-GFP from Bloomington) expressing
GFP in salivary glands. DNA was amplified using Qiagen’s RepliG
kit. qPCR was performed in triplicate as described in the Supple-
Staging of egg chambers
Drosophila ovaries were fixed and DAPI-stained and were analyzed
by microscopy on a Nikon Eclipse Ti. At least 250 egg chambers
from ovarioles from at least four different ovaries per genotypewere
stage to the total number of chambers counted.
Follicle cell DNA preparation
Ovaries were isolated from OrR or SuUR mutant Drosophila ovaries
fattened on wet yeast for 2 d. Genomic DNA was prepared from
16C FACS sorted follicle cell nuclei and prepared as for aCGH
(Claycomb et al. 2004).
DAPI microdensitometry for ploidy quantification
Third-instar larval salivary glands were fixed with 4% formalde-
DAPI intensities of the experimental nuclei were measured and
comparedto the intensities of the 2C larval antennaldisk nucleito
determine ploidy level, using a Nikon Eclipse Ti deconvolution
microscope. At least 30 nuclei from two biological replicates were
used for quantification.
SUUR tethering experiment
An SUUR fusion was created by cloning SUUR into the C terminus
of the hsp70:GAL4 construct described by Aggarwal and Calvi
(2004), and transgenic lines were generated by P element–mediated
transformation. These were crossed to strains with UAS P elements
inserted at 29C, 42A, 52D, 58D8, and 82F7. Larvae bearing both
constructs were induced daily for 30 min at 37°C in a water bath,
starting 6 h after egg laying, and were compared with non–heat-
shocked controls. Salivary glands were dissected when larvae be-
came committed wandering third instars and were processed for
qPCR or ChIP as described above.
For the follicle cell experiment, flies with the GAL4DBD-
SUUR fusion construct were crossed to the EY01493 UAS insertion
was induced by a 1-h heat shock at 37°C. After a 3-h recovery,
amplification levels were tested across the amplicon in stage-13
egg chambers in the UAS-containing and balancer sibling controls
by qPCR as described (Claycomb et al. 2002).
The data sets in this article have been submitted to the NCBI Gene
Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/)
under the reference series GSE31900. The specific experiments are
as follows: (1) GSE31895-ChIP with anti-ORC2 antibody to iden-
tify regions of ORC binding in third-instar salivary glands of wild-
find differences between third-instar salivary glands of the wild
type and SuUR; (3) GSE31897-ChIP with anti-H3K27me3 to com-
pare binding in salivary glands of wild-type and SuUR Drosophila;
(4) GSE31898-aCGH to ascertain levels of genomic DNA in third-
instar salivary glands of various mutant Drosophila; (5) GSE31899-
ChIP-seq of ORC2 bound to third-instar salivary gland DNA in
wild-type and SuUR mutant Drosophila, analyzed by Illumina se-
quencing; (6) GSE33017-RNA-seq expression profiles of wild-type
and SuUR mutant third-instar larval salivary gland tissue.
We thank Helena Kashevsky for doing the pilot aCGH studies,
Yingdee Unhavaithaya for help with ploidy quantification, Tony
Lee for advice, Tom Volkert and Sumeet Gupta of the Whitehead
Genome Technology Core for help with ChIP-seq and RNA-seq,
Tom DiCesare for help with graphics. Steve Bell generously pro-
vided the anti-ORC2 antibodies, the Bloomington Stock Center
and Deborah Andrew (John Hopkins Medical School) provided
stocks, Maki Asano (Ohio State University) provided the orc1 mu-
tant, and Flybase was critical for genomic information (Tweedie
et al. 2009). Steve Bell, Allan Spradling, Richard Young, and Daniel
Sher provided helpful comments on the manuscript. N.S. was
supported by a postdoctoral fellowship from the American Cancer
Society; J.N. by the Damon Runyon Cancer Research Foundation.
This work was initiated by support from the Mathers Charitable
Foundation, and the aCGH studies were funded by NIH grant
1U01HG004279 as part of the modENCODE project. The project
was supported by NIH grant GM57960 and by a Research Professor
grant from the American Cancer Society to T.L.O.-W.
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Received May 7, 2011; accepted in revised form October 17, 2011.
Developmental control of origins and fork movement