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INTRODUCTION |
Origins of DNA replication are the chromosomal regions where DNA
replication forks for bidirectional duplication of replicons are
established. The large and discontinuous genomes of eukaryotes require
a large number of origins that are distributed throughout the genome to
guarantee a complete replication within the limited time of the S phase
in a cell division cycle (for reviews, see Refs. 1-9. The best
characterized eukaryotic origins are those of the budding yeast
Saccharomyces cerevisiae because they function as sites of
replication initiation in extrachromosomal plasmid DNA and are,
therefore, amenable to detailed molecular analyses (10, 11). Prototypic
budding yeast origin (autonomously replicating sequences
(ARSs)1) are composed of
100-200 base pairs and contain several essential sequence elements
including a domain A with the AT-rich ARS consensus sequence and three
short stimulatory elements, B1-B3, which are functionally important but
divergent in sequence (12). The ARS consensus sequence and the
adjacent B1 domain element constitute a binding site for proteins of
the origin recognition complex (ORC), whereas the B3 domain element
forms a binding site for the transcription factor Abf1 in some, but not
all yeast origins (13).
ORC is a multimeric protein complex composed of six essential subunits
(Orc1p-Orc6p) that associate in an ATP-dependent manner with ARSs (13-15). The major known function of ORC appears to be the
recruitment of factors such as Cdc6, Mcm proteins, and others for the
formation of functional pre-replication complexes (16-19).
Origins of replication in multicellular organisms can usually not be
investigated by ARS assays. Therefore, biochemical procedures such as
two-dimensional gel electrophoresis (20, 21) or nascent strand length
or nascent strand abundance assays (22, 23) were established to
determine the sites where bidirectional genome replication initiates.
Using these experimental strategies, a small number of mammalian
origins have been identified. One major conclusion of these experiments
is that, whereas the replication of genomes in differentiated mammalian
and other metazoan cells begins at specific genomic loci that are quite
stably inherited from one cell division cycle to the next, individual
origins of a given organism differ greatly in size and sequence and are
clearly less uniform and more complex in structure than budding yeast origins (3-5, 9, 10, 24). Many known mammalian origins are found to be
located between transcribed regions and frequently in the vicinity of
active transcriptional start sites (25-28). One reason for a preferred
location of origins at transcriptional start sites might be the more
loosely organized chromatin structure, allowing initiator replication
proteins better access to their target DNA binding sites (for a recent
review, see Ref. 29). It has been shown by footprinting analyses
that the well studied lamin B2 origin is protected in a cell
cycle-specific manner; however, it has not been directly demonstrated
whether these or related sequence elements in differentiated metazoan
cells are binding sites for ORC (30, 31).
A Drosophila melanogaster ORC localizes in vivo
to the chorion gene amplification control element, which is active in
ovarian follicle cells and determines the amplification of chorion gene clusters by repeatedly initiating DNA replication (32). Interestingly, amplification control element-bound ORC is in close contact with transcription factor E2F, which together with the Rb protein, regulates
the initiation of replication (33). An ORC binding site has also been
identified in the Epstein-Barr virus genome. The 165-kbp viral
chromosome replicates as an episome in latently infected human cells in
a regulated once-per-cell cycle manner dependent upon a functional
bipartite viral origin. This origin binds the viral initiator protein,
EBNA-1, in addition to proteins of the human ORC, which appear to be
essential for viral genome replication (34, 35).
Using a modified version of the chromatin immunoprecipitation (ChIP)
protocol in combination with quantitative real-time PCR, we have
recently identified an ORC binding site between two divergently transcribed human genes in a region that coincides with a start site
for bidirectional DNA synthesis (36). We have now used the ChIP
technique to investigate another transcription unit in the human
genome, the TOP1 gene, which occupies ~100 kbp of the chromosome 20 sequence (37). The TOP1 gene promoter
co-localizes with a CpG island and contains an A+T-rich element (38).
In addition, the gene has two well characterized matrix attachment regions (MARs; Ref. 39). MARs are believed to connect chromatin loops
to the non-chromatin ribonucleoprotein network known as the nuclear
matrix (recently reviewed in Ref. 40). Several reports suggested that
sites of DNA synthesis may be linked to the nuclear matrix (41, 42).
Thus, TOP1 offers an interesting opportunity to
determine whether MARs are binding sites for ORC proteins and whether
they function as replication origins. We have also used the ChIP assay
to isolate and clone DNA sequences from immunoprecipitated ORC
protein-bearing chromatin fragments and identified ORC binding regions
in heterochromatic parts of the human genome.
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MATERIALS AND METHODS |
Cell Culture and Nucleoprotein
Preparation--
Asynchronous HeLa-S3 cells were cultivated on
145-mm dishes in Dulbecco's modified Eagle's medium supplemented with
5% fetal calf serum. Formaldehyde (Merck) was diluted to 1% in
prewarmed medium (37 °C) and added to monolayers of 108
cells for 4 min if not otherwise indicated. After removal of the
medium, cells were washed 3 times with cold phosphate-buffered saline
(PBS), scraped off, washed twice in PBS and resuspended in hypotonic
RSB buffer (10 mM Tris, pH 8.0, 3 mM
MgCl2, 10 mM sodium bisulfite, pH 8.0) for 10 min on ice. All centrifugation steps were carried out at 600 × g for 5 min at 4 °C. Cells were disrupted by Dounce
homogenization (15 strikes). After centrifugation, nuclear material was
washed twice in RSB buffer and once in high salt SNSB buffer (1 M NaCl, 10 mM Tris, pH 7.4, 0.1% Nonidet P-40, 1 mM EDTA, 10 mM sodium bisulfite, pH 8.0) and
subsequently incubated on ice for 5 min. Finally, the nuclear material
was resuspended at physiological salt concentration in NSB buffer (0.1 M NaCl, 10 mM Tris, pH 7.4, 0.1% Nonidet P-40,
1 mM EDTA, 10 mM sodium bisulfite, pH 8.0)
and loaded onto gradients consisting of 1.3, 1.5, and 1.75 mg/ml
CsCl diluted in gradient buffer (0.5% sarcosyl, 1 mM EDTA,
20 mM Tris, pH 8.0). Ultracentrifugation was carried out at
37,000 rpm for 24 h at 18 °C. The nucleoprotein fraction was
collected from the gradients followed by overnight dialysis against
Tris-EDTA (10 mM Tris, pH 7.4, 1 mM EDTA)
supplemented with 10 mM sodium bisulfite, pH 8.
Nucleoprotein complexes were sonicated by a total number of 100 short
pulses on ice. The concentration of nucleoproteins was determined
(A260) and adjusted to 2 µg/µl with
Tris-EDTA buffer. Nucleoprotein fragments <1 kb were obtained
by treatment with micrococcal nuclease (MBI Fermentas) at 10 units/mg
of nucleoprotein in the presence of 3 mM CaCl2
for 15 min at 37 °C. The reactions were stopped by adding 20 mM EDTA and analyzed on a 1% agarose gel.
Chromatin Immunoprecipitations--
Immunoprecipitations were
performed with 1 mg of nucleoprotein in NET buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40). Affinity-purified antibodies
were added at 15 µg (
-ORC1, IgG) and 10 µg (-ORC2, -SP1,
-p60/CAF-1) followed by 2-h incubation at 20 °C on a rolling
platform. Immunocomplexes were collected by adding 50 µl of 50%
protein A-Sepharose and further incubated for 2 h. Coupled protein
A-Sepharose beads were washed 8× with radioimmune precipitation buffer
(50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS), 3× in LiCl2
washing buffer (10 mM Tris, pH 8.0, 250 mM
LiCl2, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA), and 5× in Tris-EDTA buffer. All buffers were
supplemented with 10 mM sodium bisulfite, pH 8.0, as
protease inhibitor. Beads were transferred to fresh tubes after each
buffer change to reduce contamination of unspecific DNA sticking to the
tube walls. The washed beads were divided for protein and DNA
extraction, respectively.
Protein and DNA Extraction--
For Western blotting
experiments, proteins were eluted with 2% SDS, H2O for 10 min at 37 °C. For a reversal of cross-links, nucleoproteins were
incubated for 30 min at 65 °C and extracted with methanol/chloroform
(43). Input and supernatant samples were treated accordingly. Proteins
were separated by SDS page, transferred onto polyvinylidene difluoride
membranes, and treated with specific antibodies.
Antibodies against human ORC1 and ORC2 have already been described
(44). Monospecific antibodies against human ORC3-ORC6 were raised in
rabbits using the N-terminal part of the ORC3 protein and the
full-length ORC4 protein recombinant expressed in bacteria. Human ORC5
and ORC6 were expressed full-length in insect cells. Antibodies were
characterized by immunoblotting using recombinant expressed proteins.
To minimize the signal to noise ratio in the PCR, further extensive
washing of the coupled protein A-Sepharose beads was crucial.
Therefore, the whole washing procedure was repeated as described above.
Finally, nucleoproteins were eluted with 1% SDS, Tris-EDTA at 37 °C
for 10 min, and proteins were digested with 200 µg/ml proteinase K
overnight at 37 °C. DNA was extracted by the standard
phenol-chloroform procedure, ethanol-precipitated, and dissolved in 40 µl of Tris-EDTA.
Cloning of ORC DNA--
Precipitated and extracted DNA was
amplified by ligation-mediated-PCR (see Mueller and Wold (45))
using two overlapping linker oligonucleotides (5 pmol/µl/oligonucleotide) 5'-GCGGTGACCCGGGAGATCTGAATTC-3' and
5'-GAATTCAGATC-3', which were first annealed to double-stranded DNA by
stepwise cooling from 90 °C. For blunt-end ligation, purified DNA
fragments were first exposed to the exonuclease activity of the Klenow
enzyme (2 units, 5 min, 37 °C). DNA synthesis reactions were started
by adding a nucleotide mix (2.5 mmol each of dATP, dCTP, dGTP, dTTP)
and further incubated for 30 min. The Klenow enzyme was inactivated at
70 °C for 20 min. DNA fragments were dephosphorylated with 1 unit of
alkaline phosphatase at 37 °C for 1 h. Linker ligations were
performed at 18 °C for 15 h using 1 unit of T4-DNA ligase and 2 µl of the double-stranded linker oligonucleotides. PCR reactions were
performed in the presence of 10 mM each dideoxynucleotide,
dATP, dCTP, dGTP, dTTP, 3 units of Pfu DNA polymerase, 6%
glycerol, and 25 pmol of each oligonucleotide. The PCR was performed in
a thermocycler at 30 cycles consisting of 1 min at 94 °C, 2 min at
63 °C, and 3 min at 72 °C.
Amplified DNA fragments were directly cloned in the pCR-BluntII-TOPO
cloning system (Invitrogen) according to the manufacturer's manual.
Plasmid DNA was extracted from bacteria, purified, and analyzed by
sequencing and PCR methods.
Nascent Strand Abundance Assay--
Approximately 1 × 108 HeLa S3 cells were trypsinized and washed twice in
ice-cold phosphate-buffered saline and once in RBS (10 mM
Tris, pH 7.4, 10 mM NaCl, 3 mM
MgCl2). Centrifugations were carried out at 600 × g for 10 min. Cells were resuspended in RBS on ice at about
5 × 106 cells/ml for 5 min. The same volume of 1%
Nonidet P-40, RBS was added, and cells were further incubated on ice
for 10 min. The nuclei were pelleted, washed twice in RBS, and
resuspended at 5 × 107 nuclei/ml. The same volume of
lysis buffer (20 mM Tris, pH 8.0, 20 mM EDTA,
2% SDS, 500 µg/ml proteinase K) was added and incubated overnight at
56 °C. Total genomic DNA was extracted with phenol/chloroform, precipitated with isopropanol, and dissolved in Tris-EDTA buffer at 2 µg/µl. DNA was denatured at 85 °C for 10 min followed by rapid
cooling on ice and loaded on 5-30% (w/v) linear neutral sucrose
gradients in Tris-EDTA buffer (plus 0.1 M NaCl). In a parallel tube, double-stranded size marker DNA (1-kb latter, MBI Fermentas) was loaded as a reference. Gradients were centrifuged at
20 °C in a Beckman SW28 rotor for 20 h at 26,000 rpm. Fractions of 1 ml were collected from top to bottom. The distribution of size
markers in the gradient fractions was determined by agarose gel
electrophoresis. DNA fractions corresponding to an average of 1-kb size
(nascent DNA strands) and 2-10 kb were collected and precipitated with
ethanol. The abundance of nascent DNA strands in the preparation was
determined by quantitative real-time PCR.
Quantitative Real-time PCR--
Real-time PCR was performed with
the Light Cycler instrument (Roche Molecular Biochemicals) using a
ready-to-use "hot start" reaction mix. The mix contains
Taq DNA polymerase and a fluorescent dye, SYBR Green I, for
real-time detection of double-stranded DNA. Reactions were set up in 10 µl including 0.5 mM each primer. PCR reactions were
performed at 50 cycles routinely, using the standard settings
recommended by Roche Molecular Biochemicals. Annealing temperatures of
individual primers are indicated in Table I. Standard DNA samples
(human genomic DNA) were serially diluted to 30 ng, 3 ng, 300 pg, 30 pg, and 3 pg. After PCR, the x axis crossing point of each
standard sample was plotted against the logarithm of concentration to
produce a standard curve. Genomic equivalents of DNA samples were
determined by extrapolation from the standard curve (36).
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RESULTS |
Human Orc Proteins Are Recovered on Cross-linked
Chromatin--
Covalent cross-linking of chromatin proteins to DNA
in vivo, and the isolation of cross-linked chromatin was
performed as described (46-48). Isolated chromatin was sonicated and
further trimmed by micrococcal nuclease to produce fragments with DNA of 0.2-1-kbp lengths (Fig.
1A). Chromatin fragments were
either directly prepared for polyacrylamide gel electrophoresis and
immunoblotting (input, Fig. 1) or first immunoprecipitated
with specific antibodies (precipitate, Fig. 1) and then
analyzed by immunoblotting.
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Fig. 1.
Efficiency of cross-linking and ChIP.
A, preparation of chromatin fragments. Cross-linked
chromatin was first sonicated and then further trimmed by micrococcal
nuclease (MNase). The extracted DNA was analyzed by agarose
gel electrophoresis and ethidium bromide staining. B, time
of cross-linking and specificity of ChIP. HeLa cells were treated with
formaldehyde for 2, 4, and 6 min as indicated. Chromatin (100 µg) was
prepared, trimmed (see A), and directly processed for
Western blot analysis (input) using the antibodies indicated
at the right. Another part of the same preparation was
treated with Orc2-specific antibodies, immunoprecipitated, and then
investigated by Western blotting (-ORC2 precipitate).
C, co-immunoprecipitations. Cross-linking (4 min) and
immunoprecipitations were performed as in B. Input, Western blotting before
immunoprecipitations.-ORC2 precipitate, Western blots of
immunoprecipitated chromatin using the antibodies indicated on the
right.
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Immunoblots showed that a cross-linking time of 4 min was sufficient to
covalently link hOrc2p to DNA (44). This was also an optimal
cross-linking time for other proteins such as the chromatin-associated fraction of the single strand-specific DNA-binding protein RPA and the
scaffold attachment factor scaffold attachment factor A (SAF-A), an
abundant nuclear protein that is bound to MAR elements in
vivo (Ref. 49; Fig. 1B, input).
Orc2-specific antibodies efficiently precipitated chromatin fragments
with covalently bound hOrc2p, but these precipitates contained little
if any RPA and no detectable SAF-A (Fig. 1B, precipitate), indicating that hOrc2p and SAF-A were not
cross-linked to the same chromatin fragments and, therefore, do most
probably not reside at closely adjacent chromatin sites in
vivo.
Fig. 1C shows the cross-linking to DNA of other nuclear
proteins such as the p60 subunit of the chromatin assembly factor CAF1
(50) and transcription factor Sp1 (51). Immunoprecipitations with
Orc2-specific antibodies indicate that the p60 subunit was not present
in these immunoprecipitates (Fig. 1C). Interestingly, a
fraction of transcription factor Sp1 always co-precipitated with
hOrc2p-bearing chromatin (Fig. 1C), probably indicating that Sp1 and hOrc2p were occasionally cross-linked to the same chromatin fragments (see below).
Vashee et al. (53) and Dhar et al. (52) show that
human ORCs including subunits Orc1p-Orc5p can be extracted at high salt from HeLa cell chromatin and that human proteins Orc2p-Orc5p form a
core complex to which proteins hOrc1p and hOrc6p are more loosely bound. It was, therefore, of interest to determine whether the other
ORC proteins could be immunoprecipitated together with hOrc2p by
Orc2-specific antibodies.
We first determined whether the six ORC proteins in asynchronously
proliferating human cells could be identified by the available antibodies. For that purpose, HeLa cells were fractionated to yield
cytosol (Cy), soluble nuclear proteins (Nu), and
chromatin, which was treated with increasing salt (Fig.
2A). The six ORC proteins were
detected on chromatin and could be mobilized with 0.1-0.25
M NaCl. However, a fraction of hOrc1p appeared to be more
stably bound to chromatin since higher salt concentrations were
required for an efficient elution. We also detected significant amounts
of hOrc6p in soluble protein fractions (Fig. 2A).
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Fig. 2.
Intracellular distribution and cross-linking
of human Orc proteins. A, cell fractionation. Cytosolic
proteins (Cy), soluble nuclear proteins (Nu),
chromatin proteins in salt eluates (100, 250, and 450 mM
NaCl), and insoluble nuclear pellet (P) are shown. Orc
proteins were identified by polyacrylamide gel electrophoresis and
Western blotting using the antibodies indicated on the
right. B, cross-linking. Input,
cross-linked chromatin before immunoprecipitation (IP).
IP -ORC2, immunoprecipitated chromatin (with
Orc2-specific antibodies). IP IgG, immunoprecipitation with
unspecific control antibodies. Immunoblotting was performed as in
A. The star indicates the heavy or light chains
of the respective antibody.
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Next we investigated whether the ORC subunits could be cross-linked to
DNA. On isolated chromatin we detected significant amounts of subunits
hOrc1p (Fig. 1) as well as hOrc2p-hOrc5p but reduced amounts of hOrc6p
(Fig. 2B, input), which is in agreement with
published data, suggesting that hOrc6p may not be a regular component
of human ORC (52, 53).
Cross-linked chromatin was then immunoprecipitated with Orc2p-specific
antibodies. The precipitates clearly contained hOrc1 as well as
hOrc2p-hOrc4p (Fig. 2B) but very little, if any, hOrc6p (Fig. 2B). The Orc5p band overlapped to a significant extent
with the IgG heavy chain band in the experiment. However, in
independent experiment we clearly detected an Orc5p band in the
immunoprecipitate. Comparing the input and supernatant, we estimate
that between 30 and 50% of the cross-linked input sample could be
immunoprecipitated. No ORC proteins were precipitated with unspecific
IgGs (Fig. 2B) or with p60-specific antibodies (not shown).
We have also tested antibodies against Orc3p, Orc4p, and Orc5p and
found that the antibodies did not efficiently precipitate cross-linked
proteins (not shown). Therefore, we used the Orc1p- and Orc2p-specific antibodies for the experiments reported below. Thus, ORC proteins could
be covalently linked to chromatin and most likely occurred at the same
chromatin sites either as one large complex or, alternatively, as subcomplexes.
An ORC Binding Site in the Human TOP1 Gene Is Located at an
Upstream Promoter Site but Not in Matrix Attachment Regions--
Next
we addressed the question of whether ORC binds to particular genomic
regions. The well characterized TOP1 gene locus appeared to
be a interesting region because it contains several features that have
frequently been found in known origins such as A+T-rich elements, MARs,
nuclease hypersensitive sites, and a higher than average G+C content in
the gene promoter.
The TOP1 gene is composed of 21 exons (37) and contains MAR
I, located at an intronic site immediately after exon 2, and MAR II,
which occurs further downstream, between exons 13 and 14 (Ref. 39; Fig.
3). MAR I and MAR II specifically attach
to components of the nuclear matrix and exhibit specific binding sites
for the SAF-A both in vitro and in vivo (39,
49).
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Fig. 3.
The TOP1
gene. A, overview. Upper line, exons
(vertical strokes with arabic numbers), CpG
island, and MARs as indicated. Bent arrows, transcription
initiation of TOP1 and the adjacent gene PLC-148.
The DNase-I-hypersensitive sites have been mapped by Kunze et
al. (38). Middle line, genomic sequences complementary
to the PCR primers used (Table I). Bottom line, distances
(in kbp) in both directions from the TOP1 transcriptional
start site. B, sequence around the major transcriptional
start site (bent arrow). An upstream A+T-rich element is
shown in bold letters, with a region homologous to the yeast
ARS consensus sequence underlined. The Sp1 binding sites are
indicated as are the coding part of exon 1 and the (small) exon 2 (gray boxes). The arrows indicated the positions
corresponding to the forward and reverse primer sets Prom
and TopC4 (see A and Table I).
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The TOP1 promoter is composed of several elements that
function as binding sites for transcription factors including the
ubiquitous Sp1 protein (54). The promoter has a G+C content of 67% and co-localizes with a CpG island. This is of interest because it is
assumed that origins of DNA replications are predominantly located in
the vicinity of CpG islands (55-57). The upstream promoter region also
contains an A+T-rich track as found in the vicinity of replication
origins (2). For the identification of ORC binding sites, chromatin
immunoprecipitations were performed with Orc2p-specific antibodies as described above.
Nucleoproteins were extracted from the immunoprecipitates for Western
blotting, and DNA was extracted for an analysis by a quantitative
real-time PCR procedure that allows for the detection of sequences that
were specifically precipitated (see "Materials and Methods" (36)).
For PCR analyses we used eight primer sets corresponding to different
parts of the genomic section investigated (Fig. 3). The primers gave
approximately equal amplification results with unsheared genomic DNA
(fragment size greater than 20 kbp) as template under the conditions of
quantitative PCR (not shown; see Fig. 7).
As a positive control, cross-linked chromatin was immunoprecipitated
with antibodies against the transcription factor Sp1. Because the
TOP1 promoter contains two G+C boxes serving as Sp1 binding
sites (54), we expected an enrichment of promoter sequences in the
immunoprecipitate. Indeed, quantitative PCR revealed the presence of
almost 6000 copies of the promoter sequence but only very few copies of
other sequences in and around the gene (Fig. 4A).
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Fig. 4.
DNA sequences in immunoprecipitated
chromatin. Cross-linked chromatin was immunoprecipitated as
described. Insets, to control the reaction, specific
proteins were determined before (input) and after
immunoprecipitation (precipitate) with specific antibodies
or unspecific control antibodies (IgG). Columns, DNA
was extracted from the immunoprecipitates and investigated by
quantitative real-time PCR. For each primer pair, a standard
calibration curve with serially diluted genomic DNA was constructed.
The data obtained are expressed as genomic units, where one genomic
unit corresponds to one amplifiable DNA copy in the sample examined.
The antibodies used for the immunoprecipitations were either unspecific
control antibodies (C) or specific for transcription factor
Sp1 (A), chromatin assembly factor p60/CAF1 (B),
hOrc1p (D), or hOrc2p (E).
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As negative controls, immunoprecipitations with antibodies against the
p60/CAF1 protein (see Fig. 1) as well as with unspecific control
antibodies were performed. The p60/CAF1-specific antibodies efficiently
precipitated the p60 protein (precipitate, Fig.
4B), but the associated DNA could not be amplified to a
significant extent with the primer sets used (Fig. 4B).
Similarly, control antibodies failed to precipitate any chromatin
fragments with specific DNA sequences (Fig. 4C; values of
<50 copies in real-time PCR reactions are considered to be
non-significant).
In contrast, chromatin immunoprecipitated with Orc1 or with Orc2
antibodies contained DNA sequences corresponding to the upstream promoter region of the TOP1 gene (Fig. 4, D and
E). More precisely, we estimated about 800 copies of
amplifiable promoter sequences in the Orc2 precipitate and about 260 copies in the Orc1 precipitate. The difference in copy numbers between
precipitates with Orc1 and Orc2 antibodies was observed in all
experiments of this kind and has been noted before in studies on ORC
binding sites around another human gene (36). However, the elements MAR
I and MAR II appear to be much reduced in immunoprecipitated
cross-linked chromatin relative to promoter sequences (Fig. 4). This
seems to indicate that ORC is bound to a promoter site in the
TOP1 gene but not to the MAR elements.
It is possible though that promoter sequences were densely covered by
cross-linked proteins and, therefore, more protected against shearing
and nuclease digestion than MAR elements. This would result in an
increase of the copy numbers of promoter sequences in input
cross-linked chromatin and, consequently, also in the immunoprecipitates.
To investigate this possibility, isolated cross-linked chromatin was
sheared to DNA fragment sizes between <0.25 and 2 kbp and further
digested by micrococcal nuclease (Fig.
5A). DNA was extracted from
fragmented chromatin before and after nuclease digestion and analyzed
by quantitative PCR assays. The data indicate a 2-fold higher abundance
of promoter fragments compared with control DNA in sheared chromatin.
In addition, promoter DNA was more resistant than control DNA to
nuclease digestion (Fig. 5B).
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Fig. 5.
Protection of TOP1 promoter
proximal elements against shearing and nuclease attack.
A, DNA fragmentation. Sheared cross-linked chromatin was
treated with increasing amounts of micrococcal nuclease
(MNase). The DNA was extracted and visualized by ethidium
bromide after agarose gel electrophoresis. B, differential
protection. DNA fragments in A were analyzed by quantitative
PCR using primers for the TOP1 promoter region
(Prom; Fig. 3) and a control region (TopC1; Fig.
3).
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We note, however, that similar experiments with other genes (36) showed
less protection of promoter elements and conclude that the degree of
protection by cross-linked proteins varies among ORC binding sites (not
shown). Thus, the digestion step is crucial in these experiments, and
it was therefore of interest to confirm the results of Fig. 4 comparing
the number of sequence copies before and after immunoprecipitation to
estimate the enrichment achieved. This was done in the experiment of
Fig. 6, where DNA from cross-linked
chromatin fragments (0.3-1 kbp) was analyzed by quantitative PCR. The
input sample (before immunoprecipitation) contained several thousand
copies of amplifiable promoter and promoter-proximal DNA sequences but
fewer copies of distal sequences (about 1000 copies or less; Fig.
6A). Orc2-specific antibodies immunoprecipitated ~800
copies of the promoter sequence, 200 copies of the closely adjacent
TopC4 sequence, and clearly less than 100 copies of more distal
sequences (Fig. 6B). Control antibodies immunoprecipitated
no significant amounts of promoter or adjacent sequences (see Fig.
4C). The values of Fig. 6B were used to determine the ratios of precipitated over input DNA and showed that up to 10% of
the promoter sequences but less than 2% of more distal sequences could
be recovered from the immunoprecipitates (Fig. 6C).
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Fig. 6.
Enrichment of promoter-proximal sequences by
immunoprecipitation. A, before immunoprecipitation. DNA
was extracted from cross-linked chromatin and amplified by quantitative
PCR using the primer sets indicated (see Fig. 3). The results are
expressed in genomic units relative to serially diluted genomic control
DNA. B, after immunoprecipitation with hOrc2p-specific
antibodies. The PCR analyses were performed as in A. C, enrichment. Ratios of precipitated over input amplifiable
DNA are plotted against primer sites on the TOP1 map.
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Thus, in this particular experiment, immunoprecipitated TOP1
gene promoter sequences were at least 5-fold enriched over more distal
sequences. Although these numbers are lower than the enrichment achieved in the immunoprecipitates of ORC-bearing Epstein-Barr virus
fragments (35) and also lower than the enrichment for an ORC binding
region in the MCM4 gene promoter (36), it clearly confirms
that an ORC binding site is located in the promoter region of the
TOP1 gene and excludes ORC binding sites in MARs.
The ORC Binding Region Coincides with an Origin of
Replication--
We next addressed the question of whether the
identified ORC binding site in the TOP1 gene promoter region
coincides with an active replication origin. For this purpose, the
nascent strand abundance assay of Giacca et al. (22) was
used. This assay determines the abundance of DNA strands of about 1 kbp
in length extracted from denatured genomic DNA. These strands
exclusively occur in the vicinity of replication start sites because
leading strands at more advanced replication forks are much longer, and
lagging strands consist of smaller Okazaki fragments of 0.1-0.2-kbp lengths.
Denatured genomic DNA from proliferating HeLa cells was centrifuged
through sucrose gradients, and fractions containing DNA of fragments
sizes of 1-2 kbp and of 2-10 kbp were collected. To verify that the
1-kbp fraction of DNA strands was enriched for nascent DNA, primer
pairs corresponding to the well characterized lamin B2 origin and, as
controls, primer pairs for more distal regions on both sides of the
lamin B2 origin were used (58). Quantitative PCR confirmed a higher
abundance of origin over flanking sequences (Fig.
7A). Therefore, the
preparation of denatured DNA fragments was suitable for a determination
of nascent DNA strands.
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Fig. 7.
Start of bidirectional replication as
determined by a nascent strand abundance assay. A,
control. Sequences corresponding to the well established human lamin B2
origin are more abundant than more distal sequences in the 1-kb-size
fraction of denatured DNA. B, TOP1 sequences. PCR
analyses were performed with denatured DNA of the 1-kb-size class and
the 2-10-kb-size class. The data are expressed as the percent of
maximal copy number in a given size class because the two size classes
contain different amounts of DNA (maximally ~250 copies in the 1-kp
class and maximally ~3000 copies in the 2-10-kb class). The data
with genomic DNA (fragments larger than 20 kb) are included to
demonstrate that all primers function with essentially equal
efficiencies in the PCR assays.
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Accordingly, DNA strands in the 1-2- and 2-10-kbp sucrose gradient
fractions were used as templates for quantitative PCR with the eight
TOP1 gene primer sets described above (Fig. 3). The results
clearly showed a higher abundance of promoter-proximal over more distal
sequences (Fig. 7B), suggesting that a replication start
site is located in the upstream promoter region of the TOP1 gene. As expected for nascent DNA strands, the enrichment of
promoter-proximal DNA sequences relative to distal sequences was higher
in the 1-2-kbp size fraction than in the 2-10-kbp size fraction (Fig.
7B) because larger nascent DNA strands include not only
sequences from the replicative start but from adjacent regions as well.
We thus conclude that an origin of DNA replication is located in the
promoter region of the human TOP1 gene, most likely in close
association with the ORC binding site.
ORC Binding Sites Occur in Genomic Regions with Repetitive
DNA--
So far we have demonstrated that the ChIP protocol is useful
in detecting ORC binding sites in the vicinity of active genes of known
nucleotide sequences. Next, we wished to determine whether the method
is suitable for the identification of ORC binding sites elsewhere in
the genome. For that purpose, DNA was extracted from immunoprecipitated
chromatin (with Orc2-specific antibodies) and cloned in plasmid vectors.
To confirm that a cloned sequence serves as an ORC binding site
in vivo, independent immunoprecipitations were performed and investigated by quantitative PCR using primers complementary to the
sequence in question. A given sequence was considered to be specifically associated with cross-linked ORC if it occurred in enrichments of more than 5-fold greater than control sequences.
Twenty different ChIP clones were thus isolated and partially sequenced
(average insert length, 1250 bp). Two of the sequences were detected at
high copy numbers in independent immunoprecipitates of cross-linked chromatin.
One of the sequences corresponded to a region close to the gene
TOM1 on human chromosome 22q13.1 (59). The cloned sequence revealed a higher than average G+C content (51%), with consecutive CpG
dinucleotides and is, therefore, related to mapped ORC binding regions
at active genes. However, we have not yet determined whether the
TOM1 ORC binding site coincides with a replication origin.
The second sequence is homologous to the human alphoid repetitive
satellite DNA, which occurs in the centromeric regions of human
chromosomes (for a review, see Ref. 60). Several independent ChIP and
quantitative PCR assays were performed to investigate the association
of hOrc2p with the alphoid satellite DNA sequence (Fig.
8C). For this purpose, we used
primers corresponding the cloned satellite DNA and to a genomic control
region (primer set TopC1; see Table I).
Sheared and digested cross-linked chromatin contained about twice as
much control DNA than alphoid satellite DNA (Fig. 8A,
input), but hOrc2p-specific antibodies clearly precipitated more chromatin-containing satellite DNA than control DNA, whereas antibodies against p60/CAF-1 and SP1 did not (compare Fig.
8, B and C). We found that satellite
DNA was about 8-fold enriched in the hOrc2p precipitate but not in the
control precipitates (Fig. 8D). Thus, these data suggest
that at least hOrc2p is associated with alphoid satellite DNA in
vivo.
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Fig. 8.
Chromatin immunoprecipitations of an alphoid
satellite DNA region. A, input. Sheared and digested
cross-linked chromatin was deproteinized and investigated by
quantitative real-time PCR to determine the abundance of alphoid
satellite (primers ASD) and control DNA sequences (primers TopC1)
before immunoprecipitation. B, alphoid satellite DNA in
immunoprecipitates. The chromatin preparation in A was
immunoprecipitated with specific antibodies as indicated. The abundance
of alphoid satellite DNA in the precipitates was determined by
quantitative PCR. C, control DNA in immunoprecipitates. Same
experiment as in B but with the TopC1 primers. D,
enrichments. Ratios of the amounts of precipitated over input DNA are
shown.
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To determine whether this particular ORC site could serve as an origin
of replication, nascent strand abundance assays were performed (see
Fig. 7), but an enrichment of clone alphoid DNA sequences in the 1-kbp
fraction of nascent DNA was not detected (not shown).
We can, therefore, not decide whether hOrc2p on alphoid repetitive DNA
marks a replication origin. It might instead serve to establish the
special chromatin conformation at the centromeres of chromosomes just
as Orc proteins are involved in the organization of chromatin in yeast
and Drosophila genomes (for a review, see Ref. 61). Clearly,
this point deserves further investigation.
The number of clones investigated is certainly too low for a
statistical evaluation on the abundance of ORC-bearing fragments in
immunoprecipitated chromatin. However, the results show that cloning of
DNA fragments in immunoprecipitated chromatin could be an interesting
method of identifying ORC binding sites in the mammalian genome.
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DISCUSSION |
We have searched for an ORC binding site in a 100-kbp human genome
section that includes a typical housekeeping gene, TOP1, encoding a type I DNA topoisomerase (37). We provide evidence by ChIP
and quantitative PCR that an ORC binding site is located at the
upstream promoter region of the TOP1 gene.
The antibodies used for chromatin immunoprecipitation are directed
against hOrc2p and, therefore, precipitated chromatin fragments with
covalently linked hOrc2p. The same chromatin fragments also carried
other Orc proteins such as hOrc1p, hOrc3p, hOrc4p, and most probably,
hOrc5p, suggesting that these proteins form an ORC when bound to
chromatin. Interestingly, hOrc6p was barely detectable in these
complexes, consistent with results obtained with biochemically isolated
human ORC that is composed of a core of proteins hOrc2p-hOrc5p, to
which hOrc6p is bound only loosely (52, 53).
Although hOrc1- and hOrc2-specific antibodies precipitate chromatin
fragments with the same DNA sequence, the copy numbers in the
Orc1-specific precipitates were always one-half or less of the copy
numbers in the hOrc2p-specific precipitates (Fig. 4). This could be
explained by the observation that hOrc2p remains on chromatin during
all phases of the HeLa cell cycle, whereas hOrc1p is present in reduced
amounts on chromatin in S-phase cells as previously described and
discussed in detail (36, 62).
ORC in Promoter Regions--
Although the resolution of the ChIP
method is limited and the ORC binding site cannot be located precisely,
our data are consistent with the possibility that ORC is located close
to the binding sites of the transcription factor Sp1. We conclude this
because Orc2-specific antibodies precipitated chromatin fragments that carried not only hOrc2p and other Orc proteins but also significant amounts of the transcription factor Sp1 (Fig. 1). Moreover, antibodies against transcription factor Sp1 precipitated the same DNA sequences that were precipitated by Orc antibodies, suggesting that ORC and Sp1
most probably reside at closely adjacent sites within the
TOP1 promoter. It cannot be excluded though that Sp1 forms physical contacts with ORC.
We note, however, that Sp1 precipitates usually contained 7-8 times
more promoter copies than Orc2-precipitates (Fig. 4). This difference
could be due to properties of the respective antibodies or to different
accessibilities of the cross-linked proteins. Furthermore, the
PCR-amplified sequence contains several potential Sp1 binding sites but
probably only one ORC site. However, another and potentially more
interesting possibility is that, whereas most or all TOP1
promoters contain transcription factor Sp1, only a fraction of the
promoters may contain ORC, implying that the TOP1 origin is
not regularly established in HeLa cells. If this could be substantiated
by more direct experiments, it may indicate that ORC binding sites are
rather flexible genetic elements in mammalian genomes (29).
The ORC site in the TOP1 gene coincides with an origin of
replication as indicated by the results of the nascent strand abundance assay. This conclusion agrees well with earlier reports showing that
many origin sequences have been mapped in the vicinity of genes and
frequently in promoter/enhancer regions (for review, see Ref. 55).
Furthermore, many sequences that replicate early after the entry of
cells into S phase are rich in CpG dinucleotides that are frequently
found in the upstream promoter regions of housekeeping genes (57).
Indeed, it is well established that genomic sections with housekeeping
genes always replicate early in S phase as do regulated genes in cells
expressing these genes, whereas regulated genes replicate late in the S
phase of differentiated cells that do not express these genes (63).
Thus, given the close correlation of early DNA replication with active
transcription, it is quite likely that other housekeeping genes also
carry ORCs in their upstream regions.
It is conceivable that the more open chromatin structures that
characterize active promoters may facilitate an access of ORC to DNA.
Indeed, the acidic transcriptional activation domain of BRCA 1 alters
the local chromatin structure and thereby stimulates chromosomal DNA
replication (64). In addition the chromatin accessibility complex,
CHRAC, clears the simian virus 40 origin of nucleosomes and facilitates
an interaction of the viral replication initiator, T antigen, with the
origin (65).
Furthermore, the transcription factor Sp1 appears to stimulate manyfold
the function of the viral initiator, T antigen (66-68). Similar
results have been described for other viral systems (69, 70). In yeast,
transcription factor Abf1 on the B3 element of ARSs (13) and the
recruitment of an RNA polymerase II transcription complex (71) activate
replication. The localization of ORC to the Drosophila
chorion gene amplification unit depends on direct interactions with the
E2F transcription complex (33). Future experiments may show that ORC
and transcription factors such as Sp1 not only bind to adjacent DNA
sites but also interact functionally to promote replication initiation.
Wyrick et al. (72) recently used a ChIP procedure to
determine the genome-wide distribution of ORC binding sites in the budding yeast genome with its densely packed genes (72). Most yeast
ORCs are located at ARS origins within intergenic sequences, in
particular in long terminal repeats of transposable elements which
contain transcription and termination signals that may establish chromatin domains suitable for replication initiation, a conclusion that is consistent with the interpretation discussed above.
MAR and Replication--
We have chosen the TOP1 gene
for the determination of ORC binding sites because it contains two well
mapped MAR elements. The nuclear matrix has frequently been found to be
associated with nascent DNA, and it has been proposed that MAR elements
are sites where DNA replication is initiated (for recent reviews, see
Refs. 7, 24, and 73).
We have now shown that SAF-A, a major MAR binding protein, was not
cross-linked to the same chromatin fragments that carry Orc proteins.
In addition, the two MAR elements in the TOP1 gene could not
be specifically precipitated with ORC-specific antibodies and are,
therefore, not associated with cross-linked ORC, and these MAR elements
do not function as replication origins, as suggested by the results of
the nascent strand abundance assay. However, MARs may be important to
spatially structure replicons through their association with the
nucleoskeleton and to form links between sites of DNA replication and
the chromosomal architecture (74).
ORC in Late-replicating Heterochromatin--
The dense packing of
heterochromatin excludes the possibility that ORCs are guided to DNA
via promoter DNA sequences and existing open chromatin conformations.
However, replication of early chromatin regions may change the
structure of chromatin such that previously inaccessible chromatin
opens up and can then engage replication complexes.
We have now detected hOrc2p at centromeric alphoid satellite DNA that
is probably organized as heterochromatin in vivo, but we
were unable to demonstrate by the nascent strand abundance assay that
this region functions as the replication origin. Thus, hOrc2p on human
satellite DNA sequences could have functions in heterochromatin
assembly just like Orc proteins in Drosophila (75) and yeast
(76). But it remains an important task to use ChIP or other techniques
to locate Orc proteins in late-replicating human heterochromatin and to
determine the exact role for human ORC in these regions.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.:
49-7531-884238; Fax: 49-7531-884036; E-mail:
Christian.Keller@uni-konstanz.de.
