Regulation of DNA Replication Timing on Human
Chromosome by a Cell-Type Specific DNA Binding
Masako Oda, Yutaka Kanoh, Yoshihisa Watanabe, Hisao Masai*
Genome Dynamics Project, Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
Background: Replication timing of metazoan DNA during S-phase may be determined by many factors including
chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be
determined by Mb-domain structures, termed as ‘‘replication domains’’, and recent findings indicate that replication timing
is under developmental and cell type-specific regulation.
Methodology/Principal Findings: We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and
non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating
DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the
locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR)
between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1),
specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of
the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork
movement may be slowed down by SATB1.
Conclusions: Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the
Citation: Oda M, Kanoh Y, Watanabe Y, Masai H (2012) Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding
Protein SATB1. PLoS ONE 7(8): e42375. doi:10.1371/journal.pone.0042375
Editor: Marco Muzi-Falconi, Universita’ di Milano, Italy
Received March 22, 2012; Accepted July 4, 2012; Published August 7, 2012
Copyright: ? 2012 Oda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by Grant-in-Aid for Scientific Research (A) (to HM) and Grant-in-Aid for Scientific Research on Priority Area
‘‘Chromosome Cycle’’ from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to HM). No additional external funding received for this
study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
DNA replication occurs once and only once during S phase of
cell cycle . This strict regulation is achieved by multiple layers of
mechanisms. Once DNA replication is initiated, three billion base-
pair human genome is replicated in 7,8 hrs . Origins on the
eukaryotic chromosomes can be classified according to the timing
at which they are programmed to fire during S phase . Studies
in yeasts have established that intra-S checkpoint negatively
regulates the firing of late or dormant origins . This is also the
case in mammalian cells as well [5–8]. Other factors including
histone modifiers have also been shown to regulate the timing of
origin firing [9–11]. On larger genomes of higher eukaryotes,
replication is initiated from tens of thousands of loci on the
genome . It is known that clusters of origins are fired more or
less simultaneously, and they may constitute distinct chromosome
domains that may be referred to as ‘‘replication (timing) domain’’
[12–14]. The genome-wide landscape of replication timing
domains were shown to change during the process of differenti-
ation and different cell types may have distinct replication timing
domain structures [12,15–17]. As much as 50% of the genome
may be regulated differentially in different cell types , and
20% of the mouse genome undergoes reorganization during
development . Replication timing is generally conserved
between different species (e.g. between human and mice) .
Replication domains may be correlated with histone modification
marks and most notably with chromosome proximity organization
that can be estimated by 4C or high-C assays [13,18,19].
It would be important to identify factors that may be responsible
for cell-type specific regulation of replication timing. We have
been using the human chromosome 5 5q23.3/31.1 locus (will be
referred to as 5q23/31 hereafter) containing a cytokine cluster
region as a model locus for the study of replication program, since
extensive studies have revealed the transcription factors and
epigenetic regulation involved in cell-type-specific and simulation-
dependent regulation of transcription in this region. We previously
mapped an origin near the cytokine cluster and have shown that
replication timing regulation is not affected by the activity of local
We have compared replication timing of the 3.5 Mb segment of
5q23/31 in T cells and non-T cells, and found that the boundary
of the early and late replication domain may be differentially
regulated. Furthermore, a T cell specific-factor, SATB1 (Special
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AT-rich binding protein) [23–26], plays an important role in
determination of the location of TTR (replication timing transition
region). TTR forms a boundary between adjacent different timing
domains, is often present near a synteny boundary, and is
associated with higher frequency of SNP and increased occurrence
of mutations that are often responsible for cancer . Thus,
elucidation of mechanisms for setting of TTR on chromosomes
will also provide insight into the generation of mutation hot spots
which may ultimately lead to diseases.
Materials and Methods
Jurkat (Jurkat E6.1; Human T lymphocyte from acute T cell
leukemia) and HL-60 (Human promyelocytic leukemia cells) were
cultured in RPMI1640 (Nissui) supplemented with 10% fetal
bovine serum (FBS), sodium bicarbonate, 3.5 mM L-glutamine, 2-
mercaptethanol and antibiotics (100 U/ml Penicillin and 100 mg/
ml Streptomycin (GIBCO)). HeLa S3 (Human epithelial carcino-
ma cell line) were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% FBS, sodium bicarbonate and
5 mM L-glutamine. All the cells were cultured in a humidified
atmosphere containing 5% CO2. Jurkat and HL-60 were
purchased from ATCC, and HeLa S3 was purchased from the
Health Science Research Resources Bank (Tokyo, Japan).
Analyses of Replication Timing
Replication timing analyses were performed as described
previously [20,28,29]. Briefly, exponentially growing cells were
pulse-labeled with50 mM BrdU
Roche) for 30 min, fixed in 70% ethanol, stained with propidium
iodide (PI) and fractionated by fluorescence assisted cell sorting
(FACS Vantage, Becton & Dickinson) into six (G1, S1–S4 and G2)
cell cycle fractions, each containing 40,000 cells. After isolation of
total genomic DNA, the DNA was sheared by sonication to the
average size of 500 bp, denatured and newly replicated, BrdU-
labeled DNA was immunoprecipitated using anti-BrdU antibody
(BD). To determine the levels of target sequences in each fraction,
semi-quantitative PCR was conducted using primers for individual
loci and control loci reported previously (Table S1 or primer
sequences available on request). The band intensity of the BrdU-
labeled target loci was quantified and was normalized by that of
the BrdU-labeled mitochondorial DNA (mtDNA) in each cell
cycle fraction . PCR products were visualized on 8%
acrylamide gels stained with SYBR Green I (Molecular probes).
The obtained gel images were captured and band intensities were
determined by using LAS-1000 (Fujifilm). Taq polymerase (Sigma-
Aldrich or Genescript) or platinum Taq polymerase (Invitrogen)
was used for PCR quantification of mtDNA or the genomic target
For preparation of nuclei, cells, labeled with 30 mM BrdU for
10 min prior to harvest, were treated with 60 mM (Jurkat) or
75 mM (HL-60 and HeLaS3) potassium chloride (hypotonic
condition) for 30–40 min at 37uC and fixed in three changes of
ice-cold methanol: acetic acid (3:1). Fixed cells were then dropped
onto slides and dried at 55–60uC overnight. To denature
chromosomal DNA, slides were preheated at 70uC on a heat
block, placed in 70% formamide in 2X SSC at 72uC for 2 min,
then transferred quickly to ice-cold 70% ethanol for 5 min, then to
90 and 100% ethanol for 5 min each, and air-dried.
BAC clones for the human chromosome 5 were purchased from
Invitrogen and were used as probes. The position of each probe is
shown in figures. The cosmid clone of cCl12–140 for the human
chromosome 11 was kindly provided by Dr. K. Okumura .
Biotin-16-dUTP labeled probes were prepared by nick translation.
For each slide, 96 ng labeled probe was ethanol-precipitated
in the presence of 4 mg human Cot-1 DNA (Roche) and 8 mg
salmon sperm DNA (Nacalai tesque) as carrier. DNA was
resuspended in 20 ml of hybridization buffer (50% formamide
(v/v) and 10% dextran sulfate in 2X SSC), denatured at 80uC
for 10 min, and preannealed at 37uC for 20–30 min before
applying to the slide. The hybridization probe mixture was
applied to each slide under a coverslip, and sealed with rubber
solution. Each slide was incubated overnight at 37uC in a moist
formamide in 2X SSC for 5 min at 42uC, followed by three
washes in 0.8X SSC for 5 min at 60uC. The slides were then
incubated for 30 min in a blocking solution (3% bovine serum
albumin fraction V (Sigma) and 0.1% Tween20 in 4X SSC) at
37uC and further incubated with anti-BrdU Alexa Fluor 546
conjugate (Invitrogen), Streptoavidin Alexa Fluor 488 conjugate
(Invitrogen) and DAPI in a detection solution (1% BSA and
0.1% Tween20 in PBS) for 30 min at 37uC. Then slides were
washed three times with PBST (0.1% Tween20 in PBS) for
5 min at 42uC, mounted in an antifade solution (23.3 mg/ml of
DABCO (Sigma) and 20 mM Tris-HCl in 90% glycerol) and
sealed with nail polish. Slides were stored at 4uC in the dark
To count the numbers of signals, hybridized slides were
examined using 63Xoil objective on a Zeiss Axiophot fluorescence
microscope fitted with a filter set for DAPI, Alexa Fluor 546 and
Alexa Fluor 488. At least 200 S-phase nuclei were scored, and the
signal patterns were classified as singlet-singlet (SS)/singlet-doublet
(SD)/doublet-doublet (DD) or single (S)/double (D) in some cases.
three timeswith 50%
Gene Expression Analyses by RT-PCR
Jurkat and HL-60 were stimulated with 20 nM phorbol
myristate acetate (PMA) and 1 mM ionomysin for 6 hrs. Total
RNA (5 mg) was extracted with Trizol (Invitrogen) according to
the manufacturer’s protocol and then reverse transcribed using
a oligo(dT)12–18 primer and Expand Reverse Transcriptase
(Roche). To prepare non-coding transcripts, total RNA (4 mg)
was reverse transcribed using random primers (Takara).
PCR amplification of the genes of interest was carried out using
the first-strand cDNA. The PCR products were visualized on
agarose gel stained with SYBR Green I (Molecular probes). Gel
images were captured and band intensities, determined by LAS-
1000 (Fujifilm), were normalized to that of a housekeeping gene,
GAPDH. Primer sequences and PCR condition for each primer
set are available on request. The list of the genes examined are in
Genome Analyses through Bioinformatics
The genes used in the analysis were taken from NCBI H.
sapiens Genome (http://www.ncbi.nlm.nih.gov/). Transposable
elements were extracted from the 129.0–132.5 Mb segment of the
human chromosome 5 by RepeatMasker analysis (http://www.
Western Blotting and Antibodies
procedures. Protein samples were mixed with 5X sample buffer
(0.25 M Tris-HCl (pH6.8), 10% SDS, 50% glycerol, 11% 2-
mercaptoethanol, and 0.02% bromophenol blue), boiled, and
separated on SDS-PAGE (7.5 to 10% acrylamide). Antibodies
from commercial sources were as follows: SATB1 (ab49061,
was performedaccordingto standard
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Abcom; #61182, BD Biosciences), a-tubulin (T5168, Sigma),
peroxide-conjugated anti-rabbit IgG and anti-mouse IgG (711-
036-152 and 711-036-151, respectively, from Jackson Immu-
noResearch Laboratories, Inc.).
Poly2A(+) mRNA of Jurkat was prepared and reverse
transcribed as described above. Full-length SATB1 cDNA was
amplified by PCR using the following primers; huSATB1-N 59-
CCG CTC GAG ATG GAT CAT TTG AAC GAG GCA ACT-
39 and huSATB1-C 59-GCT CTA GAT CAG TCT TTC AAA
TCA GTA TTA AT-39. PCR-products, digested with XbaI and
XhoI, were gel-eluted and the resulting DNA fragment was inserted
at the XbaI-XhoI sites of pCSII-EF-mKO2-Cdt1DXhoI (in which
one XhoI to the 59 of mKO2 was deleted; mKO2, monomeric
Kusabira Orange 2; , generating pCSII-EF-mKO2DXhoI-
SATB1. pCSII-EF-mKO2-Cdt1DXhoI digested by XbaI/XhoI was
treated with the Klenow fragment and religated, generating
a control vector pCSII-EF-mKO2DXhoI. The final clones were
verified by sequencing.
Transfection and Sorting of Cells Expressing MKO2-SATB1
HeLaS3 cells, plated at a density of 46106cells per 10-cm plate,
were incubated for 24–30 hr prior to transfection. The plasmids
encoding mKO2 alone (pCSII-EF-mKO2DXhoI) or mKO2 fused
to SATB1 (pCSII-mKO2DXhoI-SATB1) (10 mg each) was in-
troduced into HeLaS3 cells using FuGENE HD Transfection
Reagent (Roche) according to manufacturer’s protocol. After 24–
48 hrs, cells were observed by Biozero BZ-8000 fluorescence
microscope (Keyence, Inc.) to examine transfection efficiency.
Then, the cells were labeled with 30 mM BrdU for 10 min prior to
harvesting. Cells were collected in PBS containing of 2 mg/ml
propidium iodide (SIGMA). Living cells expressing mKO2 were
sorted by FACS Aria (BD Biosciences). The sorted mKO2-positive
cells were used for replication timing analysis by FISH as
Short Hairpin RNA (shRNA)-mediated Knockdown
pRS vector or pRS-shSATB1 (ORIGENE) was introduced into
Jurkat cells (56106) by electroporation using Nucleofector
(AMAXA). The sequences of SATB1-shRNAs are pRS-SATB1-
GAGGTGTCTTCCGAAATCTACC-39. Cells were incubated
for 72 hrs and an aliquot of the transfected cells (56105) was used
for western analysis to check the repression of expression. The rest
of the cells were used for replication timing analyses by FISH as
Generation of Stable Cell Lines
The pCSII-EF-mKO2DXhoI or pCSII-mKO2DXhoI-SATB1
plasmid was transfected with the packaging plasmid (pCAG-
HIVgp) and the VSV-G/Rev-expressing plasmid (pCMV-VSV-
G-RSV-Rev) into 293T cells  using TransIT293 (Mirus).
High-titer viral solutions were collected at 3 days after tansfection,
and used for transduction into HeLaS3 cells. At 10 days after
transduction, mKO2-positive clones were selected under Biozero
BZ-8000 fluorescence microscope (Keyence, Inc.) and HeLaS3
cells expressing mKO2 or mKO2-SATB1 were established and
stored. Expression of expected polypeptides was confirmed by
Western blot analysis.
Chromatin Immunoprecipitation (ChIP)
ChIP analysis was carried out in HeLaS3 cells and HeLaS3
expressing SATB1. In brief, cells at about 80% confluency
(,16107cells) were cross-linked for 4 min at 37uC by addition of
1% formaldehyde, and 125 mM glycine was added to terminate
the reaction. The cells were washed with ice-cold phosphate-
buffered saline (PBS), harvested in buffer I (50 mM Hepes-KOH
(pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-
40, 0.2 5% Triton X-100, protease inhibitors and 1 mM
phenylmethylsulfonyl fluoride (PMSF)), and incubated at 4uC for
10 min. After centrifugation, pellets were resuspended in lysis
buffer II (10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM
EDTA, and 0.5 mM EGTA) and incubated at 4uC for 10 min.
After centrifugation, pellets were resuspended in lysis buffer III
(50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA,
0.5 mM EGTA, 1% TritonX-100, 0.1% sodium deoxycholate,
and 0.1% SDS containing protease inhibitors and 1 mM PMSF)
and sonicated in an ice-water bath until cross-linked chromatin
DNA was sheared to an average length around 500 bp. Insoluble
materials were removed by centrifugation at 15,000 rpm in
a microcentrifuge at 4uC for 5 min. The sonicated cell supernatant
was incubated with 10 ml of Protein A Sepharose beads (GE
Healthcare) bound with 32 mg of salmon sperm DNA and 2 mg of
mouse SATB1 antibody or mouse IgG1 at 4uC overnight. The
beads were harvested by centrifugation and washed six times with
RIPA wash buffer (50 mM HEPES-KOH (pH 7.0), 500 mM
LiCl, 1 mM EDTA, 0.7% sodium deoxycholate, 1% NP-40,
protease inhibitors and 1 mM PMSF) and once with TE (10 mM
Tris-HCl (pH 8.0) and 1 mM EDTA). Chromatin antibody
complexes were eluted from the beads by addition of elution
buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS).
Cross-linking was reversed by incubation at 65uC overnight in TE
containing 1%SDS. Protein K digestion was performed for 4 hrs
at 37uC in digestion buffer (10 mM Tris-HCl (pH 8.0), 1 mM
EDTA, 200 mM NaCl, 0.5 mg/ml of proteinase K, and 0.5 mg/
ml of glycogen). ChIP DNA was precipitated with ethanol and
dissolved in TE containing RNase A. After incubation for 1 hr at
37uC, ChIP DNA was purified by MinElute (QIAGEN) and used
for quantitative PCR.
Quantitative Real-time PCR Analysis
Real-time PCR was conducted using SYBR Premix EX Taq
(Takara) with LightCycler480
sequences are available upon request. The PCR products were
measured by SYBR green fluorescence.
(Roche Diagnostics). Primer
Cell Cycle Synchronization
Cell cycle synchronization was conducted as previously de-
scribed . Briefly, cells were arrested at the G1/S boundary by
incubation in the presence of 2.5 mM thymidine for 16 hrs twice
with a 9-hr interval of growth without thymidine. Arrested cells
were released into cell cycle and harvested at the indicated times.
Preparation and Purification of Genomic DNA
56106cells were resuspended in 600 ml of lysis buffer (10 mM
Tris-HCl (pH 8.0), 100 mM NaCl and 1 mM EDTA), and 30 ml
of 10% SDS and 6 ml of 20 mg/ml Proteinase K was added,
followed by gentle mix and incubation at 55uC for several hrs to
overnight with rotation. After adding 0.6 ml of phenol/chloro-
form/isoamyl alcohol, the samples were mixed and centrifuged at
15,000 rpm for 5 min. The supernatant was treated with an equal
volume of chloroform/isoamyl alcohol twice and DNA was
precipitated by adding 1/10 volume of 3 M NaOAc and an
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equal volume of isopropanol. DNA was dissolved in 0.1 ml EB
buffer (10 mM Tris-Cl (pH 8.5), QIAGEN). For quantification of
the amount of genomic DNA at a particular locus, qPCR was
conducted on a diluted set of the purified DNA solution at each
time point after release from double thymidine block.
Replication Timing of the 5q23/31 Locus on the Human
In order to analyze regulation of replication timing in different
cell types, we examined replication timing of a segment on the
human chromosome 5 by quantification of the nascent DNA in S-
phase stage-specific cell populations of Jurkat (Human T
lymphocyte from acute T cell leukemia) and HL-60 (Human
promyelocytic leukemia cells, non-T lymphocyte), which are
different types of mature leukemia cells. We focused on the 3.5-
Mb segment of 5q23/31 containing clusters of cytokine genes
whose transcription is regulated in a T cell-type specific manner
Nascent DNAs were labeled with BrdU, which is incorporated
into newly replicated DNA in place of thymidine, and then we
sorted the labeled cells into six fractions (representing G1, S2, S2,
S3, S4, and G2/M cell cycle stages) by a cell sorter. Fractionation
into expected cell cycle stage-specific populations was confirmed
by FACS analyses (Fig. 1A and B). The BrdU-labeled DNA was
immunoprecipitated and enrichment of newly replicated DNA in
each fraction was analyzed by PCR-based assays [20,28,29]. The
values were corrected by the level of BrdU-labeled mtDNA which
replicates equally throughout the cell cycle (Fig. 1C) . The
fraction containing the highest level of BrdU-labeled DNA
indicated the replication timing. As positive control, we first
analyzed replication timing of PGK1 (phosphoglycerate kinase I)
and F9 (coagulation factor IX) known to be replicated in early and
late S phase, respectively [28,38]. As expected, PGK1 and F9
replicated in the early and late S phase, respectively, in HL60 cells
(Fig. 1C). We noted that the distribution of replication timing is
generally broader and show variations at different locations in
TTR. This may suggest a possibility that the precise timing may
vary in different single cells or in two alleles, and this variation may
be larger in TTR compared to other regions. This could be due to
the variation of the fork speed when it proceeds in TTR.
We then examined the replication timing of the 3.5-Mb
segment at 5q23/31 by using locus-specific primers. We de-
termined the S phase stages showing the highest enrichment of
BrdU-labeled DNA, which represented the time of replication,
and aligned the data along the segment at 5q23/31 (Fig. 1D and
Table S1). The segment containing the cytokine cluster (130.3–
132.5 Mb on chr5) replicates early (G1–S2) in both cells, in spite of
differences in cell-types and gene expression profiles. On the other
hand, the adjacent segment (129.0–129.9 Mb on chr5) replicates
late (S3, S4) in both cell types (Fig. 1D). Thus, early- and late-
replicating domains are basically conserved on the 3.5-Mb
segment. A replication timing transition region (TTR) is present
between the early- and late-replicating domains. The detailed
analyses of this region revealed that TTR in two cell types are
offset by about 200 kb. This suggests cell type-specific mechanisms
may be involved in determination of TTR.
Cell-type Specific Replication Timing Transition Region
In order to more precisely determine TTR in HL-60 and
Jurkat, we analyzed replication timing in more details. The
locations of timing boundary are offset by 180-kb in the two cell
types (Fig. 1D); they are located between the coordinates 129.52
and130.16 Mbp in HL-60 (non-T cell) and between 129.98–
130.22 in Jurkat (T cell) (Fig. 1E and Fig. S1). On the basis of the
locations that give a mid-S timing (a blue dotted line in Fig. 1D),
we have tentatively defined the segments at 129.95–130.05 Mb or
130.15–130.25 Mb as TTR-H or TTR-J, respectively.
We next conducted FISH analyses to investigate replication
timing at this locus (Fig. 2A). We counted the fluorescence signals
in at least 200 S-phase nuclei labeled with BrdU in both cell types.
The fluorescence signals were divided into three categories, two
unreplicated dots (single-single, SS), one unreplicated and one
replicated dot (single-double, SD) and two replicated dots (double-
double, DD). By using the cCl12-140 probe representing an early
replicating region in HL-60 , we confirmed early replication
patterns at this locus in both HL-60 and Jurkat (Fig. 2B; DD,
45.0%; SD, 28.2%; SS, 36.1% in HL-60 and DD, 42.6%; SD,
30.2%; SS, 36.1% in Jurkat). We then conducted FISH analyses
using probes at the human chromosome 5q23/31 (Table S2 and
Fig. 2B). The fluorescence signals with the probes 2,6 in HL-60
only gave single (S, unreplicated) or double (D, replicated) dots,
indicating that this region in HL-60 is haploid. FISH analyses can
distinguish replicated and unreplicated chromosomes on both
diploid and haploid genome regions, and we set the threshold for
early replication in diploid or haploid locus at the DD or D value
of approximately 40% that is larger that the value for SS or S,
With the probe 6, the frequency of ’’replicated’’ signals was
higher than that of ‘‘unreplicated’’ signals both in Jurkat (DD,
39.2%.SS, 23.4%) and HL-60 (D, 55.6%.S, 44.4%), indicating
that this segment is early-replicating in both cells. With the probe
1, the frequency of ‘‘unreplicated’’ signals was higher than that of
‘‘replicated’’ signals both in HL-60 (DD, 12.8%,SS, 39.9%),
indicating that this is late-replicating in HL-60 cells. It should be
noted that probe 1 locus is replicated at mid/early in Jurkat (DD,
26.0%,SS, 32.0%) and at early in HeLaS cells. The mechanisms
behind this sort of localized difference of replication timing among
different cell types are not known. With the probes 4 in HL-60, the
frequency of D sharply increased (23.8%2.41.1%) and the
frequency of S decreased (76.2%2.58.9%) compared to the
probe 3 (Fig. 2B). These data indicated that the location between
the probes 3 and 4 marks the timing transition in HL-60. This
conclusion is consistent with that determined by semi-qPCR of the
timing-specific nascent DNA (Fig. 1). On the other hand, in
Jurkat, DD/SS at the probe 4 is 9.0%/55.2%, whereas that at the
probe 5 is 16.2%/36.9%, The values become 39.2%/23.4% at the
probe 6, indicating that the location between the probes 4 and 6
marks the timing transition in Jurkat. This result is consistent with
TTR determined by semi-qPCR of the timing-specific nascent
DNA in Jurkat (Fig. 1). Thus, these data further support the
presence of the cell type specific TTR.
Although replication timing domains are generally conserved
among different species (e.g. between human and mouse) ,
cell-type specific replication timing may be observed in as much as
50% of the human genome . Cell type-specific TTR may
contribute to generation of some of these variations in replication
AT-rich Nature and the Absence of Coding Regions
In order to understand the features of TTR, we analyzed
distribution of genome composition, GC-content (%), gene
density, and numbers of transposable elements (Fig. S2). Adjacent
to the TTR, a synteny break point is present in rat and mouse. as
is often found in other TTR (Fig. S2A; . The early replicating
region spanning the cytokine cluster segment is rich in GC content
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(.43%), as reported previously (Fig. S2B) [38,39]. In contrast,
the late replicating region has lower GC content (,43%). Analyses
with narrower window ranges (10–100 kb) indicate that TTR is
located in the segment with a lower GC content (AT-rich).
Although LINEs (long interspersed nuclear element) are rather
uniformly scattered in both early- and late-replicating regions,
higher numbers of LINEs (,30 LINEs) are located near cytokine-
cluster segment (Fig. S2C, shown by a black arrow). The highest
density of LINEs, notably L1, is located within the TTR (32
LINE1s in the 25 kb segment, 130.025–130.05 Mb; shown by
a red arrow). The frequency of SINEs (short interspersed nuclear
(Fig. S2D). Another feature of TTR is the absence of genes
(Fig. S2E). These findings suggest that TTR may reside in the
AT-rich genome segment lacking genes and containing clusters of
Multiple Potential SATB1 Binding Sequences are Present
Informatics examination of the 3.5-Mb segment of 5q23/31
revealed the presence of potential SATB1 (Special AT-rich
sequence Binding protein 1) binding sites in the vicinity of the
TTR. SATB1 binds to AT-rich regions and matrix attachment
region (MAR). SATB1 binding sites were reported in previous
studies and referred to as SBSs [40,41]. Among the sixteen types of
SBSs identified in Jurkat , sequences similar to the three types
of SBSs (SBS-2, 4 and 14) were found on the 3.5 Mb segment
(Fig. S3A), while those related to other types of SBSs (SBS-1, 3,
5,13, 15 and 16) were not identified. SBS-2, 4 and 14 were
located on the LINE1 sequence, especially on the L1P subfamily
(Table S4). Furthermore, we searched the sequences with higher
identity (§85%) and bit scores (§400), leading to ‘‘highly similar’’
SBSs (0 sequence for SBS-2, 9 sequences for SBS-4 and 39
sequences of SBS-14; Fig. S3B). Interestingly, these ‘‘highly
Figure 1. Replication timing of the human 5q23/31. A. Experimental strategy for determination of replication timing. Asynchronously
replicating cells were labeled with BrdU and sorted by FACS into six fractions (G1, S1–4, G2/M) on the basis of DNA content. Genomic DNA from cells
in each fraction was extracted, and newly replicated DNA was immunoprecipitated with anti-BrdU antibody. Semi-quantitative PCR was carried out
using the newly replicated DNA as template. Relative band intensity was quantified. The values in each fraction were normalized by the levels of
BrdU-labeled mitochondrial DNA (mtDNA; replicated equally throughout the cell cycle) used as an internal control for the recovery of DNA in each
sample. B. 40,000 cells (Jurkat and HL-60) sorted (upper) and collected on the basis of DNA content (G1, S1–4, G2/M) were stained with PI, and
analyzed by FACS (lower). C. Validation of cell cycle fractionation. The known early (PGK1) or late (F9) replicating region is enriched in appropriate
fractions in comparison with the level of mtDNA in HL-60. D. DNA replication timing on the human chromosome 5q23/31 region (3.5 Mb) containing
the cytokine cluster region in Jurkat (T cell) and HL-60 (non T cell). The 2.2 Mb segment containing the cytokine cluster (130.3–132.5) replicates in G1
or early in the S-phase (S1 and S2), whereas the 0.9 Mb segment distal to the cluster and proximal to the centromere replicates late in the S phase (S3,
S4 and G2). The mean locations of the timing transition region (TTR) are located at around 130.15–130.25 (yellow box) in Jurkat (TTR-J) and at around
129.95–130.05 (green box) in HL-60 (TTR-H), and are offset by 180 kb in the two cell types. The left boundary of early replicating region coincided
with the transition of chromosomal synteny (see Fig. S2A). E. The results of replication timing assays with fractionated cells are shown for each
location along the 3.5 Mb human chromosome. The locations of the 9 primers used are indicated along the 5q23/31 region shown to the right of the
panels. Small red solid boxes show the peak timing fraction for each probe, and large red dotted boxes show the maximum timing transition
segments for Jurkat (129.98–130.22) and HL-60 (129.52–130.16).
SATB1 Regulates Replication Timing
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similar’’ SBSs were located and clustered close to TTR
(Fig. S3C). Since SATB1 is specifically expressed in T-cells, the
results suggested a possibility that SATB1 may be involved in
generation of cell-type specific TTR.
SATB1 Expression Correlates with Replication Timing at
To explore whether replication timing can be altered by
SATB1, we examined SATB1 expression and replication timing of
the TTR in Jurkat, HL-60 and HeLaS3. SATB1 was expressed in
a cell type-specific manner, high in Jurkat, low in HL-60, and
almost none in HeLaS3 (Fig. 3A). FISH analysis was performed
by using the probe 4, because this probe permitted the detection of
cell-type specific TTRs. We found that replication timing at this
locus is late in Jurkat: the frequencies of SS/DD are 55.2/9.0%.
On the other hand, in HeLaS3 or HL60, DD or D is 46.5% or
41.1%, respectively, indicating that this region is early-replicating
or early/mid-replicating (Fig. 3B and Fig. 4E). Thus, regulation
at TTR is correlated with the expression level of SATB1,
suggesting a possibility that SATB1 may play a role in determining
the timing transition.
Replication Timing in the Vicinity of the TTR is Affected
To evaluate the possibility that SATB1 regulates TTR, we
examined the effect of modulation of the SATB1 level on the
location of TTR. We first expressed SATB1 in HeLa cells, whose
endogenous expression level is very low (Fig. 4A). The human
SATB1 cDNA was cloned into the CSII-EF-mKO2 vector
plasmid , generating huSATB1 tagged with mKO2 at its N-
terminus (Fig. 4A). At 24 and 43 hrs after transfection into
HeLaS3 cells, we observed the fluorescent signals of mKO2-
SATB1 in nuclei (Fig. 4B), whereas those from vector-transfected
cells were detected in the cytoplasm. Such patterns of localization
were identical to a previous study . Expression of the mKO2-
SATB1 protein was confirmed also by western blot analysis
(Fig. 4C). At 43 hrs after transfection, over 80% of HeLaS3 cells
transfected with plasmids were alive and the transfection efficiency
was generally around 26,41%. Although overexpression of
Figure 2. Analyses of replication timing by FISH. A. Hybridization signals of replicating cells. SS, singlet-singlet: SD, singlet-doublet; DD,
doublet-doublet. B. Replication timing analyzed by FISH at 5q23/31. Locations of DNA probes derived from 5q23/31used in this study are shown at
the top. Human BAC clones were purchased from Invitrogen. A cosmid clone on the chromosome 12, cCl12–140, was kindly provided by Dr. Okumura
(Nogami et al, 2000), and was used as a control for early replication. At least 200 BrdU-positive nuclei (S-phase) were counted for each probe. The
signal patterns were classified into SS, SD, or DD. On the haploid segment of HL-60 (probes 2,6), two signal patterns, singlet (S) and doublet (D),
were observed. Replication timing of the human 5q23/31, estimated from the FISH analyses, is consistent with that of the cell cycle fractionation
studies (see text for details). E, E/M M/L and L stand for early-, early/mid-, mid/late- or late-replicating, respectively.
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SATB1 was reported to be toxic in human cells , viability was
not significantly affected at least for 48 hrs after transfection under
the conditions employed (Fig. 4D). Thus, we used HeLaS3
transfected with CSII-EF-mKO2-SATB1 and CSII-EF-mKO2 for
further analysis of replication timing.
We collected live and mKO2-positive cells by FACS sorting,
which were analyzed by FISH to determine replication timing.
In HeLaS3, we observed two types of cells, one containing two
homologues and the other containing three homologues at
5q23/31. We first counted signal patterns in over 100 nuclei of
two homologue cells (Fig. 4E; shown by arrows). In HeLaS3
expressing mKO2-SATB1, replication timing at the probe 4
locus exhibited a dramatic change from early- to late-replicating
(DD, 46.5%2.16.9%; SS, 12.9%2.39.0%). We also analyzed
the cells with three copies of the locus, and similar change was
observed(Fig. S4; DDD,
9.9%2.22.7%). In contrast, replication timing at other probes
(probes 3 and 5) did not differ significantly between HeLaS3
and HeLaS3 expressing mKO2-SATB1. At the probes 1 and 6,
replication timing became slightly earlier when mKO2-SATB1
was expressed (DD 41.6%2.53.1% and SS 20.3%2.16.7%
for probe 1; DD 34.9%2.47.6% and SS 18.3%2.6.3% for
probe 6). As a control, we also examined replication timing in
HeLaS3 expressing mKO2 (Fig. 4E). Although slight change
was detected at the probe 4 (DD 46.5%2.34.4%, SS
12.9%2.24.0%), it was much less significant than that
observed with mKO2-SATB1. Basically, very similar results
were obtained in analyses of three homologue cells (data not
shown). These results indicate that expression of SATB1 in
HeLa cells results in change of TTR by delaying the replication
timing of the Probe 4 locus.
We next examined the effect of SATB1 knockdown on TTR
in Jurkat cells (Fig. 5A). shRNA-expressing vectors (pRS-
SATB1-shRNA1 and 2] or pRS vector) were introduced into
[mixture of pRS-
Jurkat cells by electroporation. Western blotting of the extracts
at 72 hr after electroporation confirmed the suppression of
SATB1 expression (Fig. 5B). Since efficiency of electroporation
was over 88%, we used all the cells for FISH without cell
sorting (Fig. 5C). Replication timing at the Probe 4 locus
changed from late to early after repression of SATB1 expression
inJurkat cells(DD 9.0%2.33.8
55.2%2.21.9% or 22.0%). Electroporation of pRS plasmid
only marginally affected the timing at this locus. These results
indicate that suppression of SATB1 expression in Jurkat results
in change of TTR by causing the Probe 4 locus to become
early-replicating. Taken together, above results indicate that
SATB1 protein regulates the location of TTR by somehow
delaying the replication timing of a specific genome segment.
SATB1 Binds to the Edge of an Early Replicating Domain
Adjacent to TTR
We then examined whether SATB1 binds to TTR or its vicinity
as was predicted from the informatics analyses. We employed
chromatin immunoprecipitation assays using HeLaS3 cells stably
expressing mKO2-SATB1 (Fig. 6). Since HeLaS3 cells express
SATB1 only at a very low level (Fig. 3 and 4), SATB1 binding to
DNAs in the stable clone occurred almost exclusively by
exogenous SATB1. By using the lentivirus transduction system
and mKO2-fused expression vectors , we could obtain stable
clones expressing SATB1 to a significant level without much effect
on growth rate and cell cycle (Fig. S5), in spite of previous reports
that SATB1 overexpression is toxic in human cultured cells ,
and that efficient translation may require the 442 bp SATB1
39UTR . The level of mKO2-SATB1 was comparable to that
of SATB1 in Jurkat cells (data not shown).
SATB1 binds to the 200-kb T-helper 2 (Th2) cytokine locus on
the mouse chromosome 11, which is syntenic to the human 5q23/
31 examined in this study, and regulates coordinated expression of
Il5, Il4 and Il13 in mice . In human Jurkat T cells, SATB1
Figure 3. Correlation between SATB1 expression and replication timing at TTR. A. SATB1 expression in Jurkat, HL-60 and HeLaS3. Whole
cell extracts, separated on 7.5% SDS-polyacrylamide gel, were blotted with anti-SATB1 antibody. Expression of SATB1 is high in T cell (Jurkat), and low
or non-detectable in non-T cells (HL-60 and HeLaS3). B. Replication timing of the Probe 4 (see Fig. 2B and Table S1) in Jurkat, HL-60 and HeLaS3.
This locus replicates in late-S in Jurkat (high SATB1) and in early-S in HL-60 (low SATB1) and HeLaS3 (very low SATB1).
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binds to the distal promoter region of human IL-2 and regulates
IL-2 expression, while it does not bind to the proximal promoter
region of IL-2Ra [41,44–47]. ChIP analyses using anti-SATB1
antibody showed that SATB1 bound to the promoter region of
human IL-2 (positive control) but not to that of IL-2Ra (negative
control) in Jurkat T cells [46,47]. We then examined SATB1
binding to the human 5q23/31 region using five different primers;
primer a located in late replicating region, primer b located at the
cell-type specific TTR, primer c located at the boundary of early-
replicating domain, primers d and e located in the early-
replicating region near the cytokine genes (Table S5). We found
that SATB1 specifically bound to the primer c locus, which is
located at the edge of the early-to-late transition region in Jurkat
cells (Fig. 6B). Furthermore, the primer c locus includes the
sequences highly similar to the SATB1 consensus (V$SATB1.01;
wntAATAnwnnwnn, from Genomatix, not within LINE1) con-
served among mammals (Fig. S3). These results suggest that
SATB1 regulates TTR by directly binding to the early-replicating-
SATB1 May Delay the Replication Timing at TRR by
Slowing Down the Fork Movement
Above results suggest that SATB1 delays the replication timing
in the TTR segment by directly binding to the edge of the early-
replicating domain adjacent to TTR. We therefore examined rate
of replication by directly measuring the copy number of genome
DNA in a synchronous culture. Cells (HeLaS3 and HeLaS3 stably
expressing SATB1; Fig. S5A) were synchronized at the G1/S
boundary by double thymidine block and then the same numbers
(0.56106cells) of cells were released into cell cycle in a synchro-
nous manner. Total genomic DNA was prepared at different
timepoints after release. The amount of the bulk DNA, as
measured by FACS analysis, increased with a similar time course
after release and reached 4C at 8 hrs in both cells (data not
shown), indicating that the proliferation and overall S phase
progression are not affected by expression of SATB1 (Fig. S5B
We selected 5 loci in and around TTR and changes of copy
number were monitored at 0 to 8 hrs after release. Genomic DNA
Figure 4. Effect of SATB1 expression on replication timing at TTR: expression of SATB1 in HeLaS3 cells. A. The procedure for obtaining
HeLaS3 cells overexpressing SATB1. B. At 24 hr after transfection with pEF-mKO2 (upper panels) or pEF-mKO2-SATB1 (lower panels), cells were
observed using a fluorescence microscope BioZero (KEYENCE). mKO2 fluorescence was observed mainly in the cytoplasm of HeLaS3 cells expressing
mKO2 vector or in the nuclei of those expressing mKO2-SATB1. Red, mKO2 signal. C. Whole cell extracts were examined by western blotting using
anti-SATB1 antibody. Lane 1, non-transfected HeLaS3, lanes 2 and 4, HeLaS3 transfected with pEF-mKO2; lanes 3 and 5, HeLaS3 transfected with pEF-
mKO2-SATB1, lane 6, Jurkat. Lanes 2 and 3, 24 hr after transfection; lanes 4 and 5, 43 hr after transfection. D. Sorting of mKO2 positive cells by FACS
Aria (BD Biosciences). Cells expressing mKO2-SATB1 were separated into viable (yellow) and dead (red) cells stained with PI, then mKO2 positive cells
(green) among viable cells were further separated. E. Replication timing of HeLaS3 and HeLaS3 expressing mKO2-SATB1 at 43 hr after transfection.
We analyzed replication timing of HeLaS3 transfected with mKO2-SATB1 at 24 and 43 hr after transfection. Only the data at 43 hr are shown for cells
non-transfected (left panel) or transfected with mKO2-SATB1 plasmid (central panel). At least 200 BrdU-positive nuclei (S-phase) were counted for
each probe. Replication timing in TTR (detected by Probe 4) changed from early (HeLaS3) to late (HeLaS3 expressing mKO2-SATB1) (indicated by the
arrows). Expression of mKO2 did not affect the replication timing of the transition region (right panel).
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purified at the time of release (0 hr) was used as a standard for
quantification (unreplicated DNA). Then, genomic DNAs purified
from 2–8 hrs after release were quantified by qPCR at the 5 loci.
was normalized by that at mitochondria region. To estimate the
amount of DNA synthesis at each timepoint, the levels of DNA
synthesis at 2 and 8 hrs were set at 0% and 100%, respectively
Replication timing as well as the rate of fork movement could be
estimated from this analysis. At the primer d locus, 100% DNA
synthesis is achieved at 4 hrs after release in both cells, indicating
that this segment is very early-replicating and its replication timing
is not affected by SATB1. In contrast, at the primer c, f and b loci,
the timing is still early-replicating in HeLaS3, but the 8 hrs is
needed to achieve 100% DNA synthesis in SATB1-expressing
cells. DNA synthesis appears to proceed slower in the latter cells
compared to the former cells. At the primer a locus, 8 hrs is
needed to achieve 100% DNA synthesis in HeLaS3, consistent
with this loci being late-replicating. In SATB1-expressing HeLaS3,
little DNA synthesis is observed at the primer a locus by 6 hrs, and
is completed by 8 hrs, indicating that replication timing is further
Possible mechanisms for delayed replication in TTR and the
surrounding region in the presence of SATB1 include the
suppression of origin firing in the TTR segment  and/or
inhibition of fork movement along the TTR. Since it has been
proposed that TTR may be composed of a long origin-less
segment replicated by a single unidirectional fork , and
previous mapping did not show the presence of origins in this
TTR segment [16,49–52], we speculate that it is most likely that
SATB1 slows down the replication fork movement.
Eukaryotic genomes are replicated under a program that
dictates the preferred locations for initiation and timing of
replication during S phase . In yeasts, checkpoint functions
regulate the origin firing program, suppressing the firing of some
origins, which may be late-firing or dormant under normal
conditions [4,53–56]. Histone modification and transcription
factors have also been shown to regulate the origin firing program
[10,11]. In yeast, physiological conditions can also affect the origin
selection or timing regulation, suggesting significant plasticity of
this process . Single cell or single molecule analyses indicate
that origin selection can vary in individual cells and during
successive cell cycle in a single cell, suggestive of stochastic nature
in the process [58,59].
In higher eukaryotes, timing of origin firing may be
determined by the chromosome domains of Mb size, known
as ‘‘replication (timing) domain’’ [12,13]. The replication
domain structures correlate with transcriptional activity, histone
modification, and chromatin proximity defined by Hi-C analyses
Figure 5. Effect of SATB1 expression on replication timing at TTR: suppression of SATB1 expression in Jurkat cells. A. The procedure
for repression of SATB1 expression in Jurkat. B. Whole cell extracts were examined by western blotting using anti-SATB1 antibody. Lane 1,
untransfected Jurkat; lane 2, Jurkat transfected with pRS vector; lane 3 and 4, Jurkat transfected with pRS-SATB1-shRNA1 and with pRS-SATB1-
shRNA1+ pRS-SATB1-shRNA2, respectively. Cells were harvested at 72 hr after transfection. C. Replication timing was determined by FISH across the
TTR. Only the data at the Probe 4 are shown. Replication timing in the transition region changed from late (Jurkat) to early (SATB1-depleted Jurkat)
(indicated by the arrows). At least 200 BrdU-positive nuclei (S-phase) were counted.
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Figure 6. Chromatin immunoprecipitation (ChIP) assays of SATB1 binding. A. Locations of primers (a–e) used for ChIP assays. B. ChIP
analyses were carried out by using anti-mouse SATB1 antibody (left panel) or purified mouse IgG1 control antibody (central panel) jn HeLaS3 cells
stably expressing SATB1. Chromain-immunoprecipitated DNA was purified by MinElute (QIAGEN) and used for quantitative PCR. Error bars represent
the mean and standard deviations based on four independent experiments. Relative ratio (SATB1/control) is shown as SATB1-specific binding (right
Figure 7. Genome copy number analyses of TRR in synchronized cell population. A. Locations of primers (a, b, c, d and f) used for copy
number analyses. B. The procedure for quantification of copy number. C. Time course of DNA replication at various locations in and around TTR.
Genome copy numbers of cells synchronously growing from double thymidine block were quantified at various loci at various timepoints. The level
of DNA synthesis at 2 hr or 8 hr after release was set as 0% or 100% DNA synthesis, respectively.
SATB1 Regulates Replication Timing
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(chromosome conformation capture), and may undergo major
change during the differentiation process [12,15]. As much as
50% of the entire human genome may exhibit cell-type specific
replication timing domain structures . However, mechan-
isms regulating replication timing in mammals have been largely
The human 5q23/31 locus contains clusters of cytokine genes
and presents an attractive model for the studies on regulation of
replication timing because of the availability of ample information
on transcriptional regulation and chromatin regulation. We
previously reported identification of two origins (GM-CSF ori1
and ori2) on human 5q31.1  as well as another new origin
downstream of IL-13 (oriIL-13) and crucial role of CNS (conserved
non-coding sequence essential for transcriptional regulation of
cytokine genes)  for firing of this origin, suggesting possible
coregulation of transcriptional activation and origin firing [20,22].
We also showed that the replication timing of the cytokine locus is
not affected by inactivation of this origin, showing that local origin
activity does not affect the timing regulation. In this communi-
cation, we have compared the replication timing of the 3.5 Mb
segment containing the cytokine clusters in T-cells and non-T
cells. We have identified difference in timing at the boundary
between the early-replicating and late-replicating segments.
Genomic features of TTR and its surrounding region suggest
the followings. 1) TTR is present in an AT-rich area, adjacent to
GC-rich early-replicating segments. 2) TTR coincides with the
boundary of synteny. 3) TTR resides in a gene-poor, origin-less
We noted the presence of clusters of potential binding sites for
SATB1, a T-cell specific transcription regulator, in and near TTR.
SATB1 was originally discovered as a T cell-specific AT-rich
sequence binding protein, and was shown to bind to MAR
[23,40,61,62]. It was shown to regulate T cell activation by
recruiting HDAC or HAT to chromatin [45,46,63,64]. It may
promote chromatin loop structures for coregulation of cytokine-
related genes [24,25,65,66]. It was also shown to promote breast
tumor growth and metastasis through regulation of a large number
of genes predominantly involved in cell adhesion, cellular signaling
and cell cycle . It also contributes to initiation of X
inactivation [68–70], and may regulate embryonic stem cell
differentiation through modulation of Nanog expression . We
found that the level of SATB1 correlates with the location of the
replication timing boundary. Therefore, we decided to examine
potential roles of SATB1 in determination of replication timing
domains. We conducted two experiments. The first was to
examine the effect of SATB1 expression in HeLaS3 cells which
normally do not express SATB1. The second was to repress the
expression of SATB1 in Jurkat cells which normally express
SATB1 at a high level. We have shown in both experiments that
SATB1 expression correlates with the extension of the late-
replicating segment toward the early-replicating segment, in-
dicating that SATB1 somehow enforces the late replication at
TTR. By ChIP assays, we showed that SATB1 indeed binds to the
vicinity of the TTR.
We then measured rate of DNA synthesis through S phase at
or near TTR by measuring the copy number increase and
showed that the rate of DNA synthesis is delayed at the TTR
where SATB1 binds. This could be due to failure of firing at
the origins potentially present at TTR or its surrounding region.
However, it has been known that replication timing boundary is
generally associated with origin-less segment, and previous
analyses have indicated the absence of origins in this TTR
[48,49]. Therefore, we speculate that fork progression may be
slowed down or arrested by SATB1. We do not know how
SATB1 interferes the progression of a replication fork at the
moment. SATB1 may bind to some nuclear structures (nuclear
matrix or skelton), as was previously indicated [23,40,61,62],
and this may interfere with fork progression. We have detected
change of histone modification near TTR in the presence or
absence of SATB1. Generally, active marks increase in the
absence of SATB1, whereas they decrease in the presence of
SATB1 (from UCSC genome browser), and this may be
correlating with early or late replication, respectively. However,
SATB1 is also involved in activation of transcription through
inducing histone acetylation [24,45,63,64].
What would be the physiological significance of regulation of
replication timing by SATB1? SATB1 does not seem to affect
genome-wide replication domain structures (data not shown).
Rather, it affects local replication timing where it binds. Slowed
replication in the presence of SATB1 may be a result of
chromatin modificationor altered
Alternatively, SATB1-mediated arrest/pausing of replication
fork may contribute to more error-free replication of TTR,
which tends to be associated with higher mutation rate and
more SNP. As suggested before, SATB1 could facilitate the
formation of chromatin loop structures [25,66,72–75], which
could be intimately related to transcriptional regulation as well
as to replication timing domain structures. Although SATB1
does not appear to play a major role in defining the general
replication timing domain structures (our unpublished data), it
may fine-tune the replication timing so that replication and
transcription may be coordinately regulated. Genome-wide
views on how SATB1 affects replication timing as well as on
the binding sites of SATB1 would help elucidate the precise
roles of SATB1 in regulation of DNA replication.
Replication timing assays were conducted as described in the
legend to Fig. 1. The locations of the primers used are indicated
along the 5q23/31 region shown to the right of the panels. Probes
1–6 represent those used in FISH assays (Fig. 2), and red boxes
show the peak timing fraction. See Table S1 for the locations of
Replication timing of the human 5q23/31.
region. A. Chromosomal synteny among human, rat, mouse and
chicken corresponding to the human 5q23/31 region. B.
Distribution of GC content (%). The GC contents were calculated
by sliding window analyses across the genome region shown
(sliding size, 1 kb; window size, 10–100 kb). The result indicates
that early replicating segment is located in higher GC region
(.43%). On the other hand, late replicating region as well as
transition region are located in low GC region (,43%). C.
Distribution of LINEs. A segment containing the most LINE1 was
identified near the TTR (32 LINEs in the 25 kb segment,
130.025–130.05 Mb; red arrow). D. Distribution of SINEs.
Average numbers of SINEs were calculated within the early-
and late-replicating domains and are shown as horizontal red bars
(19 SINEs/25 kb and 8 SINEs/25 kb, in early and late regions,
respectively). Data were extracted from a RepeatMasker analysis
(http://www.repeatmasker.org/) on the 129.0–132.5 Mb segment
of the human chromosome 5. The numbers of LINE1 and SINEs
were calculated by a sliding window analysis (sliding size: 25 kb,
window size: 25 kb). E. Gene density. The total numbers of genes
were calculated in non-overlapping windows of 250 kb each. The
genes used in the analysis were taken from NCBI H. sapiens
Genomic features of the human 5q23/31
SATB1 Regulates Replication Timing
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Genome (http://www.ncbi.nlm.nih.gov/). The gene density in the
early replicating region is significantly higher than that in the late
replicating region. In the vicinity of the IL-13/IL-4 gene loci, there
are 11 genes in a 250 kb segment (130–132.25). Notably, no
experimentally confirmed genes are present in the TTR. TTR-H
and TTR-J are shown by green and yellow vertical bars,
sequences (SBSs) at the human 5q23/31. A. Sequences
similar to SBS-1,16 on the 129–132.5Mb segment of human
chromosome 5 were searched by using NCBI Blast (http://www.
ncbi.nlm.nih.gov/BLAST/). Sequences similar to SBS-2 (514 bp),
24 (465 bp) and 214 (387 bp) were found, and ‘‘bit scores’’ were
plotted against identity. B. Sequences with high identity (§85%)
and bit scores (§400) as ‘‘highly similar’’ SBSs were selected (0
sequence for SBS-2, 9 sequences for SBS-4 and 39 sequences of
SBS-14). Locations and numbers of these potential SBSs are
shown on the human 5q23/31 3.5-Mb segment. C. Among them,
nine pairs of SBS-4 and -14 that were found to be present on one
Line1P (highlighted in yellow in Table S4) are indicated (blue,
green and yellow small rectangles). Five of them were identified to
be present close to the TTR (shown within the orange box). These
findings lead us to suggest a possibility that the SBS clusters within
and close to TTR may play a role in defining the replication
timing boundary. Different colors for the rectangles indicate
different orientations of SBS-4 and -14 on each Line1P. TTR-H
and TTR-J are shown by green and yellow vertical bars,
Distribution of potential SATB1 binding
timing at TTR in HeLaS3 cells (with three chromosomes
at the 5q locus). Replication timing of HeLaS3 and HeLaS3
expressing mKO2-SATB1 at 43 hr after transfection. We
analyzed replication timing of HeLaS3 transfected with mKO2-
SATB1 at 24 and 43 hr after transfection. Only the data at 43 hr
are shown for cells non-transfected (left panel) or transfected with
mKO2-SATB1 plasmid (right panel). At least 200 BrdU-positive
nuclei (S-phase) containing three chromosomes at the 5q locus
were counted for Probe 3 and 4. Replication timing in TTR
(detected by Probe 4) changed from early (HeLaS3) to mid/late
(HeLaS3 expressing mKO2-SATB1) (indicated by the arrows).
Effect of SATB1 expression on replication
HeLaS3 cells stably expressing mKO2-SATB1. A. HeLaS3
and HeLaS3 cells stably expressing mKO2-SATB1 were observed
by FSX100 (OLYMPUS). Red, mKO2 signal. B. Growth rate of
HeLaS3 and stable mKO2-SATB1 HeLaS3. C. Stable mKO2-
SATB1 HeLaS3 cells were synchronized at the G1/S boundary by
double thymidine block, and then synchronously released into cell
cycle and were collected at the times indicated (0, 2, 4, 6, 8 and
9 hrs). Collected cells were fixed in 70% ethanol, and DNA
contents were analyzed by FACS. Growth rate and cell cycle
distribution are not affected by expression of SATB1 in HeLaS3
Cell growth and cell cycle of HeLaS3 and
replication timing at the human 5q23/31. Primers located further
to the right of position (9) are not listed in this table.
Locations of PCR primers used to determine
BAC clones used for replication timing analyses by
their map positions and gene sizes.
The names of the genes at the human 5q23/31. and
SATB1 binding sites located on L1P of the LINE-1 subfamily.
Yellow columns indicate the Line1P sequences that carry both
SBS-4 and 14, which are shown in Fig. S3C.
Locations of sequences highly similar to known
SATB1 binding and for copy number analyses.
Locations of the primer sets used for ChIP analyses of
We thank Dave Gilbert and Ichiro Hiratani for helpful discussion and
critical comments on the manuscript. We would like to thank Rino
Fukatsu, Naoko Kakusho and Ai Ishii for excellent technical assistance.
Conceived and designed the experiments: MO HM. Performed the
experiments: MO YK HM. Analyzed the data: MO YK HM. Contributed
reagents/materials/analysis tools: MO YK YW HM. Wrote the paper:
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