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. Results Replication Timing of the 5q23/31 Locus on the Human Chromosome 5 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 [34–37]. 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) [30]. 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 SATB1 Regulates Replication Timing 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 [31], 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, respectively. 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) [13], cell-type specific replication timing may be observed in as much as 50% of the human genome [16]. Cell type-specific TTR may contribute to generation of some of these variations in replication timing domains. AT-rich Nature and the Absence of Coding Regions Characterize TTR 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; [27]. The early replicating region spanning the cytokine cluster segment is rich in GC content PLoS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42375