SATB1 Regulates Replication Timing 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). doi:10.1371/journal.pone.0042375.g001 (.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 element) is generally higher in early replicating regions (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 LINE1. Multiple Potential SATB1 Binding Sequences are Present Near TTR 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 [40], 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 PLoS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42375