(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 [16]. However, mechan- isms regulating replication timing in mammals have been largely elusive. 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 [21] 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) [60] 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 region. 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 [67]. It also contributes to initiation of X inactivation [68–70], and may regulate embryonic stem cell differentiation through modulation of Nanog expression [71]. 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 Regulates Replication Timing 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 modification or altered transcriptional activity. 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. Supporting Information Figure S1 Replication timing of the human 5q23/31. 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 the primers. (TIF) Figure S2 Genomic features 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 PLoS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e42375