SATB1 Regulates Replication Timing 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. doi:10.1371/journal.pone.0042375.g002 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 TTR 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 by SATB1 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 [32], 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 [42]. 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 PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42375