SATB1 Regulates Replication Timing 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). doi:10.1371/journal.pone.0042375.g004 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- domain-proximal boundary. 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 (0.56106 cells) 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 and C). We selected 5 loci in and around TTR and changes of copy number were monitored at 0 to 8 hrs after release. Genomic DNA PLoS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e42375