SATB1 Regulates Replication Timing Figure 3. Correlation between SATB1 expression and replication timing at TTR. A. SATB1 expression in Jurkat, HL-60 and HeLaS3. Whole cell extracts, separated on 7.5% SDS-polyacrylamide gel, were blotted with anti-SATB1 antibody. Expression of SATB1 is high in T cell (Jurkat), and low or non-detectable in non-T cells (HL-60 and HeLaS3). B. Replication timing of the Probe 4 (see Fig. 2B and Table S1) in Jurkat, HL-60 and HeLaS3. This locus replicates in late-S in Jurkat (high SATB1) and in early-S in HL-60 (low SATB1) and HeLaS3 (very low SATB1). doi:10.1371/journal.pone.0042375.g003 SATB1 was reported to be toxic in human cells [43], viability was not significantly affected at least for 48 hrs after transfection under the conditions employed (Fig. 4D). Thus, we used HeLaS3 transfected with CSII-EF-mKO2-SATB1 and CSII-EF-mKO2 for further analysis of replication timing. We collected live and mKO2-positive cells by FACS sorting, which were analyzed by FISH to determine replication timing. In HeLaS3, we observed two types of cells, one containing two homologues and the other containing three homologues at 5q23/31. We first counted signal patterns in over 100 nuclei of two homologue cells (Fig. 4E; shown by arrows). In HeLaS3 expressing mKO2-SATB1, replication timing at the probe 4 locus exhibited a dramatic change from early- to late-replicating (DD, 46.5%2.16.9%; SS, 12.9%2.39.0%). We also analyzed the cells with three copies of the locus, and similar change was observed (Fig. S4; DDD, 32.5%2.12.7%; SSS, 9.9%2.22.7%). In contrast, replication timing at other probes (probes 3 and 5) did not differ significantly between HeLaS3 and HeLaS3 expressing mKO2-SATB1. At the probes 1 and 6, replication timing became slightly earlier when mKO2-SATB1 was expressed (DD 41.6%2.53.1% and SS 20.3%2.16.7% for probe 1; DD 34.9%2.47.6% and SS 18.3%2.6.3% for probe 6). As a control, we also examined replication timing in HeLaS3 expressing mKO2 (Fig. 4E). Although slight change was detected at the probe 4 (DD 46.5%2.34.4%, SS 12.9%2.24.0%), it was much less significant than that observed with mKO2-SATB1. Basically, very similar results were obtained in analyses of three homologue cells (data not shown). These results indicate that expression of SATB1 in HeLa cells results in change of TTR by delaying the replication timing of the Probe 4 locus. We next examined the effect of SATB1 knockdown on TTR in Jurkat cells (Fig. 5A). shRNA-expressing vectors (pRS- SATB1-shRNA1, pRS-SATB1-shRNA1+2 [mixture of pRS- SATB1-shRNA1 and 2] or pRS vector) were introduced into Jurkat cells by electroporation. Western blotting of the extracts at 72 hr after electroporation confirmed the suppression of SATB1 expression (Fig. 5B). Since efficiency of electroporation was over 88%, we used all the cells for FISH without cell sorting (Fig. 5C). Replication timing at the Probe 4 locus changed from late to early after repression of SATB1 expression in Jurkat cells (DD 9.0%2.33.8 or 42.1%, SS 55.2%2.21.9% or 22.0%). Electroporation of pRS plasmid only marginally affected the timing at this locus. These results indicate that suppression of SATB1 expression in Jurkat results in change of TTR by causing the Probe 4 locus to become early-replicating. Taken together, above results indicate that SATB1 protein regulates the location of TTR by somehow delaying the replication timing of a specific genome segment. SATB1 Binds to the Edge of an Early Replicating Domain Adjacent to TTR We then examined whether SATB1 binds to TTR or its vicinity as was predicted from the informatics analyses. We employed chromatin immunoprecipitation assays using HeLaS3 cells stably expressing mKO2-SATB1 (Fig. 6). Since HeLaS3 cells express SATB1 only at a very low level (Fig. 3 and 4), SATB1 binding to DNAs in the stable clone occurred almost exclusively by exogenous SATB1. By using the lentivirus transduction system and mKO2-fused expression vectors [32], we could obtain stable clones expressing SATB1 to a significant level without much effect on growth rate and cell cycle (Fig. S5), in spite of previous reports that SATB1 overexpression is toxic in human cultured cells [43], and that efficient translation may require the 442 bp SATB1 39UTR [42]. The level of mKO2-SATB1 was comparable to that of SATB1 in Jurkat cells (data not shown). SATB1 binds to the 200-kb T-helper 2 (Th2) cytokine locus on the mouse chromosome 11, which is syntenic to the human 5q23/ 31 examined in this study, and regulates coordinated expression of Il5, Il4 and Il13 in mice [25]. In human Jurkat T cells, SATB1 PLoS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e42375