SATB1 Regulates Replication Timing Figure 5. Effect of SATB1 expression on replication timing at TTR: suppression of SATB1 expression in Jurkat cells. A. The procedure for repression of SATB1 expression in Jurkat. B. Whole cell extracts were examined by western blotting using anti-SATB1 antibody. Lane 1, untransfected Jurkat; lane 2, Jurkat transfected with pRS vector; lane 3 and 4, Jurkat transfected with pRS-SATB1-shRNA1 and with pRS-SATB1- shRNA1+ pRS-SATB1-shRNA2, respectively. Cells were harvested at 72 hr after transfection. C. Replication timing was determined by FISH across the TTR. Only the data at the Probe 4 are shown. Replication timing in the transition region changed from late (Jurkat) to early (SATB1-depleted Jurkat) (indicated by the arrows). At least 200 BrdU-positive nuclei (S-phase) were counted. doi:10.1371/journal.pone.0042375.g005 purified at the time of release (0 hr) was used as a standard for quantification (unreplicated DNA). Then, genomic DNAs purified from 2–8 hrs after release were quantified by qPCR at the 5 loci. The amount of genomic DNA from 2–8 hrs after release at each loci was normalized by that at mitochondria region. To estimate the amount of DNA synthesis at each timepoint, the levels of DNA synthesis at 2 and 8 hrs were set at 0% and 100%, respectively (Fig. 7). Replication timing as well as the rate of fork movement could be estimated from this analysis. At the primer d locus, 100% DNA synthesis is achieved at 4 hrs after release in both cells, indicating that this segment is very early-replicating and its replication timing is not affected by SATB1. In contrast, at the primer c, f and b loci, the timing is still early-replicating in HeLaS3, but the 8 hrs is needed to achieve 100% DNA synthesis in SATB1-expressing cells. DNA synthesis appears to proceed slower in the latter cells compared to the former cells. At the primer a locus, 8 hrs is needed to achieve 100% DNA synthesis in HeLaS3, consistent with this loci being late-replicating. In SATB1-expressing HeLaS3, little DNA synthesis is observed at the primer a locus by 6 hrs, and is completed by 8 hrs, indicating that replication timing is further delayed. Possible mechanisms for delayed replication in TTR and the surrounding region in the presence of SATB1 include the suppression of origin firing in the TTR segment [48] and/or inhibition of fork movement along the TTR. Since it has been proposed that TTR may be composed of a long origin-less segment replicated by a single unidirectional fork [40], and previous mapping did not show the presence of origins in this TTR segment [16,49–52], we speculate that it is most likely that SATB1 slows down the replication fork movement. Discussion Eukaryotic genomes are replicated under a program that dictates the preferred locations for initiation and timing of replication during S phase [2]. In yeasts, checkpoint functions regulate the origin firing program, suppressing the firing of some origins, which may be late-firing or dormant under normal conditions [4,53–56]. Histone modification and transcription factors have also been shown to regulate the origin firing program [10,11]. In yeast, physiological conditions can also affect the origin selection or timing regulation, suggesting significant plasticity of this process [57]. Single cell or single molecule analyses indicate that origin selection can vary in individual cells and during successive cell cycle in a single cell, suggestive of stochastic nature in the process [58,59]. In higher eukaryotes, timing of origin firing may be determined by the chromosome domains of Mb size, known as ‘‘replication (timing) domain’’ [12,13]. The replication domain structures correlate with transcriptional activity, histone modification, and chromatin proximity defined by Hi-C analyses PLoS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e42375