Genome (http://www.ncbi.nlm.nih.gov/). The gene density in the early replicating region is significantly higher than that in the late replicating region. In the vicinity of the IL-13/IL-4 gene loci, there are 11 genes in a 250 kb segment (130–132.25). Notably, no experimentally confirmed genes are present in the TTR. TTR-H and TTR-J are shown by green and yellow vertical bars, respectively. (TIF) Figure S3 Distribution of potential SATB1 binding sequences (SBSs) at the human 5q23/31. A. Sequences similar to SBS-1,16 on the 129–132.5Mb segment of human chromosome 5 were searched by using NCBI Blast (http://www. ncbi.nlm.nih.gov/BLAST/). Sequences similar to SBS-2 (514 bp), 24 (465 bp) and 214 (387 bp) were found, and ‘‘bit scores’’ were plotted against identity. B. Sequences with high identity (§85%) and bit scores (§400) as ‘‘highly similar’’ SBSs were selected (0 sequence for SBS-2, 9 sequences for SBS-4 and 39 sequences of SBS-14). Locations and numbers of these potential SBSs are shown on the human 5q23/31 3.5-Mb segment. C. Among them, nine pairs of SBS-4 and -14 that were found to be present on one Line1P (highlighted in yellow in Table S4) are indicated (blue, green and yellow small rectangles). Five of them were identified to be present close to the TTR (shown within the orange box). These findings lead us to suggest a possibility that the SBS clusters within and close to TTR may play a role in defining the replication timing boundary. Different colors for the rectangles indicate different orientations of SBS-4 and -14 on each Line1P. TTR-H and TTR-J are shown by green and yellow vertical bars, respectively. (TIF) Figure S4 Effect of SATB1 expression on replication timing at TTR in HeLaS3 cells (with three chromosomes at the 5q locus). 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 (right panel). At least 200 BrdU-positive nuclei (S-phase) containing three chromosomes at the 5q locus were counted for Probe 3 and 4. Replication timing in TTR (detected by Probe 4) changed from early (HeLaS3) to mid/late (HeLaS3 expressing mKO2-SATB1) (indicated by the arrows). (TIF) References 1. Arias EE, Walter JC (2007) Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Dev 21: 497–518. 2. Masai H, Matsumoto S, You Z, Yoshizawa-Sugata N, Oda M (2010) Eukaryotic chromosome DNA replication: where, when, and how? Annu Rev Biochem 79: 89–130. 3. Gilbert DM (2010) Evaluating genome-scale approaches to eukaryotic DNA replication. Nat Rev Genet 11: 673–684. 4. Hayano M, Kanoh Y, Matsumoto S, Renard-Guillet C, Shirahige K, et al. (2012) Rif1 is a global regulator of timing of replication origin firing in fission yeast. Genes Dev 26: 137–150. 5. Merrick CJ, Jackson D, Diffley JF (2004) Visualization of altered replication dynamics after DNA damage in human cells. J Biol Chem 279: 20067–20075. 6. Seiler JA, Conti C, Syed A, Aladjem MI, Pommier Y (2007) The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses. Mol Cell Biol 27: 5806–5818. 7. Chastain PD, Heffernan TP, Nevis KR, Lin L, Kaufmann WK, et al. (2006) Checkpoint regulation of replication dynamics in UV-irradiated human cells. Cell Cycle 5: 2160–2167. 8. Karnani N, Dutta A (2011) The effect of the intra-S-phase checkpoint on origins of replication in human cells. Genes Dev 25: 621–633. 9. Wu PY, Nurse P (2009) Establishing the program of origin firing during S phase in fission Yeast. Cell 136: 852–864. SATB1 Regulates Replication Timing Figure S5 Cell growth and cell cycle of HeLaS3 and HeLaS3 cells stably expressing mKO2-SATB1. A. HeLaS3 and HeLaS3 cells stably expressing mKO2-SATB1 were observed by FSX100 (OLYMPUS). Red, mKO2 signal. B. Growth rate of HeLaS3 and stable mKO2-SATB1 HeLaS3. C. Stable mKO2- SATB1 HeLaS3 cells were synchronized at the G1/S boundary by double thymidine block, and then synchronously released into cell cycle and were collected at the times indicated (0, 2, 4, 6, 8 and 9 hrs). Collected cells were fixed in 70% ethanol, and DNA contents were analyzed by FACS. Growth rate and cell cycle distribution are not affected by expression of SATB1 in HeLaS3 cells. (TIF) Table S1 Locations of PCR primers used to determine replication timing at the human 5q23/31. Primers located further to the right of position (9) are not listed in this table. (TIF) BAC clones used for replication timing analyses by Table S2 FISH. (TIF) Table S3 The names of the genes at the human 5q23/31. and their map positions and gene sizes. (TIF) Table S4 Locations of sequences highly similar to known SATB1 binding sites located on L1P of the LINE-1 subfamily. Yellow columns indicate the Line1P sequences that carry both SBS-4 and 14, which are shown in Fig. S3C. (TIF) Table S5 Locations of the primer sets used for ChIP analyses of SATB1 binding and for copy number analyses. (TIF) Acknowledgments We thank Dave Gilbert and Ichiro Hiratani for helpful discussion and critical comments on the manuscript. We would like to thank Rino Fukatsu, Naoko Kakusho and Ai Ishii for excellent technical assistance. Author Contributions Conceived and designed the experiments: MO HM. Performed the experiments: MO YK HM. Analyzed the data: MO YK HM. Contributed reagents/materials/analysis tools: MO YK YW HM. Wrote the paper: MO HM. 10. Aparicio JG, Viggiani CJ, Gibson DG, Aparicio OM (2004) The Rpd3-Sin3 histone deacetylase regulates replication timing and enables intra-S origin control in Saccharomyces cerevisiae. Mol Cell Biol 24: 4769–4780. 11. Knott SR, Peace JM, Ostrow AZ, Gan Y, Rex AE, et al. (2012) Forkhead transcription factors establish origin timing and long-range clustering in S. cerevisiae. Cell 148: 99–111. 12. Hiratani I, Ryba T, Itoh M, Yokochi T, Schwaiger M, et al. (2008) Global reorganization of replication domains during embryonic stem cell differentiation. PLoS Biol 6: e245. 13. Ryba T, Hiratani I, Lu J, Itoh M, Kulik M, et al. (2010) Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types. Genome Res 20: 761–770. 14. Birney E, Stamatoyannopoulos JA, Dutta A, Guigo´ R, Gingeras TR, et al. (2007) Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447: 799–816. 15. Hiratani I, Ryba T, Itoh M, Rathjen J, Kulik M, et al. (2010) Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis. Genome Res 20: 155–169. 16. Hansen RS, Thomas S, Sandstrom R, Canfield TK, Thurman RE, et al. (2010) Sequencing newly replicated DNA reveals widespread plasticity in human replication timing. Proc Natl Acad Sci U S A 107: 139–144. PLoS ONE | www.plosone.org 12 August 2012 | Volume 7 | Issue 8 | e42375