Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1 Masako Oda, Yutaka Kanoh, Yoshihisa Watanabe, Hisao Masai* Genome Dynamics Project, Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Abstract Background: Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as ‘‘replication domains’’, and recent findings indicate that replication timing is under developmental and cell type-specific regulation. Methodology/Principal Findings: We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. Conclusions: Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome. Citation: Oda M, Kanoh Y, Watanabe Y, Masai H (2012) Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1. PLoS ONE 7(8): e42375. doi:10.1371/journal.pone.0042375 Editor: Marco Muzi-Falconi, Universita’ di Milano, Italy Received March 22, 2012; Accepted July 4, 2012; Published August 7, 2012 Copyright: 2012 Oda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by Grant-in-Aid for Scientific Research (A) (to HM) and Grant-in-Aid for Scientific Research on Priority Area ‘‘Chromosome Cycle’’ from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to HM). No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: masai-hs@igakuken.or.jp Introduction DNA replication occurs once and only once during S phase of cell cycle [1]. This strict regulation is achieved by multiple layers of mechanisms. Once DNA replication is initiated, three billion base- pair human genome is replicated in 7,8 hrs [2]. Origins on the eukaryotic chromosomes can be classified according to the timing at which they are programmed to fire during S phase [3]. Studies in yeasts have established that intra-S checkpoint negatively regulates the firing of late or dormant origins [4]. This is also the case in mammalian cells as well [5–8]. Other factors including histone modifiers have also been shown to regulate the timing of origin firing [9–11]. On larger genomes of higher eukaryotes, replication is initiated from tens of thousands of loci on the genome [2]. It is known that clusters of origins are fired more or less simultaneously, and they may constitute distinct chromosome domains that may be referred to as ‘‘replication (timing) domain’’ [12–14]. The genome-wide landscape of replication timing domains were shown to change during the process of differenti- ation and different cell types may have distinct replication timing domain structures [12,15–17]. As much as 50% of the genome may be regulated differentially in different cell types [16], and 20% of the mouse genome undergoes reorganization during development [12]. Replication timing is generally conserved between different species (e.g. between human and mice) [13]. Replication domains may be correlated with histone modification marks and most notably with chromosome proximity organization that can be estimated by 4C or high-C assays [13,18,19]. It would be important to identify factors that may be responsible for cell-type specific regulation of replication timing. We have been using the human chromosome 5 5q23.3/31.1 locus (will be referred to as 5q23/31 hereafter) containing a cytokine cluster region as a model locus for the study of replication program, since extensive studies have revealed the transcription factors and epigenetic regulation involved in cell-type-specific and simulation- dependent regulation of transcription in this region. We previously mapped an origin near the cytokine cluster and have shown that replication timing regulation is not affected by the activity of local origins [20–22]. We have compared replication timing of the 3.5 Mb segment of 5q23/31 in T cells and non-T cells, and found that the boundary of the early and late replication domain may be differentially regulated. Furthermore, a T cell specific-factor, SATB1 (Special PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42375