Embryonic stem cell culture
BAF250a flox/flox; Mer-Cre-Mer ESC lines were established from day 3.5 blastocysts obtained by crossing BAF250a flox/+; Mer-Cre-Mer with BAF250a flox/flox and maintained on feeder MEFs in the presence of leukemia inhibitory factor (LIF) as described previously . Mer-Cre-Mer mice were purchased from the Jackson Laboratory; Bar Harbor, ME USA (stock number: 008463). Brg1 flox/flox; Actin-CreER and BAF53a flox/-; Actin-CreER ESC lines were maintained as described previously [33, 34]. To generate mutant ESCs, these ESC lines were treated with 1 μM 4-hydroxytamoxifen (OHT) for 24 h and harvested 48 h later, unless otherwise indicated. As a control, cells were treated with ethanol.
Somatic cell reprogramming
MEF cells derived from BAF250a flox/flox; Mer-Cre-Mer and BAF250a +/+; Mer-Cre-Mer were infected with four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc, OSKM) . Early passage fibroblasts (less than passage 5) were cultured in 6-well dishes and about 4 × 104 cells in each well were infected overnight with viral supernatants freshly prepared by transfection of the retroviral packaging Plat-E cell line (Lipofectaine 2000, Invitrogen, Life Technologies, Carlsbad, CA, USA) containing the cDNAs of the mouse reprogramming factors. Three days after infection, cells were passaged into new wells and tamoxifen was added for three days (Days 3 to 5) or other time windows to ablate BAF250a. Control iPSC-like colonies (BAF250a +/+, OHT treatment or BAF250a flox/flox; Mer-Cre-Mer, no OHT treatment) were typically picked 21 days after infection and iPSC-like colonies from BAF250a flox/flox; Mer-Cre-Mer, OHT treated fibroblast culture were typically picked 30 days post infection. Genotyping of BAF250a was performed by PCR. We used the primer sequences 5′-GTAATGGGAAAGCGACTACTGGAG-3′ and 5′-TGTTCATTTTTGTGGCGGGAG-3′, which amplify a 632-bp fragment from the WT locus, an 812-bp fragment from the floxed locus and a 298-bp fragment from the knockout locus, respectively. PCR reactions were carried out with 40 cycles (30 sec at 94°C, 30 sec at 59°C, 1 min at 72°C). For alkaline phosphatase (AP) staining, culture wells containing iPSC-like colonies were washed with PBS and cells were fixed with 4% paraformaldehyde in PBS for 2 min at 20°C. Fixed cells were then rinsed twice with 0.5 ml of TBST (TBS plus 0.05% Tween-20) and incubated with fresh AP staining solution (4.5 μl 50 mg/ml nitro blue tetrazolium, 3.5 μl 50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris–HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2) in the dark room at 25°C for about 15 min. Stained cells were rinsed with PBS and kept at 4°C.
ChIP was performed as previously described . Two million cells were harvested and fixed in 1% formaldehyde for 10 min at 25°C, then stop fixation in 0.125 M glycine. Fixed cells were sonicated to produce chromatin fragments 300 to 700 bp in length. Chromatin fragments were then immunoprecipitated with anti-Brg1 antibody . The precipitated DNAs were then purified by ethanol precipitation after phenol-chloroform extraction. Quantitative PCR reactions were performed to detect the occupancy of Brg1 at multiple sites within the chromosome 4 and 7 EtoL domains. Quantitative PCR reactions included the following: 4 μl of ChIP product (200 μl per ChIP assay), 10 μl of 2X SYBR green PCR master mix (Applied Biosystem, Carlsbad, CA, USA, 4309155) and 25 nM of each primer. QPCR reactions were tripled and performed in ABI StepOnePlus system through 50 cycles (15 sec at 95°C, 45 sec at 60°C). Ct values were generated by ABI software. Standard errors in Figure 4C were generated from six individual ChIP-qPCR experiments. Concentration of the ChIP samples was calculated as percent of input. QPCR was performed using primers for Oct4 promoter (forward, 5′-AGTGAGAAGGGCAGGAGGAT-3′; reverse, 5′-CCTACTTGCTCACACCACCA-3′), Nkx2.5 promoter (forward, 5′-CCACCCCCAACCCTGCGTTT-3′; reverse, 5′-AGGGGCCGCGACACATTTGG-3′), Chr4 site-1: 104,654,835-104,654,965 (forward, 5′- CAACAACCAACCTAGCTTTCCT-3′; reverse, 5′-GAGAGGATCGGTGGGAGGTC-3′), Chr4 site-2: 104,668,986-104,669,071 (forward, 5′- TCTGAGGGGGTTGGCATAGA-3′; reverse, 5′-GATGTGTGCAAATGGGACCG-3′), Chr4 site-3: 104,693,231-104,693,309 (forward, 5′-TCCCTTACGTCACCGTCTGA-3′; reverse, 5′-AAACACCTTGACCAGAGGGC-3′), Chr 4 site-4: 104,713,676-104,713,776 (forward, 5′-GTTGGCGCTTGTGAACTGAG-3′; reverse, 5′-GTTAGGCAATGGCAGGAGGT-3′), Chr7 site-1: 82,610,306-82,610,419 (forward, 5′-TCCTCGGGAACCTACTCCAG-3′; reverse, 5′-TACAGACACCGACTGAGGCT-3′), Chr7 site-2: 82,647,473-82,647,844 (forward, 5′-GCTCGGGTCTCTGTGTCTGTC-3′; reverse, 5′-CGGGTGGGAGAAAGTGGAAGA-3′), Chr7 site-3: 82,660,145-82,660,243 (forward, 5′-CTCTGCAGCCTGTAAGTGGT-3′; reverse, 5′-ATGTACCACCAGCACACCAG-3′), and Chr7 site-4: 82,662,755-82,662,863 (forward, 5′-CTGATGCCCTGTAGTGCCTT-3′; reverse, 5′-TACAGGGTGGAGGTGGCTTT-3′).
ES cells grown on culture dishes were collected by trypsinization, cytospun onto glass slides, fixed with 4% paraformaldehyde in PBS (10 min, 25°C), washed, and then permeabilized with 0.5% Triton X-100 in PBS (10 min, 25°C). For immunostaining, the samples were incubated in blocking solution (3% BSA, 0.1% Tween 20, 4 × SSC) for 30 min at 37°C to reduce nonspecific binding, and then in detection solution containing primary antibodies (1% BSA, 0.1% Tween 20, 4 × SSC) for 1 h at 37°C. After three washes with 4 × SSC, the samples were incubated in detection solution containing secondary antibodies. For Nanog immunostaining, cells were fixed with formalin/acetic acid and then treated with methanol for 20 min at -20°C. The primary antibodies were: anti-BAF250a mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-20701) diluted 1:50, anti-Oct4 mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 611202) diluted 1:200, anti-Nanog rabbit polyclonal antibody (Chemicon, Temecula, CA, USA, MAB3448) diluted 1:20. Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, Life Technologies, Carlsbad, CA, USA, A11017) and Alexa Fluor 555 goat anti-rabbit IgG (Molecular Probes, Life Technologies, Carlsbad, CA, USA, A21430) were the secondary antibodies. Before imaging, the slides were counterstained with DAPI (200 ng/ml), washed with 4X SSC, and then mounted in 90% glycerol containing antifade reagent.
RNA FISH was performed as described previously . To generate RNA FISH probes, Rex1 genomic DNA fragments were amplified, cloned into pBluscript, and labeled by nick translation. Cells were treated with 0.5% Triton X-100 in CSK buffer (100 mM NaCl, 300 mM sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl2, 1 mM EGTA) for 30 sec at 4°C, fixed with 4% paraformaldehyde, and then immersed in 70% ethanol for 5 min at -20°C, dehydrated through a 90% and 100% ethanol series, and the denatured FISH probe mixture was hybridized to slides at 37°C for 16 h in a moist chamber. Slides were washed three times with 50% formamide in 2X SSC at 43°C and three times with 0.8X SSC at 60°C. Slides were then incubated for 30 min in a blocking solution (3% BSA, 0.1% Tween 20 in 2X SSC) at 37°C and incubated in a detection solution (in 1% BSA, 0.1%Tween 20 in 2X SSC) containing anti-digoxigenin-conjugated rhodamine (Roche, Nutley, New Jersey, USA, 11207750910) for 30 min at 37°C. Then slides were washed three times with 4X SSC, 0.1% Tween 20 for 5 min at 43°C. Before imaging, the slides were counterstained with DAPI (200 ng/ml), washed with 4X SSC, and then mounted in 90% glycerol containing antifade reagent.
Replication timing profiling by microarray
Replication timing analysis was performed as described previously [6, 7]. In brief, cells were labeled with 50 μM BrdU for 2 h, washed twice with ice-cold PBS, trypsinized, and then were fixed in 75% ethanol. These cells were resuspended in PBS containing 1% FBS, stained with propidium iodide (50 μg/ml) for 30 min in the presence of RNaseA (0.5 mg/ml), and then were sorted into early and late S phase fractions by flow cytometry. After phenol-chloroform extraction of DNA, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA, 555627) was performed in each fraction to enrich BrdU-substituted replicating DNA. Isolated early and late replicating DNA were amplified by whole-genome amplification (WGA) (Sigma-Aldrich, St Louis, MO, USA, GenomePlex), labeled with Cy3 and Cy5, and hybridized to a mouse whole-genome microarray (NimbleGen Symtems, Madison WIS, USA, 2006-07-26_MM8_WG_CGH or 100718_MM9_WG_CGH_HX3). Sample labeling, hybridization and data extraction were performed according to standard NimbleGen Systems procedures. Data analyses were performed using R/Bioconductor (http://www.r-project.org; http://www.bioconductor.org). Obtained raw datasets were normalized using the limma package in R/Bioconductor and loess-smoothed over a 300-Kb window size. These smoothed datasets were used to generate replication-timing plots in figures. For some analyses, datasets were averaged into 200-Kb windows (fixed position) and replication timing differential (that is, OHT ratio - mock ratio) was determined for each 200-Kb segment. In order to determine the significant replication timing switching domains that are independent of changes between replicates, we determined Euclidian distance at 10,974 200-Kb segments between groups (that is, mock versus OHT) and within groups (that is, mock replicate-1 versus mock replicate-2), which was used to calculate P values at each 200-Kb genomic segment. Statistical significance was then calculated using the qvalue package in R/Bioconductor, which yields a q-value for each segment that reflects the proportion of false-positives (False Discovery Rate; FDR) among segments deemed to have significant replication timing (RT) changes. High confidence replication timing switching domains were selected with a q-value cutoff of 0.01, corresponding to an overall FDR of 1%. A q-value cutoff of 0.05 was also used to identify a set of lower confidence domains. To examine alignment of timing switching domains to developmental domains, replication timing data from 9 cell types (ESC/iPSC, EBM3/EPL, EBM6/EpiSC, NPC, Mesoderm, Endoderm, partial iPSC, MEF, and Myoblast) were assembled from the ReplicationDomain.org database  and plotted together with the data from BAF250a mock and OHT. Timing switching domains from chromosome 1 (largest-sized) and chromosome 10 (middle-sized) were selected and their alignment to developmental domains was judged by visual inspection in Figure 1E. Indeed, when we examined statistical significance of replication timing changes of BAF250a OHT compared to other cell lines, most domains examined in Figure 1E were not significantly different from at least one of nine cell types, even with a q-value cutoff of 0.2 (42/52 EtoL domains and 42/44 LtoE domains). The size of switching domains was determined using a segmentation algorithm in the DNAcopy package in R/Bioconductor as described previously . Unsmoothed datasets consisting of replication timing (BAF250a OHT ratio - mock ratio) for all probes were processed for switching domain segmentation and the resultant EtoL and LtoE segment sizes were shown in Figure 1F. Replication timing datasets are downloadable from ReplicationDomain (http://www.replicationdomain.org).
Imaging system and measurement
Images were collected using a Nikon Ti-U Eclipse fluorescence microscope equipped with a 60x, 1.40 NA lens and a cooled charge-coupled device camera (C4742-95-12ER, Hamamatsu Photonics, Hamamatsu, Japan), controlled by a windows computer running the software program MetaMorph (Molecular Devices, Sunnyvale CA, USA).