H1 12/12+ﬂox cells were cultured in the presence or absence of OHT for 4 days. Then, the treated and untreated cells were mixed in a 10:1 ratio and monitored for GFP positive (H1R-eGFP depleted) population by ﬂow cytometric analysis. Detection of chromosomal aberration Measurement of chromosomal aberration was performed as previously described (20,21). Brieﬂy, 5 ml of a 2 105 cells/ml suspension was treated with 0.1 mg/ml of colcemid for 1.5 h prior to analysis. Cells were subsequently har- vested and then incubated in 1 ml of 0.9% sodium citrate for 15 min after which 4 ml of Carnoy’s solution (methanol:acetic acid = 3:1) was added. After centrifugation for 5 min (150 g), cells were further incubated in 5 ml of Carnoy’s solution for 30 min at room temperature. Cells were centrifuged again for 5 min (150 g), resuspended in 200 ml of fresh Carnoy’s solution, and dropped onto glass slides prewetted with 50% ethanol. After drying overnight, slides were immersed into 3% Giemsa solution diluted in 50 mM phosphate buﬀer (pH 6.4) for 10 min, washed with tap water, completely dried at room temperature, and mounted with Entellan New. Genomic aberrations were counted (11 macro-chromosomes and Z-chromosomes/sample) following the criteria of ISCN. Aberration examples are shown in Supplementary Figure S2. Chromosomes were analyzed using a Zeiss AxioPhoto microscope (100 objective lens). Preparation of acid-extracted histones Cells (4–5 107) were washed once with 1 PBS(–) and suspended in 1 ml of HM buﬀer [10 mM HEPES (pH 7.0), 2 mM MgCl2], containing 0.2% Triton-X100. Nuclei were collected by centrifugation at 3000 r.p.m. for 5 min and washed with HM buﬀer. Then, nuclei were washed with HM buﬀer containing 150 mM NaCl, and collected by centrifugation at 15 000 r.p.m. for 5 min. Nuclei were resuspended with 0.2 M H2SO4 and incubated for 1 h at 4 C to extract the histone fraction. After centrifugation at 15 000 r.p.m. for 15 min, the supernatant was collected and divided into two tubes. To prepare the H2SO4 histone extract, trichloroacetic acid (TCA) was added to one tube at a ﬁnal concentration of 20%. To prepare HClO4 histone H1 extract, perchloric acid was added to another tube at a ﬁnal concentration of 5% and incubated for 1 h on ice before the addition of TCA. After further incuba- tion on ice for 1 h, the tubes were centrifuged, and the precipitates thus obtained were washed with acetone– HCl and twice with acetone before being suspended in 40–50 ml of 8 M Urea – 1 SDS buﬀer (13,23). Western blot analysis The relative levels of HMG1 and HMG2 proteins in total cell lysate and chromatin fraction were estimated among wild-type and H1 mutant DT40 cells by western blot analysis. Chromatin-bound fraction was prepared by treating cells with 0.2% Triton-X 100 followed by extrac- tion with 0.2 M H2SO4, as described above. Diﬀerent amounts of protein samples (0.5–4 105 cell equivalent/ Nucleic Acids Research, 2010, Vol. 38, No. 11 3535 lane) were separated by SDS-PAGE before western blotting using anti-HMG1/2 monoclonal antibody (clone FBH7; 1:2000), which cross-reacts with both chicken HMG1 and HMG2 (23). H1R-eGFP was detected using anti-GFP (MBL; 1:1000). MNase digestion and NRL measurement Nuclear preparation for MNase digestion was performed as described previously (4). Cells were harvested, washed with PBS(–), and suspended in 0.5% NP-40 in RSB [10 mM Tris–Cl (7.5), 10 mM NaCl, 1 mM CaCl2, 3 mM MgCl2 and 0.5 mM phenylmethylsulfonyl ﬂuoride] (2 107/ml) for 20 min on ice. Then, the cells were homogenized with 15 strokes of pestle A in a Dounce homogenizer, centrifuged for 6 min at 1000 g, and the nuclear pellets were resuspended in RSB (2 107/100 ml). Nuclei were digested with MNase (Takara) for 5 min at 37 C (0.05 and 0.4 U MNase for 4 106 nuclei/20 ml). Reactions were terminated by addition of 5 mM ethylene-diamine-tetra-acetic acid. Then, chromatin was treated with proteinase K (100 mg/ml) for 90 min at 56 C, extracted with phenol–chloroform, precipitated with alcohol in the presence of 0.3 M Na-acetate, and the pellet was resuspended in TE buﬀer containing RNase. NRL measurement was done in 1% agarose gel in Tris acetate buﬀer. DNA sizes of diﬀerent nucleosome units were calculated from their gel migration distance using the following formula: [DNA length] = A e B [mobility] + C (where the values of A, B and C were obtained by ﬁtting to a curve obtained using 100-bp ladder standards). Then NRL was calculated using the following formula: [DNA size] = [NRL] [number of nucleosome] + D (where D is a variable number). The mean square displacement, r2, ranged between 0.9989 and 0.9995. All ﬁtting analyses were done using PRISM 5 (GraphPad Software, Inc.). Expression array analysis Exponentially growing DT40 cells cultured in medium containing 100 nM Z-VAD-FMK were harvested, and total RNA was prepared using Sepasol RNA-I (Nakalai Tesque, Kyoto, Japan) following the manufacturer’s instructions and further puriﬁed with RNeasy (QIAGEN). Puriﬁed total RNA (5 mg) was used to syn- thesize cRNA with a one-cycle labeling kit (Aﬀymetrix) according to the manufacturer’s instructions. cRNAs were hybridized to a chicken genome array (Aﬀymetrix). Fluorescent intensities were measured with a GeneChip Scanner 3000 and analyzed with GeneChip Operating Software v3.5 (Aﬀymetrix); data were further analyzed with GeneSpring GX v7.2 (Agilent Technologies). Duplicated array hybridizations were performed for cRNAs from H1 12/12 + ﬂox (clone name: K11), H1 12/12 #1 (clone name: K11-5) and wild-type DT40 cells. Duplicate expression array data were deposited with the NIH Gene Expression Omnibus (http://www.ncbi.nlm. nih.gov/projects/geo/: accession number GSE8483). Each raw data point was normalized to 1.000 per chip. Probes other than absent-tagged probes, with a raw signal of less than 100.0 among all three genotypes, were rejected.