3534 Nucleic Acids Research, 2010, Vol. 38, No. 11 micro-nuclei, respectively, and the micronuclear mitotic chromosome structure is slightly less condensed in MLH-deﬁcient cells (8). Among higher eukaryotes, the domain structure of histone H1 is conserved, but multiple variants (six for chickens and eight for mice) exist (11,12). So far, knockout (KO) phenotypes of single and multiple histone H1 variants have been reported (4,13–17). Single histone H1 variant KO studies show that none of the histone H1 variants appears to be essential for cell growth and that the expression of only a subset of genes is aﬀected. However, triple H1 (H1c, H1d and H1e) KO mice are embryonic lethal (4). Phenotypes generated in knockdown studies in mammalian cell lines are slightly diﬀerent from those in KO studies, showing that mouse H1.2 and H1.4 are involved in cell cycle and cell death (18). Knockdown of H1 in Drosophila also induces embry- onic lethality, suggesting that H1 is generally important in embryogenesis (4,19). To further elucidate the role of linker histone H1 in higher eukaryotes, particularly under nonredundant con- ditions, we established completely histone H1-deﬁcient mutant cells using the chicken B lymphocyte line DT40. Herein we describe the phenotype of histone H1-null mutant cells and discuss the potential role(s) of histone H1 in stabilizing chromatin structure, growth rate and chromosomal aberrations, in addition to its function as a global transcription regulator. MATERIALS AND METHODS Cell lines, cell culture and plasmid construction Generation of H1 10/12 H1-DT40 cells (clone name: 17-3-41-25) and H1 11/12 DT40 cells (clone name: 17-3-41-25-13) has been previously described (13). Cells were cultured in RPMI 1640 medium supplemented with 10 mM 2-mercaptoethanol, 10% fetal calf serum (FCS) (Gibco) and 1% chicken serum (Gibco) at 39.5 C. DNA transfection was carried out as previously described (20). Brieﬂy, for each transfection, 1 107 cells were suspended in 0.5 ml phosphate-buﬀered saline (PBS) containing 25 mg of linearized plasmid and electroporated with a Gene Pulser apparatus (BioRad) at 550 V and 25 mF. Following electroporation, cells were transferred into 20 ml of fresh medium and incubated for 24 h. Cells were then divided into four 96-well microtiter plates (0.2 ml/well) and selected with either 0.25 mg/ml Zeocin or 0.5 mg/ml puromycin. To generate the bleomycin-resistant targeting construct, pD02H1/Bleo, the DNA fragment with the bleomycin-resistant gene driven by the beta-actin promoter was subcloned into the BclI site of the plasmid containing the 02H1 gene (02H1 ORF) and linearized by BamHI. The ﬂoxed C-terminal-enhanced green ﬂuorescence protein (eGFP) fusion histone H1R variant H1R-eGFP expression vector, Flox2-H1R-eGFP-IRES-puro, was generated as follows. The BglII/NotI fragment of H1R-eGFP (20) was subcloned into the pCAGGS-IRES-puromycin- expressing vector (a kind gift from Hitoshi Niwa at RIKEN), and loxP sequences were introduced into the 50 region of the ATG start site and the 30 region of the polyA sequences. The ﬂoxed H1R-eGFP expression vector was linearized by AhdI. The Mer-Cre-Mer expression vector (a kind gift from Michael Reth) was linearized by ScaI. The genome-integrated ﬂoxed H1R-eGFP transgene in H1 12/12+ﬂox (clone name: K11) was deleted by treat- ment with 4-OHT (100 nM) for 5 days. Southern blot analysis Southern blotting was performed as previously described (13,17,20). Flow cytometry analysis For cell cycle analyses, cells were labeled for 10 min with 20 mM bromodeoxyuridine (BrdU) (Amersham). They were then harvested and ﬁxed at 4 C overnight with 70% ethanol and successively incubated as follows: (i) in 4N HCl, 0.5% Triton X-100 for 30 min at room temper- ature; (ii) with FITC-conjugated anti-BrdU antibody (Pharmingen, San Diego, CA, USA) for 1 h at room tem- perature; and (iii) in 5 mg/ml propidium iodide (PI) in PBS. Between each incubation, cells were washed with PBS con- taining 5% FCS and 0.1% sodium azide. Subsequent ﬂow cytometric analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA, USA). Fluorescence data were displayed as dot plots using the Cell Quest software (Becton Dickinson). For measurement of mitotic index, cells exposed to 2.3 Gy IR or unirradiated cells were cultured in the presence or absence of colcemid (0.1 mg/ml), and the cells were collected at 1-h intervals. The fraction of mitotic cells was measured by ﬂow cytometry using an antibody directed against histone H3 serine 10 phosphorylation (H3S10ph) as follows. Cells were washed in PBS once, ﬁxed in 3.5% formaldehyde containing PBS for 10 min at 37 C, and reﬁxed in 90% methanol for 30 min on ice. The ﬁxed cells were washed in incubation buﬀer (0.5 mg/ ml of BSA containing 1 PBS) twice, and incubated with rabbit anti-phospho-histone H3 Serine 10 antibody (1:100; Cell Signaling) in incubation buﬀer for 30 min. Cells were then incubated with FITC-conjugated anti-rabbit IgG (1:100; Santa Cruz) for 30 min. Cells were ﬁnally stained with 1 mg/ml of PI and treated with 10 mg/ml of RNase A. Nuclear volume measurement Cells were ﬁxed with 4% paraformaldehyde and stained with 6-diamino-2-phenylindole (DAPI), and z-stack images were collected at 1-mm intervals using a FV-1000 confocal microscope (Olympus). The nuclear area in each section was measured using ImageJ software (NIH), and the total nuclear volume was obtained by summing the area of all sections showing DAPI signal. At least 27 interphase nuclei were analyzed in each cell line. Cell growth assay Cells were passed every 3 or 4 days (2.5–5 103 cells/ml for wild-type or 5–10 103 cells/ml for H1 mutant cells) and the total number of cells were counted at each passing time. For a growth competition assay, H1 11/12+ﬂox and