3536 Nucleic Acids Research, 2010, Vol. 38, No. 11 Probes showing more than two sigma diﬀerence between two duplicate chips were also rejected. Then, we excluded selectable marker gene probes and internal control probes. The total number of probes used was 7442 probes per chip. RESULTS Generation of histone H1-null DT40 mutant cells The chicken genome encodes six histone H1 variants (a total of 12 H1 alleles). We previously established a DT40 mutant cell line, H1 11/12 (D57kbhomo, 0.10H1–/–, 02H1+/–; clone name: 17-3-41-25-13), in which 11 out of the 12 histone H1 variant alleles were deleted and only the one 02H1 variant allele was intact (12,13). Since the last 02H1 allele in this cell line could not be deleted by simple homol- ogous gene targeting (13), we developed a diﬀerent strategy using the 4-hydroxytamoxifen (OHT)-inducible Cre/loxP deletion system to establish completely histone H1-deﬁcient DT40 cells. We ﬁrst introduced two expression vectors into H1 11/12 cells (Figure 1A); one expressed a Cre-estrogen receptor fusion molecule, Mer-Cre-Mer, and the other contained the C-terminal- eGFP H1R (H1R-eGFP) gene ﬂanked by two loxP sequences. It has been reported that C-terminal GFP tagging to histone H1 does not aﬀect H1 protein behavior in vitro or in living cells (24). Once the transfectant H1 11/12+ﬂox (D57kbhomo, 0.10H1–/–, 02H1+/–, MerCreMer+, ﬂox2-H1R-eGFP+; clone name: 17-3-41-25-13-10) was established, the last 02H1 allele was successfully deleted by homologous recombina- tion, generating a completely endogenous histone H1-null DT40 cell line, H1 12/12+ﬂox (D57kbhomo, 0.10H1–/–, 02H1–/–, MerCreMer+, ﬂox2-H1R-eGFP+; clone name: K11), with a targeting eﬃciency of 1/215. Southern blot analysis conﬁrmed that the last 02H1 allele was replaced in the pD02H1/Bleo targeting vector since the 13-kb HindIII fragment corresponding to the last 02H1 wild-type allele was converted to a 8.5-kb fragment (bleomycin targeted allele) in H1 12/12+ﬂox cells (Figure 1B and C). Furthermore, Coomassie brilliant blue (CBB) staining of the sodium dodecyl sulfate (SDS)-polyacrylamide gel conﬁrmed the complete absence of endogenous histone H1 protein in H1 12/ 12+ﬂox cells (Figure 1D). Endogenous histone H1 proteins separate as two bands: the upper and lower bands correspond to two (H1L and H1R) and four (01H1, 02H1, 03H1, and 0.10H1) variants, respectively (13). Both H1 11/12 and H1 11/12+ﬂox cells expressed endogenous 02H1 alone, displaying only the lower band. In addition, the 50-kDa exogenous H1R-eGFP fusion protein was detected in the H1 11/12+ﬂox cells (Figure 1E). In H1 12/12+ﬂox cells, the lower 02H1 band was lost, while the H1R-eGFP band remained. H1 12/ 12+ﬂox cells were further treated with 4-OHT to delete the exogenous H1R-eGFP transgene by Cre/loxP recom- bination. Five days after OHT treatment, the ﬂuorescence signals of H1R-eGFP were nearly diminished (data not shown). We next subcloned the eGFP-negative clones H1 12/12 #1 and #2 (clone names: K11-5 and K11-7) by a limiting dilution. Both H1 12/12 #1 and #2 cells lost H1R-eGFP expression as well as endogenous histone H1 expression (Figures 1D and E), indicating the establish- ment of completely histone H1-null mutant DT40 cells. In chromatin acid extracts, we noticed that the amounts of proteins that migrated slightly faster than H1 increased in H1 mutant DT40 cells, particularly in H1 12/12+ﬂox and H1 12/12 #1 and #2 cells (Figure 1D, asterisks). These proteins were likely to be high mobility group (HMG) proteins, since histone H1 and HMG protein doses are known to be compensated by each other and they share the same DNA-binding sites (25). Therefore, we deter- mined HMG protein contents in these cells by western blot analysis using anti-HMG1/2 antibody. In total cell lysates, the levels of HMG1/2 were similar among wild-type and H1 mutant cells (Figure 2A), whereas HMG1/2 in Triton-insoluble chromatin fraction was increased in H1 12/12+ﬂox and H1 12/12 cells (Figure 2B). Thus, in wild-type DT40 cells, the linker DNA appears to be occupied preferentially with H1 even in the presence of a large pool of HMG. When the level of H1 is substan- tially decreased, or completely depleted, in mutant cells, HMG could become more bound to DNA. However, the amount of chromatin-bound HMG in H1 mutant cells appears still lower than that of H1 in wild-type (Figure 1D), and only a small fraction of HMG was recovered in Triton-insoluble chromatin fraction (Figure 2B). These results suggest that H1 and HMG have diﬀerent properties and HMG cannot fully compen- sate for depletion of H1, even though they might have overlapping functions. Growth defect of H1-null DT40 cells As histone H1-null mutant DT40 cells could not be obtained from H1 11/12 cells by using a simple gene targeting strategy (13), we speculated that the complete depletion of histone H1 could aﬀect cell growth or survival. The growth rate of H1 12/12+ﬂox cells was indeed reduced after OHT treatment. After H1 11/12+ﬂox and H1 12/12+ﬂox cells were cultured in the presence or absence of OHT for 4 days, treated and untreated cells were mixed in a 10:1 ratio and further cultured. Figure 3A shows the fractions of GFP (H1R-eGFP)- positive cells among the total population at diﬀerent time points. No enrichment of GFP+ cells among H1 11/12+ﬂox cells was observed. In contrast, the propor- tion of GFP+ cells continuously increased in H1 12/ 12+ﬂox during the cultivation of the mixed population, suggesting that GFP negative (H1 null) cells grew slowly and were selected out (Figure 3A). However, it should be noted that cell lines missing 11 out of the 12 H1 variants, i.e. H1 11/12, H1 11/12+ﬂox and H1 12/12+ﬂox, had already shown signiﬁcant growth defects compared to the wild-type parental cell line (Figure 3B and data not shown), and the complete deletion of H1 further decreased the growth rate. Based on a separate culture experiment, we calculated that the doubling times of wild-type cells, H1 11/12 and H1 12/12 were 9.9 h, 12.6 h and 13.9 h, respectively (Figure 3B, Supplementary Figure S1, and Supplementary Table S1).