3538 Nucleic Acids Research, 2010, Vol. 38, No. 11 Figure 1. Continued. not diﬀer between wild-type and H1 12/12 cells (Figure 4D, right). Then, we examined ionizing radiation (IR)-induced G2/M checkpoint activation. After exposing the colcemid-containing culture to 2.3 Gy of IR, the accu- mulation rate of an H3S10ph-positive population was signiﬁcantly impaired for 2 h in both wild-type and H1 12/12 cells (Figure 4D, right). In the absence of colcemid, the H3S10ph-positive population was reduced for 2 h in both cell types (Figure 4D, left). Therefore, IR-induced G2/M checkpoint activation appeared to occur in H1-null DT40 cells as it did in wild-type cells. These data suggest that the growth defect in H1-null cells resulted mostly from an extended single cell cycle, but not from a speciﬁc cell cycle stage. Reduction of nucleosome repeat length in H1-mutant DT40 cells A reduction of 50% in histone H1 content in mouse H1c, H1d and H1e triple KO embryonic stem (ES) cells (0.25 H1 per nucleosome) resulted in a reduction of nucleosome repeat length (NRL) due to micrococcal nuclease (MNase) digestion (4,14). Therefore, we analyzed the NRL for the wild-type, H1 11/12, H1 11/ 12+ﬂox, H1 12/12+ﬂox and two histone H1-null mutant cells (Figure 5A and B). Wild-type DT40 cells exhibited an average NRL of 181 bp. Both H1 11/12 and H1 11/ 12+ﬂox cells showed similar lengths with a reduction of 13 bp: 167 and 169 bp, respectively. H1 12/12+ﬂox and the two H1-null mutant cells, H1 12/12 #1 and #2, displayed an even shorter average NRL: 163, 162 and 163 bp, respectively. These NRL diﬀerences seemed to depend on the total amount of histone H1 in a cell. The NRL of H1 12/12+ﬂox and H1 12/12 mutant cells did not change as much as in the case of the H1-null mutants because the amount of exogenous H1R-eGFP protein was very small compared to the amount of endogenous H1 protein (Figure 1D). The H1c, H1d and H1e triple KO ES cells have an average NRL of 174 bp (4,14), and the baseline NRL without histone H1 is predicted to be 166 bp (28). Therefore, the average NRL calculated in the histone H1-null DT40 cells was mostly