Nucleic Acids Research, 2010, Vol. 38, No. 11 3539 Figure 2. Chromatin bound HMG1/2 is increased in H1-null DT40 cells. Total cell lysate (A) and Triton-insoluble chromatin fraction (B) were separated by 14% SDS-PAGE, blotted onto PVDF membrane, followed by Ponceau S-staining to verify equal protein loading (lower), and then HMG1 and 2 were immunodetected by chemiluminescence (upper). Figure 3. Growth defect of H1-null DT40 cells. (A) Slower growth of H1-null cells. Cells harboring H1R-eGFP with or without 02H1 gene (H1 11/12+ﬂox or H1 12/12+ﬂox) were treated with OHT to remove H1R-eGFP gene, and mixed with OHT-untreated cells with a ratio of 10:1. The percentages of eGFP-positive fractions were monitored using ﬂow cytometry for 19 days. The accumulation rate of GFP positive population in every 4 days from Day 3 to 19 (% average ± SD; n = 4) was 0.91 ± 0.97 for H1 11/12+ﬂox and 14.15 ± 3.19 for H1 12/12+ﬂox. Diﬀerence of two values is statistically signiﬁcant (Student’s t-test, P-value <0.0005). (B) Growth curve of wild-type and H1 12/12+ﬂox cells. The cell division number in every 4 days (average ± SD; n = 7) was 9.69 ± 1.34 for wild-type and 6.16 ± 1.11 for H1 12/12+ﬂox. Diﬀerence of two values is statistically signiﬁcant (P-value <0.0005). comparable with the predicted NRL in the total absence of H1. Linker histone H1 is involved in interphase chromatin compaction We examined the MNase sensitivity of histone H1-null mutant cells in comparison with wild-type and H1 11/12 cells, but no signiﬁcant diﬀerence was observed (data not shown). Since Tetrahymena H1-deﬁcient strains exhibit enlarged nuclei (8), we examined the nuclear volumes of linker histone H1-mutant DT40 cells. Cells were stained with DAPI, and serial z-section images covering the whole nucleus were taken by confocal microscopy. The volume of each nucleus was then calculated using ImageJ software [National Institutes of Health (NIH), Bethesda, MD, USA] (Figure 6). The median nuclear volume (MNV) of wild-type cells was 369.5 mm3 (n = 47). On the contrary, MNV of H1 11/12 and H1 12/12 cells increased by >20%, i.e. 461.9 mm3 (n = 27) and 450 mm3 (n = 28), respectively. These data also suggest that histone H1 is involved in interphase chromatin compaction in chicken DT40 cells. Increased chromosomal aberrations in histone H1-null mutant DT40 cells Among the six chicken histone H1 variants, H1R is speciﬁcally involved in DNA damage response, and chro- mosomal instabilities are induced in the H1R-deﬁcient DT40 cells (20). Therefore, we compared the rate of