3542 Nucleic Acids Research, 2010, Vol. 38, No. 11 Table 1. Frequency of spontaneous chromosomal aberrations in the wild-type and H1 mutant DT40 cells Genotype Wild typeref[20] H1 11/12 H1 11/12+ﬂox H1 12/12+ﬂox H1 12/12 #1 H1 12/12 #2 Break 0.5 2.0 0.0 1.5 5.0 4.7 Gap 1.5 1.5 0.0 1.0 6.0 2.0 Break 0.0 1.5 0.0 0.0 2.5 4.0 Cells were treated with colcemid for 1.5 h to enrich mitotic cells. aData are presented as the nuﬃmﬃﬃﬃﬃbﬃﬃeﬃr of aberrations per 100 cells. bS.E. is culculated as x/N±px=N (x: total chromosomal aberrations and N: the number of cell. Table 2. Summary for the aﬀected genes in H1 12/12+ﬂox and H1 12/12 cells Isochromatida Chromatida Exchangea Aberration (per cell±S.E.b) No. of analyzed Mitotic cells Expression array analysis among wild-type, H1 12/12+ﬂox and H1 12/12 cells Wild type versus H1 12/12+ﬂoxa Wild type versus H1 12/12a overlap with H1 12/12+ﬂoxa,b Gap 0.0 2.0 0.5 1.5 0.5 2.7 215e 643f 58 115 22 46 200 200 200 200 200 150 (45.1) (58.5) (50) (31.3) (72.7) (26.1) 0.0 0.5 0.0 1.0 0.5 0.7 (2.9) (8.6) (0.8) (1.5) (0.3) (0.6) 0.02 ± 0.007 0.08 ± 0.014 0.01 ± 0.004 0.05 ± 0.011 0.15 ± 0.019 0.14 ± 0.025 97 363 29 36 16 12 >2-fold up <1/2-fold down >4-fold up <1/4 fold down >8-fold up <1/8-fold down 172c 1206d 44 96 20 19 (0.3) (0.3) (0.3) (0.3) (0.3) (0.3) aThe number of probes aﬀected in H1 12/12+ﬂox or H1 12/12 #1 cells in comparison with wild-type cells as base line; values in parenthesis denote % based on the total number of expressed probes 7442 as 100%. bThe number of aﬀected probes in H1 12/12 #1 cells which were already aﬀected in H1 12/12+ﬂox cells in comparison with wild-type cells as base line; values in parenthesis denote % based on the number of aﬀected probes in H1 12/12 #1 cells as 100%. c–fGenelist is provided as Supplementary Table S3–6. while H1 11/12+ﬂox or any single H1 (except H1R)-deﬁ- cient cells did not diﬀer from the wild type (17,20). This markedly high chromosomal aberration rate may be the cause of the growth defect phenotype of the histone H1-deﬁcient cells. Global impact of H1 depletion on transcription Another critical issue is whether histone H1 in higher eukaryotes functions as a global transcriptional repressor. Studies of chicken and mouse H1-KO cells suggest that histone H1 is not a global transcriptional repressor; however, a deﬁnitive conclusion needs to be made in this regard by using H1-null cells (4,13,17). We performed a gene expression microarray analysis using wild-type cells, H1 12/12+ﬂox, and H1-null mutant H1 12/12 #1 (accession number GSE8483; summarized in Table 2 and Supplementary Table S2). Among the 32 773 target probe sets assayed, 7442 targets (23%) showed signiﬁcant expression in at least one of the DT40 cell lines analyzed with GeneSpring software (Agilent). The comparison of H1 12/12+ﬂox cells and wild-type cells revealed that 1378 probes showed changes that were more than 2-fold (172 up and 1206 down; Supplementary Tables S3 and S4 and Supplementary Figure S3). Among these, 44 (0.6%) and 20 (0.3%) were 4- and 8-fold upregulated, and 96 (1.3%) and 19 (0.3%) were 4- and 8-fold downregulated, respectively. These data demonstrated that signiﬁcant reduction in histone H1 content in H1 12/12+ﬂox cells has a global, but relatively mild, impact on transcription (mostly with less than 4-fold dif- ferences). More importantly, most of the changes were due to suppression (87.5%) instead of activation, as would be expected if H1 caused transcriptional repression. Similar changes were observed in the histone H1-null clone H1 12/ 12 #1 cells, with slightly higher numbers of 2-, 4- and 8-fold upregulated probes, i.e. 215 (2.9%), 58 (0.8%) and 22 (0.3%), respectively (Supplementary Table S5). The number of probes that were downregulated 2-, 4- and 8-fold were 643 (8.6%), 115 (1.5%) and 46 (0.6%), respectively (Supplementary Table S6). Again, 75.9% (643/858) of the probes with more than 2-fold changes in H1 12/12 #1 were downregulated. Notably, 50% of those aﬀected probes (45.1% and 58.5%) were also up or downregulated in H1 12/12+ﬂox cells. We con- ﬁrmed the expression levels of genes in H1 12/12+ﬂox, H1 12/12 #1 and H1 12/12 #2 cells that were up or downregulated by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis (Supplementary Figure S3A). Among the genes that were up or downregulated 8-fold in H1 12/12 #1 cells, no clustering toward speciﬁc gene families or functions was