detected, and the ontology of the whole probe and that of the 8-fold changed genes were quite similar (Supplementary Figure S3B). These results are consistent with the similar phenotype in H1 12/12+ﬂox and H1 12/12 cells. Even though H1 12/12 cells showed more growth defects than H1 12/12+ﬂox cells, this diﬀerence was relatively small, and this phenomenon may be because the amount of exogenous H1R-eGFP protein expressed in H1 12/12+ﬂox cells was small compared to that of endogenous H1 expression. It was observed that the NRL of H1 12/12+ﬂox and H1 12/12 cells were indistinguishable; therefore, H1 12/12+ﬂox cells may behave like H1 12/12 cells in some respects. DISCUSSION This report describes the ﬁrst histone H1 complete KO cells in vertebrates. Our results also indicate that linker histone H1 is not essential for mitotic chromatin conden- sation and global transcriptional repression in higher eukaryotes, but it does play important roles in nucleosome spacing and interphase nuclear compaction. Histone H1 and cell growth Since histone H1-null mutant DT40 cells could not be obtained from H1 11/12 cells with a conventional gene tar- geting strategy (13), we speculated that complete depletion of histone H1 could aﬀect cell growth or survival. Indeed, completely H1-deﬁcient cells, which were successfully generated from conditional lines, displayed growth defects. However, we recently reported that DT40 cells deﬁcient in one of the chicken H1 variants, H1R, showed a reduction in homologous gene targeting eﬃ- ciency (20) and that 02H1-targeting eﬃciency in H1R-deﬁ- cient H1 11/12 cells was still extremely low (1/215). Using the same targeting vector, wild-type cells were targeted with 3/18 eﬃciency (data not shown). Therefore, it appears that both the mild growth defect due to complete H1 depletion and the defect in the homologous recombination (HR) pathway of H1 11/12 cells probably contributed to the failure to establish completely H1-deﬁcient cells by conventional gene targeting in H1 11/12 cells. A recent report described growth defects in human cell lines caused by single histone H1 variant depletion (18). H1.2 depletion induced the expression of a number of normally repressed cell cycle genes and led to G1-phase arrest, and H1.4 knockdown-induced cell death. Drosophila histone H1 knockdown-induced develop- mental defect and histone H1 triple-KO mice were also embryonic lethal, indicating that histone H1 is essential for development (4,14,19). In chicken DT40 cell lines, we also observed that depletion of multiple H1 variants induced growth defects. However, although H1-null mutant cells are viable, they grow slowly. Thus, histone H1 is not essential for cell growth and survival, at least for the chicken DT40 cell line. However, H1R and completely H1-null mutant cells showed an increased chromosomal aberration rate. Since the p53-mediated cell cycle and DNA damage checkpoint pathway are known to be Nucleic Acids Research, 2010, Vol. 38, No. 11 3543 inactivated in DT40 cells (29), we speculate that complete depletion of histone H1 in cells that have an intact p53 pathway may cause more severely impaired growth phenotypes. Histone H1, chromatin structure, and genome integrity Histone H1 (or H1-like protein) integration into chromatin structure and dynamics has been extensively discussed (30–32). So far, experimental data in unicellular organisms has shown that only the micronuclear mitotic chromosome structure is slightly less condensed in the MLH-deﬁcient Tetrahymena cells (8). In multicellular organisms, normal mitotic chromatin condensation was observed in H1-depleted Xenopus egg extracts (27), while defects in chromosome condensation and segregation were seen in replicated chromatin in the absence of H1 in cycling extracts (26). In our experiments as well, histone H1 appeared to be dispensable for mitotic chromatin compaction and segregation in DT40 cells. Nucleosomal architecture was clearly aﬀected by H1 depletion, as shown by the NRL measurements, and the nuclear volume was also increased. While the mechanism by which H1-depletion causes nuclear enlargements remains unknown, the local compaction of polynucleosomes might be aﬀected by electrostatic repulsion of phosphate groups of DNA in the absence of positively charged linker histones. The altered chromatin structure may aﬀect gene expression and chromosomal stability by allowing the access of transcription factors and increasing DNA damage during replication. However, such subtle eﬀects on interphase chromatin might be overwhelmed by strong forces for further chromosome condensation during mitosis, as global metaphase chromatin structure did not appear to be altered in the absence of H1 at the light microscope level. Investigating many mitotic H1-null DT40 cells and chromosome spreads, we never detected drastic morphological changes, similar to extended ﬁbers, seen in H1-depleted Xenopus cycling extracts (26). While further higher-resolution and quantitative studies (e.g. by using atomic force microscopy and live cell analysis) will be required to fully evaluate the eﬀect of H1 depletion on mitotic chromosome structure, our data have so far sup- ported earlier work reporting little eﬀect of H1 in Xenopus M-phase extract system (27). Alternatively, HMG proteins might compensate for the function of H1 during mitosis, which should also be examined in future. We also showed that the chromatin aberration rate was increased in H1-null DT40 cells by cytochemical staining of metaphase chromosomes. How is H1 involved in genome integrity? Since the mobility of H1R, but not H1L, in the nucleus decreased after methane methylsulfonate treatment, we speculate that H1R specif- ically integrates into HR-mediated repair pathways at the chromosome structure level (20). H1 12/12 #1 and #2 (H1-null) showed even higher chromosomal aberration rates than that of H1 11/12, suggesting that H1 has addi- tional functions besides its involvement in the H1R-mediated pathway for global genome integrity.