Histone supply regulates S phase timing and cell cycle progression.
Bottom Line: We used a histone mutation of Drosophila melanogaster to show that histone supply levels, provided by a defined number of transgenic histone genes, regulate the length of S phase during the cell cycle.Lack of de novo histone supply not only extends S phase, but also causes a cell cycle arrest during G2 phase, and thus prevents cells from entering mitosis.Our results suggest a novel cell cycle surveillance mechanism that monitors nucleosome assembly without involving the DNA repair pathways and exerts its effect via suppression of CDC25 phosphatase String expression.
Affiliation: Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.Show MeSH
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Mentions: The histone mutation in D. melanogaster, called Df(2L)HisC, lacks all genes encoding the canonical histones (Günesdogan et al., 2010). Df(2L)HisC homozygous mutant animals (hereafter referred to as HisC mutants) that are derived from heterozygous parents contain only maternal histone mRNA and proteins, which are sufficient to complete the first 14 cell division cycles of the embryo (Günesdogan et al., 2010). HisC mutant embryos arrest before the onset of mitosis in cycle 15 (M15) (Günesdogan et al., 2010). This highly uniform phenotype is likely due to the degradation of maternal histone mRNAs during the first G2 phase of embryogenesis in cell cycle 14 (Marzluff et al., 2008; O'Farrell et al., 1989) combined with the complete lack of zygotic histone gene expression during S phase of cell cycle 15 (S15) (Günesdogan et al., 2010). In order to verify that the lack of histone transcription also results in a diminished pool of histone proteins in S15, we compared the protein levels of histone H2B and H3 of wild type embryos that are in S15 at 4–5 hr after egg laying (AEL) to sorted HisC mutant embryos that are still in S15 at 5.5–6.5 hr AEL (see below and Günesdogan et al., 2010) by quantitative Western blotting (Figure 1A,B). The approximate twofold reduction in the histone levels of HisC mutant embryos is consistent with the fact that these embryos lack synthesis of new histones in S15 but still contain parental histones from chromatin that was assembled during cycle 14. To test whether the reduced supply of histones in HisC mutant embryos leads to a decrease in nucleosome formation, we carried out Micrococcal Nuclease (MNase) digestion assays on chromatin from sorted HisC mutant and wild type sibling embryos (Figure 1C,D, Figure 1—figure supplement 1). The results show that chromatin from HisC mutant embryos is more accessible to MNase than control chromatin, leading to a more rapid generation of mononucleosomal DNA fragments and reflecting a decrease in nucleosome occupancy in chromatin of the HisC mutants.10.7554/eLife.02443.003Figure 1.Nucleosome density is affected in HisC mutant cells.
Affiliation: Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.