Investigation of the Mechanism of Meiotic DNA Cleavage by VMA1-Derived Endonuclease Uncovers a Meiotic Alteration in Chromatin Structure around the Target Site

Detection of VDE binding to the VRS. (A) Diploid cells homozygous for VMA1⁻- expressing VDE or DNA-binding-deficient VDE (R90A) were sampled at the indicated times during meiosis. Cells were treated with formaldhyde, and then the cell extract was prepared, sonicated to shear the chromatin, and immunoprecipitated with anti-VDE antibody. DNA from input samples and in the immunoprecipitates (IP) was amplified by multiplex PCR with primers for the 3′ region adjacent to the VRS (VRS3), the 5′ region adjacent to the VRS (VRS5), and the telomeric region of chromosome VI (TEL). (B) The ratios of the VRS signals to the TEL signal were normalized to those of the input samples at each time point. Error bars denote the standard deviations among three independent experiments. (C) A ChIP assay with anti-VDE antibody was performed for the indicated mutants. The signals obtained for the 4.5-h samples are shown as in panel B.

Chromatin configuration at the VMA1⁻ locus during meiosis. (A) Chromatin was prepared in diploid cells homozygous for VMA1⁻ at various times of incubation in SPM, treated with 0, 5, 10, or 20 U/ml of MNase, and redigested with BamHI and SphI. Naked DNA was also treated with 0.2, 0.4, or 0.8 U/ml of MNase. MNase-sensitive sites were detected by indirect end-labeling using a probe for the sequence adjacent to the BamHI site. The vertical gray arrow indicates the position of the coding region of the VMA1⁻ locus. The arrowheads show MNase-sensitive sites near the VRS that became prominent during meiosis. Numbers indicate the positions in nucleotides relative to the VDE cutting site that is shown by the horizontal arrow. Meiotic progression was monitored by DNA content, which was analyzed by a fluorescence-activated cell sorter (B), and by nuclear division, which was analyzed by microscopy of 4′,6′-diamidino-2-phenylindole-stained cells (C).

Chromatin alteration during meiosis occurs independently of VDE expression and is dependent on meiotic progression. Results of chromatin analysis of VMA1⁻ homozygous cells expressing wild-type VDE (A), endonuclease-defective VDE (D326V) (B), and VDE defective in DNA binding (R90A) (B) are shown. Samples were treated with 0, 5, or 10 U/ml of MNase. Indirect end-labeling was performed, and MNase-sensitive sites were analyzed as described in the legend to Fig. 3. The arrowheads indicate the chromatin alteration site. (C) Chromatin structure of the haploid cell in SPM. Chromatin was prepared in VMA1⁻ haploid cells sampled at the indicated times of incubation in SPM and treated with 0, 5, or 10 U/ml of MNase. The sir2Δ mutation permits haploid cells to enter the meiotic program. (C) Chromatin structure around the VRS in mutant cells defective in meiotic progression. Chromatin was prepared from VMA1⁻ homozygous diploid cells after 1.5 and 4.5 h of incubation in SPM and subjected to indirect end-labeling.