Loss of YY1 results in polyploidy and chromosome structural aberrations. (a) Metaphase spread from YY1^f/f MEFs plus Cre, showing polyploidy. Wild-type and YY1^f/f MEFs were transduced with Ad-Cre. (b) Metaphase spread from the YY1^f/f MEFs plus Cre with multiple structural abnormalities (arrowheads). (c–e) Enlargements of the typical quadriradial structure (c), triradial structure (d) and chromatid break (e). (f) Percentages of aneuploid and polyploid chromosomes, which differ significantly between YY1^f/f and YY1^f/f plus Cre cultures. Difference between wild-type (WT) and YY1^f/f plus Cre cultures was also statistically significant but is not shown. n indicates the number of metaphase spreads scored. (g) Percentages of abnormal chromosome structures, which differ significantly between YY1^f/f and YY1^f/f plus Cre cultures.

YY1 interacts with INO80 subunits in vitro and in vivo. (a) Co-immunoprecipitation between YY1 and INO80 upon DNA damage. HeLa cells transfected with Flag-INO80, HA-YY1 or both were radiated with UV (30 J M⁻²) and lysed 2 or 4 h after stress. Lysates were immunoprecipitated with Flag or HA affinity beads, probed with anti-Flag and reprobed with anti-HA. Lysate western blots show the protein levels of Flag-INO80 (v), HA-YY1 (vi), γH2AX (vii) and GAPDH (viii) in 10% input. IP, immunoprecipitation; IB, immunoblot. (b) Diagrams of human INO80 full-length protein and fragments used to map the YY1-binding domain. Abilities of indicated INO80 fragments to bind GST-YY1 protein are summarized on the right. (c,d) In vitro binding of indicated proteins to YY1 protein. Upper gels, amounts of ³⁵S-labeled, in vitro–translated proteins retained on glutathione-agarose beads after pull-down assay, compared to the corresponding input (10%). Lower gels, Coomassie blue staining of GST and GST-YY1 proteins used for this experiment. In d, p53 and exportin 1 were used as positive and negative binding controls, respectively. (e) GST-YY1 pull-down assay with bacterially purified Flag-TIP49A and Flag-TIP49B. Top gel, pulled down samples analyzed by western blotting with the Flag antibody. Bottom gel, GST fusion proteins visualized by Coomassie blue staining.

Impaired homology-directed repair of a chromosomal DSB in YY1- and INO80-deficient 293T and HT-1080 cell lines. (a) Schematic showing how, in the Neo direct-repeat substrate, I-SceI–induced DSBs can result in gene-conversion or deletion products. Neo direct repeats of 1.4 kilobases (open boxes) flank the Gpt gene. Transcription of the upstream Neo is driven by the TEAD1 (here called SV40) promoter and an I-SceI recognition site interrupts the reading frame, whereas the downstream Neo lacks a promoter. Shaded box, SV40 promoter; green arrow, functional Neo gene after repair. (b) Plasmid expressing shRNA constructs was cotransfected with I-SceI plasmid or control plasmid (SceI⁻ plasmid; ‘None’ in key) into HR-293T cells. Total HRR frequencies were calculated as the number of G418-resistant colonies per viable cell plated in selective medium. Blue bars, spontaneous homologous recombination events; purple bars, I-SceI–induced events. Error bars indicate s.e.m. (c) Gene-conversion frequencies, calculated as the number of colonies resistant to both G418 and mycophenolic acid per viable cell plated. (d) Structure of inverted puromycin (Pac) repeats flanking the cytomegalovirus (CMV) Bsd promoter in the HT1080–1885 cell line. The upstream Pac is driven by the mouse Pgk promoter and inactivated by insertion of an I-SceI site in the coding sequence; the downstream donor Pac lacks a promoter. (e) Total HRR frequencies in Pac inverted-repeat HT1080–1885 strain. shRNA constructs were cotransfected with I-SceI plasmid or control plasmid into HT1080–1885 cells. Total HRR frequencies were calculated as the number of puromycin-resistant colonies per viable cell plated in selective medium. Error bars indicate s.e.m.

YY1 associates with mammalian Ino80 complex. (a) Silver staining of the YY1 complex separated on 4%–12% SDS-PAGE gel. The YY1 complex was immunopurified from nuclear extract prepared from HeLaS cells stably expressing Flag-HA-YY1. Subunits of INO80 complex are labeled in gray. (b) The double-purified YY1 complex in a was further fractionated by 10%–40% glycerol-gradient sedimentation. Fractions were visualized by silver staining (top) and immunoblotting with indicated antibodies (bottom). Input (3%) and molecular weight (MW) marker were also included on the silver-staining gel. (c) YY1 interacts with subunits of INO80 complex in vivo. Total HeLa cell lysates were immunoprecipitated with BAF53, TIP49A and TIP49B antibodies. YY1 levels in immunoprecipitates and 15% input were compared by western blotting with the YY1 antibody.

Knockdown of TIP49B and INO80 leads to UV hypersensitivity. (a) U2OS cells were transfected with plasmids expressing shRNAs (sh) that target mGFP (negative control) (Supplementary Fig. 1g), YY1 or TIP49B, or both YY1 and TIP49B, for 48 h and treated with indicated doses of UV light. Survival fraction is the number of live cells after irradiation divided by the number of live cells after mock treatment and is expressed as a percentage. About 4,000 cells were counted in at least three independent experiments. Data are presented as means ± s.e.m. (b) As in a, cells were transfected with plasmids expressing shRNAs that target YY1, INO80 or both.

YY1 binds a recombination-intermediate structure in vitro. (a) Purified YY1 complex was incubated with biotinylated Holliday-junction DNA (probe), and the binding reactions were resolved on 4% nondenaturing PAGE gels. Reactions contained 10 nM probe and YY1 complex at 0, 10, 20, 40, 60, 80, 100 and 200 nM per reaction in lanes 1–8, respectively. The positions of unbound probe, gel origin and YY1 band-shift complexes I–V are indicated. (b) Specificity determination using YY1 supershift and unlabeled competitor DNA binding assays. Lane 1, probe only; lane 2, 40 nM YY1 complex; lane 3, supershift with YY1 antibody; lane 4, supershift with control antibody; lane 5, excess unlabeled Holliday junction (50 nM); lane 6, excess unlabeled dsDNA (50 nM); lane 7, CtBP complex. Reactions contained 10 nM probe. (c) Purified His-YY1 was incubated with biotinylated Holliday junction DNA (probe) or dsDNA probes, and the binding reactions were resolved on 4% nondenaturing PAGE gels. Reactions contained 1 nM probe and His-YY1 at 0, 100, 200, 400, 500, 600, 700, 800 and 900 nM in lanes 1–9, respectively. The positions of unbound probe and His-YY1 band-shift complexes I–V are indicated. (d) Specificity determination. Unlabeled DNA structures were titrated against biotinylated Holliday junction probe (1 nM). Lanes 1, 6 and 11 contain 600 nM His-YY1. The type of unlabeled competitor DNA is indicated above each of the three titration series (lanes 2–5, 7–10 and 12–15); concentrations of competitor DNA in each series were 5-fold, 10-fold, 25-fold and 50-fold molar excess.