Fig 2: Knockdown of NIPBL, SMC3 and CTCF. (A) Real-time quantification of NIPBL mRNA levels relative to those for the mRNA of NADH dehydrogenase (ubiquinone) 1 alpha (NDUFA1) in untreated cells, in mock transfected cells and in cells transfected with random siRNAs or with NIPBL-specific siRNA. All values are normalized to levels in untreated cells (value of 1). All analysis performed in triplicate. (B) Western blot for NIPBL and actin in untreated cells and in cells after siRNA knockdown for CTCF, NIPBL and SMC3. Data for transfection with a random siRNA control are also shown. (C) Quantification of NIPBL protein levels based on western results. Samples normalized with actin levels and relative to the ratio in untreated cells. (D) As in (A) but for SMC3 knockdown. (E) As in (B) but for SMC3 and PCNA. (F) As in C but SMC3 protein levels normalized to those of PCNA. (G, H and I) As in (D), (E) and (F) but for CTCF knockdown.

Fig 3: Sex Body Formation(A) Spermatocyte chromosome spreads of Smc1ß+/+, Smc1ß-/-, and Smc1ß-/-,1a mice, stained with anti-SYCP3 (green) for AEs/LEs and with anti-phosphorylated H2AX (?H2AX) for DNA damage.(B) Spermatocyte chromosome spreads of Smc1ß+/+, Smc1ß-/-, and Smc1ß-/-,1a mice, stained with anti-SYCP3 (red) for AEs/LEs and with anti-phosphorylated RNA POLII (green). Asterisk indicates sex chromosomes.(C) Graphical representation of relative expression of ZFY1 (Smc1ß-/-,1a 0.9158 ± 0.10584, Smc1ß-/-: 2.292 ± 1.212 SD; n = 3; p < 0.0001) and ZFY2 (Smc1ß-/-,1a 1.924 ± 0.904, Smc1ß-/- 3.243 ± 1.052 SD; [n = 3; p = 0.0023]). Relative expression was normalized to Smc1ß+/+ levels.(D–K) Spermatocyte chromosome spreads of Smc1ß+/+ and Smc1ß-/-,1a mice, stained with anti-SYCP3 (red) for AEs/LEs and with anti-SYCP1 (green) (D and F–H) and with anti-phosphorylated SMC3 (E and I–K).(D, E, F, and I) Normal sex chromosome morphology.(G and J) No obvious sex chromosome PAR region.(H and K) Abnormal XY axis morphology.See also Figure S7.

Fig 4: Cohesin acetylation restricts the length of architectural stripes and chromatin loops.a, Immunoblot analysis of ?ESCO1 cells. The ?ESCO1 cell line displays reduced levels of acetylated SMC3 (AcSMC3). This experiment was performed three times, with similar results. b, Immunoblot analysis of ?HDAC8 cells; these cells have increased levels of acetylated SMC3. This experiment was performed three times, with similar results. c, Hi-C contact matrices for G1 cells of the indicated genotypes. A locus at chromosome 4 is shown at 20-kb resolution. Matrices were normalized to 100 million contacts per sample. The arrows indicate examples of architectural stripes whose length has changed in ?ESCO1 and ?HDAC8 cells. d, Aggregate stripe analysis to quantify signal enrichment emanating from CTCF sites at 100-kb resolution. Typically, interactions are formed close to the diagonal and decay over distance. These so-called expected contacts were obtained from a distance-normalized contact matrix; we then calculated enrichment of the observed (obs) contacts over the expected (exp) contacts at 100-kb resolution. This method reveals the presence of architectural stripes emanating from CTCF sites. ?ESCO1 cells are enriched for long interactions while ?HDAC8 cells display shorter interactions. e, Cartoon illustrating the difference between primary and extended loops. f, APA for extended loops. Differential APA plots for extended loops compared with wild type (WT). ?ESCO1 (?1) cells show an increase in extended loops while ?HDAC8 (?8) cells show a decrease.Source data

Fig 5: The Extent of Kinetochore Fragmentation Correlates with the Degree of Cohesion Loss(A) Scheme of the partial Trim-Away method in metaphase-II eggs. The degree of cohesin loss is fine-tuned by varying the amount of microinjected antibody.(B) Representative live spindles from TRIM21-overexpressing young MII eggs microinjected with either a control IgG antibody or various concentrations of the anti-Smc3 antibody. Chromosomes (red, H2B-mRFP) and microtubules (blue, MAP4-MTBD-Snap647). The scale bar represents 10 µm.(C) Representative examples of the three chromosome categories aligned at the equator of the same metaphase-II spindle following partial depletion of Smc3 by Trim-Away, as in Figure S5E.(D) Occurrence of different MII kinetochore architectures based on CREST signal in the three chromosome categories defined by Hoechst signal, as in (C), following partial depletion of Smc3 (41 young MII eggs, 4 experiments).(E) Representative kinetochores in chromosomes (left panels) and single chromatids (all other panels) in MII eggs from aged mice (=60 weeks) or women of all ages.(F) Kinetochore architectures shown in (E) and their occurrence on intact chromosomes and single chromatids (50 metaphase-II eggs from aged mice, 5 experiments).(G) Kinetochore architectures shown in (E) and their occurrence on intact chromosomes and single chromatids in 29 human MII eggs.Scale bars, 2 µm in overview, and 0.5 µm in insets. Chromosomes (blue, Hoechst) and kinetochores (magenta, CREST) are shown in (C) and (E). In (C), microtubules are additionally labeled (green, a-tubulin).p values are designated as *p < 0.05 and ****p < 0.0001. p values were calculated with Fisher’s exact test. Error bars show SEM. Arrows: white point to compact kinetochores, and yellow point to lobes within a fragmented MII kinetochore.See also Figure S5 and Video S3.