Fig 2: Chromosome association of condensin II is decreased by CAP-D3 deletion but is increased by CAP-G2 deletion.(A) (Left) Mouse sperm nuclei were incubated with ?cond I/II extracts that had been supplemented with control buffer (buffer), recombinant condensin II holocomplex (holo[WT]) or its subcomplexes (?G2[WT], ?D3[WT], and ?D3?G2[WT]) at 200 nM. After 150 min, reaction mixtures were fixed, and labeled with anti-hCAP-H2 antibody. DNA was counterstained with DAPI. The images of the hCAP-H2 signal were captured at different relative exposure times (1 × and 1/4 ×) to show non-saturated signal intensities of chromosome structures produced by ?G2(WT). Shown here is a representative image from over 15 chromosome clusters examined per condition. Scale bar, 10 µm. (Right) Schematic diagrams of the holocomplex and subcomplexes. (B) Signal intensities of hCAP-H2 from the experiment described in (A) were divided by DAPI signal intensities and the mean values were normalized relative to the value by holo(WT). The mean ± SD is shown (n=15 chromosome clusters). The p-values listed were assessed by two-tailed Welch’s t-test. (C) Mouse sperm nuclei were incubated with ?cond I/II extracts that had been supplemented with holo(WT) or ?G2(WT) at concentrations indicated. After 150 min, reaction mixtures were fixed and processed for immunofluorescence as in (A). Shown here is a representative image from over 15 chromosome clusters examined per condition. The right panels show cropped images of the orange sections of the left panels. Scale bar, 10 µm (left) and 5 µm (right). (D) DAPI and hCAP-H2 intensities from the experiment described in (C) were measured along lines drawn perpendicular to the chromosome to create line scan profiles for the width of chromosomes assembled in ?cond I/II extract with 200 nM holo(WT) or either 25 nM or 200 nM ?G2(WT) (n=20 chromosomes). The mean and standard deviation were normalized individually to the DAPI intensities (arbitrary unit [a.u.]) at the center of chromosome axes (distance = 0 µm) within each set. Intensities of hCAP-H2 were normalized relative to the central value by 200 nM holo(WT) addition. A dataset from a single representative experiment out of three repeats is shown. WT: wild-type. Figure 2—source data 1.Microsoft excel of non-normalized data corresponding to Figure 2B. Figure 2—source data 2.Microsoft excel of non-normalized data corresponding to Figure 2D.

Fig 3: Model of condensin II regulation.(A) Comparison between condensin I and condensin II. In condensin I, the two HEAT subunits, CAP-D2 and CAP-G, contribute to axis assembly and bulk loading, respectively, and their balancing acts regulate proper chromosome assembly. In condensin II, although CAP-D3 and CAP-G2 contribute to axis assembly and bulk loading, respectively, their balancing acts are modified differently from condensin I: CAP-D3’s role in axis assembly is dominant over CAP-G2’s role in bulk loading, whereas CAP-G2’s function to negatively regulate CAP-D3’s function is prominent (bar-headed arrow shown in red). The CAP-D3 C-tail could negatively regulate CAP-D3’s action through CAP-G2-dependent (blue arrow shown in blue) and CAP-G2-independent mechanisms (bar-headed arrow shown in blue). (B) A schematic representation of the chromosome morphologies formed by holo(WT), ?G2(WT), and holo(D3-dC) in Xenopus egg extracts. Holo(WT) added at 200 nM localizes at the axis and forms chenille-like chromosomes with large loops. ?G2(WT) and holo(D3-dC) form similar chenille-like chromosomes at lower doses (at 25 nM and 50 nM, respectively). At 200 nM, ?G2(WT) greatly accumulates at the axis and elongates the axis while keeping the chenille-like morphology. In contrast, holo(D3-dC), when added at 200 nM, also associates with non-axial regions to support lateral compaction, resulting in the formation of a rod-like chromosome.

Fig 4: Recombinant condensin II holocomplex can functionally replace endogenous condensin II in Xenopus egg extracts.(A) A schematic diagram of the condensin II holocomplex that is composed of two structural maintenance of chromosomes (SMC) subunits (SMC2 and SMC4), two HEAT subunits (CAP-D3 and CAP-G2), and a kleisin subunit (CAP-H2). (B) Mouse sperm nuclei were incubated with ?mock, ?cond I, or ?cond I/II extracts that had been supplemented with a control buffer (buffer) or recombinant (rec) condensin II holocomplex, holo(WT), at a final concentration of 200 nM. After 150 min, reaction mixtures were fixed, split, and labeled with an antibody against either Xenopus CAP-H2 (XCAP-H2) or human CAP-H2 (hCAP-H2). DNA was counterstained with DAPI (4',6-diamidino-2-phenylindole). Shown here is a representative image from over 15 chromosome clusters examined per condition. Scale bar, 10 µm. (C) A schematic representation of the chromosome structures observed in ?mock, ?cond I, or ?cond I/II extracts. Rod-shaped chromosomes and chenille-like chromosomes are assembled in ?mock extract and ?cond I extract, respectively. ?cond I/II extract produces an amorphous mass of chromatin (‘clouds’). The addition of recombinant condensin II (rec cond II) to the same extract restores its ability to assemble chenille-like chromosomes. (D) A single chromosome assembled in ?cond I extract with buffer and ?cond I/II extract with 200 nM holo(WT) from samples from experiment (B) in comparison to a chenille stem. Scale bars for chromosomes, 2 µm. (E) DAPI intensities from the experiment described in (B) were measured along lines drawn perpendicular to chromosome axes to create line scan profiles for the width of chromosomes assembled in the ?cond I extract with buffer and ?cond I/II extract with 200 nM holo(WT) (n=20 chromosomes). The mean and SD were normalized individually to the DAPI intensity (arbitrary unit [a.u.]) at the center of chromosome axes (distance = 0 µm) within each set. A dataset from a single representative experiment out of three repeats is shown. Figure 1—source data 1.Microsoft excel of non-normalized data corresponding to Figure 1E.