Fig 2: Condensin II supports interaction and expression of histone gene clusters.(A) Contact profiles of control and NCAPH2 knockdown mESCs at histone gene loci measured by 4C-seq. Knockdown of NCAPH2 reduced interactions between two histone gene loci (1.86 ± 0.25–fold change, P = 7.81 × 10-8). The green dotted line indicates the anchor point. (B) Knockdown of NCAPH2 by short hairpin RNA (shRNA) in mESCs significantly reduced the expression of histone genes in the loci shown in (A). Error bars, SEM. (C) Knockdown of NCAPH2 in HEK293 cells by siRNA significantly reduced the expression of histone genes. Error bars, SEM. (D) The size of the histone clusters in NCAPH2 knockdown mESCs is significantly smaller than the sizes of control cells, indicating that the formation of the clusters is disrupted in NCAPH2 knockdown cells. Fluorescent staining was performed with an antibody to NPAT, a protein that labels the histone clusters (31). Error bars, SD. FWHM, full width at half maximum. (E) Working model for how condensin II and TFIIIC complexes tether dense active promoters in the mammalian genome: Condensin II and TFIIIC complexes (CTS) are colocalized at densely clustered active promoters at TAD boundaries. Condensin II may support the expression of the genes within the boundaries. Some TADs associated with the lamina are less transcriptionally active. The lower right inset shows interaction between CTS that are associated with transcriptionally active promoters. The upper right inset depicts details of the CTS, including condensin II (H, NCAPH2; D, NCAPD3; G, NCAPG2), the TFIIIC complex, the B box–like sequence, and the H3K4me3 histone modification. Condensin II may be recruited to the chromatin, in part, by the HEAT domain of NCAPD3 binding to the H3K4me3 histone modification and, in part, by the interaction with TFIIIC.

Fig 3: The condensin II complex interacts with H3K4me3.(A) H3K4me3 and H3K27ac, but not H3K9me3 or H3K27me3, colocalized with CTS. (B and C) Pull-down assays were performed by mixing nuclear extracts from HEK293T cells with biotinylated histone peptides (HP) modified as indicated. Condensin II and TFIIIC-220 were pulled down with H3K4me3, but not H3K27ac, histone peptides. (D) Pull-down assays were performed by mixing full-length proteins of condensin II generated by in vitro translation with biotinylated histone peptides with and without H3K4me3 modification. NCAPD3 and SMC2 interacted with the H3K4me3 histone peptide. (E and F) Co-IP with anti-SMC2 and anti-NCAPD3 antibodies pulled down chromatin with H3K4me3, but not H3K27ac. (G) HEAT repeat domains from NCAPD3 or NCAPG2 and the C terminus of NCAPD3 were identified and cloned out as described (4). Pull-down assays were performed by mixing HEAT repeat domains from NCAPD3 or NCAPG2 or the C terminus of NCAPD3 with biotinylated H3K4me3 histone peptides. HEAT repeat domains from NCAPD3 interacted with the H3K4me3 peptide. Three independent experiments were performed for each assay.