TnT T7 Grasp Mix (25 l) was mixed with 2 l of TnT Reaction Buffer, 1 l of TnT RNA Polymerase, 2 l of Complete Amino Acid Mix (L4461, Promega), 1 l of RNasin Ribonuclease Inhibitor (N2511, Promega), 2 l of DNA plasmid, and 17 l of nuclease-free water. with stronger TAD boundaries in mESCs. fig. S14. Interactions between two histone gene loci on chr13. fig. S15. Chromatin interactions between two highly expressed gene loci were reduced upon NCAPH2 knockdown. fig. S16. Formation of histone clusters may be disrupted in NCAPH2 knockdown mESCs. table S1. Public ChIP-seq data sets analyzed. Abstract Structural maintenance of chromosome complexes, such as cohesin, have been implicated in a wide variety of Mc-Val-Cit-PABC-PNP chromatin-dependent functions such as genome organization, replication, and gene NBN expression. How these complexes Mc-Val-Cit-PABC-PNP find their sites of association and affect local chromosomal processes is not well comprehended. We report that condensin II, a complex distinct from cohesin, physically interacts with TFIIIC, and they both colocalize at active gene promoters in the mouse and human genomes, facilitated by conversation between NCAPD3 and the epigenetic mark H3K4me3. Condensin II is usually important for maintaining high levels of expression of the histone gene clusters as well as the conversation between these clusters in the mouse genome. Mc-Val-Cit-PABC-PNP Our findings suggest that condensin II is usually anchored to the mammalian genome by a combination of H3K4me3 and the sequence-specific binding of TFIIIC, and that condensin supports the expression of active gene-dense regions found at the boundaries of topological domains. Together, our results support a working model in which condensin II contributes to topological domain name boundaryCassociated gene activity in the mammalian genome. ribosomal RNA (rRNA). TFIIIC (consisting of subunits 220, 110, 102, 90, 63, and 35) has been considered as an insulator (genomes (((NCAPD3) (= 14964) for detailed analysis. TFIIIC-220 was colocalized with condensin II (NCAPH2) at RNAPIII binding sites, Mc-Val-Cit-PABC-PNP as indicated by colocalization with BRF1, a TFIIIB subunit, and RPC1 and RPC4, RNAPIII subunits (Fig. 2D). Surprisingly, TFIIIC-220 was also colocalized with condensin II at many more non-RNAPIII binding sites. The sites where condensin II and TFIIIC are colocalized are termed CTS herein. TFIIIC-220 has comparable colocalization with cohesin (SMC1A) and condensin. However, TFIIIC-220 peaks were only colocalized with CTCF at the RNAPIII binding sites (Fig. 2D). The TFIIIC binding sites at which NCAPH2 peaks were absent are termed condensin-free TFIIIC sites (CFTS) (Fig. 2D) and serve in contrast to CTS. We found that TFIIIC was necessary for condensin II association with all CTS, not just tRNA genes. Knockdown of TFIIIC-220 significantly reduced the binding of NCAPH2 to the CTS, whereas knockdown of NCAPH2 did not affect the binding of TFIIIC-220 to the CTS (Fig. 2, E and F, and fig. S2E) or CFTS (fig. S2F). Thirty-five percent of CTS were at promoters or annotated transcription start sites (TSSs) (Fig. 2G) as compared to only 4% of CFTS (fig. S2G). Considering the enrichment of CTS at promoters, we probed the correlation between CTS and expression levels. The protein-coding genes were divided into three categorieshigh, moderate, and low expressionbased on mESC RNA sequencing (RNA-seq) data. The binding intensity of both NCAPH2 and TFIIIC-220 at TSS positively correlated with gene expression levels (fig. S3, A and B). In addition, we found that CTS were correlated with both the promoters of highly expressed genes and deoxyribonuclease hypersensitive sites (figs. S3, D and E, and S4). The promoters of housekeeping genes were strongly enriched at CTS (Fig. 2H). Therefore, CTS are associated with transcriptional activity. CTS had additional features suggesting that they play a distinct role in genome topology compared to CFTS. For example, architectural proteins such as cohesin are highly enriched at CTS but not at CFTS (fig. S5A). CTCF appears slightly enriched at CTS in metagene analysis due to the colocalization at the RNAPIII genes (fig. S5A). Proteins indicative of transcriptional activity (for example, p300 and RNA polymerase II) are also enriched at CTS but not at CFTS (fig. S5, B to D). There were significantly more CpG islands, together with higher levels of GC content, around CTS than CFTS (fig. S6, A and B). CpG islands are important for initiating transcription (= 15,796) for detailed analysis. We found that TFIIIC-220 peaks were also strongly colocalized with those of NCAPH2 (Fig. 3A), allowing us to define the locations of CTS in the human genome. Similar to the mESCs, TFIIIC-220 was colocalized with condensin II (NCAPH2) at RNAPIII binding sites, as indicated by colocalization with BRF1, a TFIIIB subunit, and PRC32, a subunit of RNAPIII (Fig. 3B)..