1. The cis Element and Factors Required for Condensin Recruitment to Chromosomes 
Katsuki Johzuka, Takashi Horiuchi 
Molecular Cell 
Volume 34, Issue 1, Pages (April 2009)
DOI: /j.molcel
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2. Figure 1 Association of Condensin with Ectopic RFB Site
(A) Structure of the rDNA repeats is shown above, and enlarged depictions of the nontranscribed spacer regions, NTS1 and NTS2, between 35S rRNA coding regions, are shown below. The replication origin (rARS) and replication fork barrier (RFB) are located within the NTS2 and NTS1 regions, respectively.
(B) Strain carrying complete deletion of rDNA repeat (rdnΔΔ) was used (i). The multicopy plasmid (pKJ299) was used to support growth of rdnΔΔ strains (ii). This plasmid carries the 35S and 5S rRNA genes under the control of their native promoters, respectively.
(C) Schematic representation of the chromosomal region between ARS 606 and 607 on chromosome VI. The RFB fragment, tagged with kanMX, was inserted into the site 2.7 kb to the left of ARS607 in both the “barrier-active” and “barrier-inactive” directions. Short bars below the map (a, b, c, d, and e) represent positions of PCR fragments used for the ChIP assay. The black rectangle below the map represents the position of the probe used for 2D gel electrophoresis (2D). The BamHI sites, which are used for digestion in 2D, are also shown.
(D) The upper panel shows quantitative PCR analysis of DNA fragments coprecipitated with TAP-tagged Brn1 in FOB1+ (N2KJY463) or Δfob1 (N2KJY565) cells. PCR fragments a, b, c, and d were used as indicated at the left side of the panel. M, DNA size marker; WCE, whole-cell extract; IP, immunoprecipitates; (+), ChIP samples carried out with formaldehyde cross-linking; (−), ChIP samples without crosslinking as negative controls. The bottom graph shows the enrichment ratios of signal intensity of IP samples from three experiments. The enrichment ratio is the value of signal intensity of each fragment in the IP sample divided by that in the WCE sample (signal intensity in IP/signal intensity in WCE/500).
(E) Replication fork blockage at the ectopic RFB site in either direction, “barrier-active” and “barrier-inactive,” was observed by 2D gel electrophoresis (Brewer and Fangman, 1988). Chromosomal DNA was digested with BamHI and subjected to 2D gel electrophoresis followed by Southern hybridization with the probe shown in Figure 1C. Arrowhead indicates the replication fork arrested intermediate at the ectopic RFB site. (1) Brn1-TAP Barrier-active () (N2KJY463), (2) Brn1-TAP Barrier-inactive () (N2KJY465), (3) Fob1-TAP (N2KJY471), (4) Fob1-TAP (N2KJY473).
(F) The upper panel shows quantitative PCR analysis of DNA fragments coprecipitated with TAP tagged Brn1 or Fob1. PCR fragments a, c, d, and e were used as indicated at the left side of the panel. The bottom graph shows the enrichment ratio of signal intensity of IP samples. Strains are the same as in (E).
Molecular Cell  , 26-35DOI: ( /j.molcel )
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3. Figure 2
Isolation of Mutants Synthetically Lethal with the smc2-157 Mutation
(A) Parental strain, N2KJY546 (ade2, ade3, leu2, ura3, smc2-157), possessing pKJY335 (SMC2, URA3, ADE3), produces red colonies (ade2, ADE3) with many white sectors (ade2, ade3) due to frequent segregation of pKJ335 (1). The fob1 smc2-157 double mutant produced red colonies without any detectable white sectors (2). The #71 (3) and #134 (6) mutants were screened by selecting sectorless colonies. These mutants are confirmed as maintaining sectors formation ability by introducing the SMC2 gene (4 and 7). The CSM1 (5) and LRS4 (8) genes complement sector-forming ability of the #71 and #134 mutants, respectively.
(B) Positions of single amino acid substitutions in the Csm1 (#71 mutant) and Lrs4 (#134) proteins are shown.
(C) Complete deletion of the TOF2, CSM1, or LRS4 genes in the smc2-157 strain produces red colonies without any detectable white sectors. Strains: N2KJY616 (1), N2KJY624 (2), N2KJY628 (3).
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4. Figure 3 Sizes of rDNA Repeats in tof2, csm1, and lrs4 Mutants
Changes of the rDNA repeat size was determined by analyzing the length of chromosome XII detected by Southern hybridization with the single copy gene (SMC4) as a probe (upper panels). Ethidium bromide staining of other chromosomes shows as a quality control for the preparation (bottom panels).
(A) The size of the rDNA repeats in the original mutants, #66, #71, and #134, was analyzed. Plus (+) represents mutants carrying the SMC2 plasmid (pKJ335), and minus (−) represents survivors after FOA selection in which cells lost pKJ335. Strains: UKJY223 (lanes 1 and 3), UKJY225 (lane 2), N2KJY546 (lane 4). Strains in lanes 5–10 are original mutants.
(B) The size of the rDNA repeats in complete deletion mutants of the TOF2, CSM1, or LRS4 gene was analyzed. The smc2-157 point mutation or complete deletion of the TOF2, CSM1, LRS4 was introduced into the wild-type (UKJY224) strain, and two independent transformants were processed for CHEF electrophoresis. Strains: UKJY224 (lane 1), N2KJY602 (lane 2), N2KJY603 (lane 3), N2KJY604 (lane 4), N2KJY605 (lane 5), N2KJY606 (lane 6), N2KJY607 (lane 7), N2KJY608 (lane 8), N2KJY609 (lane 9).
(C) The size of rDNA repeats in Δtof2, Δcsm1 or Δlrs4 in combination with smc2-157 was analyzed. Complete deletion of the TOF2, CSM1, or LRS4 on the smc2-157 background were processed for CHEF. Plus (+) and minus (−) are the same as in (A). Two independent colonies grown on FOA plates, in which cells had lost pKJ335 (−), were analyzed by CHEF. Strains: UKJY223 (lane 1), N2KJY546 (lane 2), N2KJY616 (lane 3), FOAR clones selected from N2KJY616 (lanes 4 and 5), N2KJY624 (lane 6), FOAR clones selected from N2KJY624 (lanes 7 and 8), N2KJY628 (lane 9), FOAR clones selected from N2KJY628 (lanes 10 and 11).
Molecular Cell  , 26-35DOI: ( /j.molcel )
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5. Figure 4
Tof2, Csm1, and Lrs4 Are Essential for the Association of Condensin with the RFB Site
(A) Structure of the rDNA repeats is shown above, and a single unit is enlarged below. The location of the RFB and rARS are indicated. PCR fragments analyzed in ChIP are indicated as black and white bars. The fragment, Cont. (white), was used as an internal control.
(B) Graph shows the association profile (enrichment value) of Brn1-TAP within a single rDNA unit in each strain.
(C) Each panel shows the result of quantitative PCR analysis of ChIP. The number below the panel corresponds to the fragment described in (A). Multiplex PCR containing the Cont. fragment (shorter band in each lane) were carried out. The untagged control (−) was also included. WCE, whole-cell extract; IP, immunoprecipitated extract. Strains: KJY225 (−), N2KJY104 (W), N2KJY131 (f), N2KJY584 (t), N2KJY590 (c), N2KJY596 (l).
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6. Figure 5 Hierarchical Association of Factors with the RFB Site
(A) The association of Fob1 with the RFB site. Left panel shows quantitative PCR analysis of ChIP. Multiplex PCR containing fragments 7, 10, 16, and C (cont.) were carried out. The name of the PCR fragment used in this experiment corresponds to the fragment described in Figure 4A. The graph on the right shows the enrichment value (IP/WCE) at each position. Strains: N2KJY251 (W, wild-type), N2KJY588 (t, Δtof2), N2KJY594 (c, Δcsm1), N2KJY600 (l, Δlrs4), N2KJY639 (s, smc2-157), KJY225 (−, no tag).
(B) Tof2 association with the RFB site. Strains: N2KJY573 (W), N2KJY610 (f: Δfob1), N2KJY647 (c), N2KJY649 (l), N2KJY633 (s), KJY225 (−).
(C) Csm1 association with the RFB site. Strains: N2KJY577 (W), N2KJY614 (f), N2KJY655 (t), N2KJY657 (l), N2KJY637 (s), KJY225 (−).
(D) Lrs4 association with the RFB site. Strains: N2KJY575 (W), N2KJY612 (f), N2KJY651 (t), N2KJY653 (c), N2KJY635 (s), KJY225 (−).
(E) Brn1 association with the RFB site. Strains: N2KJY104 (W), N2KJY131 (f), N2KJY584 (t), N2KJY590 (c), N2KJY596 (l), KJY225 (−).
Molecular Cell  , 26-35DOI: ( /j.molcel )
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7. Figure 6
In Vivo Interaction between Csm1, Lrs4, and Condensin Evaluated by Two-Hybrid Analysis
All subunits of the condensin complex were fused with a Gal4-acidic domain. Both the Csm1 and Lrs4 were fused with the lexA protein. The point mutations csm1-P164L and lrs4-Q325stop were also fused with the lexA protein. All combinations of subunits of condensin and Csm1/Lrs4 were cotransformed into L40, which carries the lexA-lacZ reporter. No-fusion vectors lexA and Gal4-AD were used as negative controls. Expression of lacZ was monitored by detecting β-galactosidase activity.
Molecular Cell  , 26-35DOI: ( /j.molcel )
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8. Figure 7
Model for Condensin Recruitment to the RFB Site, Contributing to Maintenance of the Integrity of Long rDNA Repeats
Fob1 binds first to the RFB site in a DNA sequence-dependent fashion. Fob1 then recruits Tof2, which is required for the association of Csm1/Lrs4 with the RFB. Thereafter, Csm1/Lrs4 recruits condensin to the RFB site by physical interactions between Csm1/Lrs4 and several subunits of condensin. Once condensin is recruited to the RFB site, it may directly interact with DNA and can spread out over the RFB site, while normally restricted by transcription (Johzuka and Horiuchi, 2007). Condensin may then facilitate the construction of rDNA-specific higher order conformations, such as loop structures between periodic RFB sites, and contribute to clamping repeats, thereby repressing aberrant recombination.
Molecular Cell  , 26-35DOI: ( /j.molcel )
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