SMC FAMILY PROTEINS AND ASSOCIATED FACTORS IN MITOTIC CHROMOSOME SEGREGATION
, Head, Unit on Chromosome Structure and Function
Lidiya Dimitrova Angelova-Duleva, PhD, Visiting Fellow
Alexander Samoshkin, PhD, Visiting Fellow
Yoshimitsu Takahashi, PhD, Visiting Fellow
Pavel Butylin, MSc, Graduate Student1
Stanimir Danailov Dulev, MSc, Graduate Student1
Shephali Malhotra, BS, Postbaccalaureate Fellow
Figure 3.3
Cellular pathways and processes controlling
condensin binding to specific chromosomal sites
Arrows denote the pathways that we have already identified while boxes denote the pathways currently under investigation. Abbreviations: rARS = origin of DNA replication in rDNA; RFB = replication fork barrier; 5S = 5S gene transcribed by RNA Pol III; 35S = major rDNA transcript, precursor of 18S, 5.8S, and 25S rRNAs (transcribed by RNA Pol I); TEL = telomeres; and CEN = centromere. Dotted arrows denote hypothetical functions.
The specificity of condensin targeting to the nucleolar chromatin: interplay with RNA polymerase I transcription and sumoylation
Our previous characterization of the S. cerevisiae rDNA identified it as a main target of mitotic condensin activity, thus defining one of the key functions of condensin activity in all eukaryotes. To answer the question about the nature of the special role played by condensin in the nucleolus, we tested whether condensin is required to segregate a single rDNA repeat placed on a circular minichromosome. The analysis of such a minichromosome showed that condensin inactivation results in up to a 20 percent lower mitotic stability than either the rDNA-less minichromosome in the same mutant or the rDNA minichromosome in wild type. This finding established that even a single rDNA repeat requires condensin activity for proper segregation. It also offers a plausible explanation for the hypersensitivity of the tandem rDNA locus to condensin proficiency—the cumulative effect of several individual repeats, each with the above-mentioned decrease in segregation fidelity.
To identify the molecular determinants of the condensin requirement in rDNA, we used quantitative chromatin immunoprecipitation (ChIP) to analyze condensin binding sites in rDNA and found two sites positioned within the Pol I–transcribed rRNA gene. As Pol I transcription is highly active, its physical linkage with condensin binding could be problematic. To investigate this dilemma, we monitored condensin occupancy in parallel with 35S transcript levels in a time-course experiment. All four condensin peaks showed similar cell-cycle profiles of DNA association, but the intensity of condensin binding was inversely correlated with the level of Pol I transcript at a given time point. In direct experiments, we confirmed the counter-dependence of condensin binding and the transcription level in rDNA by modulating transcription of rDNA by both Pol I (rapamycin treatment) and Pol II (GAL promoter–controlled rDNA). Thus, condensin binding and transcription in rDNA are mutually exclusive, indicating that condensin must compete directly for binding sites with transcription machinery in rDNA.
The competition between condensin and transcription in the native rDNA locus may, however, be resolved by the compartmentalization of transcriptionally silent units and transcriptionally active repeats, which is indeed the case in budding yeast. We obtained compelling evidence for this compartmentalization hypothesis by modulating transcription levels in rDNA with simultaneous monitoring of condensin occupancy and/or rDNA segregation fidelity. Indeed, Pol I transcription and segregation proficiency of rDNA are inversely correlated, with segregation largely dependent on condensin function. Independent time-lapse microscopy showed that the segregation of chromosome XII with the minimal-size (20-repeat) rDNA array, where all repeats are actively transcribed, is significantly slower than in wild type. Thus, the probable mechanism underlying special condensin requirements in rDNA segregation in S. cerevisiae is related to the need to segregate the actively transcribed and fully assembled nucleolus, probably by counteracting the DNA overwinding in the rDNA regions flanking the actively transcribed repeats. Our study revealed a likely functional role for previously unexplained Pol I transcription heterogeneity of native rDNA repeats.
After previously demonstrating that the post-translational modification by SUMO has a potential role in condensin targeting to rDNA, we tested whether such control is mediated by direct condensin modification by SUMO/Smt3p. We confirmed that the modifications of four condensin subunits are indeed conjugated to SUMO. Our investigations of the biological role of condensin sumoylation are in line with our hypothesis that SUMO modification of condensin facilitates condensin’s targeting to the nucleolus, promoting condensin cooperativity with nucleolar pools of topoisomerase I and II, which are also sumoylated. Preliminary experiments showed that reduction of the number of SUMO-modified sites even in a single condensin subunit Smc2p (smc2-6KR allele) results in a notable growth defect, which is exacerbated by topoisomerase mutations.
Wang B, Butylin P, Strunnikov AV. Condensin function in mitotic nucleolar segregation is regulated by rDNA transcription. Cell Cycle 2006;5:2260-7.
The role of active condensin for functionality of other chromosomal sites: the centromere and DNA replication connections
Our previous ChIP-chip analysis of the genome-wide condensin binding pattern showed that the peri-centromeric regions are enriched in condensin binding, suggesting that condensin activity may promote centromere function. Other experimental facts also hint that condensin plays some role at centromeres. For example, in budding yeast, condensin mutants maintain high viability throughout the mitotic arrest, indicative of a checkpoint control, possibly mediated by kinetochores. Biochemical and cytological analysis showed that the spindle assembly checkpoint (SAC) is indeed the factor responsible for condensin mutant arrest. The dependence of condensin mutant arrest on SAC was not allele-specific, indicating that SAC monitors condensin function as a whole. Moreover, the depletion of condensin in human cells also engages the MAD2-dependent spindle checkpoint, resulting in a transient metaphase delay.
This discovery provided an opportunity to elucidate mitotic condensin function not obscured by SAC. In particular, we were interested in explaining the low mitotic stability of the rDNA-containing chromosomes in condensin mutants. After using in vivo chromosomal fluorescent tags to compare individual rates of mis-segregation for the rDNA-containing chromosome XII and two non–rDNA chromosomes, we showed that, in the smc2-8 mutant, the rDNA-containing chromosome XII was much more prone to mis-segregation than chromosomes IV or IX in SAC-proficient cells. However, we observed equally high mis-segregation rates for all three chromosomes in the SAC-deficient double mutant smc2-8/bub1Δ. Thus, condensin is essential for the segregation of the whole yeast genome. We obtained even more compelling evidence that condensin triggers SAC by showing that double condensin mutants with sgo1Δ and skp1-AA alleles, both impairing the sensor of improper inter-centromere tension, result in lethality. Moreover, direct measurement in metaphase of inter-kinetochore distance showed that it is markedly higher in condensin mutants than in wild type.
To examine the possible nature of the initial SAC trigger in the smc2 mutant, we analyzed centromere and kinetochore composition and found that the Dsn1 protein (a part of the MIND complex) and the Cse4 protein (the orthologue of the CENP—a centromeric histone) were partially delocalized from centromeres; whereas DDD, COMA, Ndc80 complexes, and inner kinetochore proteins were not visibly affected. Thus, centromeric chromatin is the likely mediator between condensin activity and the kinetochore. In addition, it is plausible that condensins contribute to the proper tension between sister kinetochores by establishing localized condensation of the centromeric region, which is enclosed between the two closest cohesion sites. Our analysis has uncovered a putative molecular interface between condensin and centromeres.
One of the unexpected outcomes of our ChIP-chip study of condensin binding was the discovery of condensin enrichment at the zones of converging DNA replication. This discovery suggested that replicon layout and condensin deposition on chromosomes are related, possibly through direct contacts between replication machinery and condensin. The putative interaction became a starting point for our investigation of condensin interface with DNA replication machinery. We are conducting a genome-scale ChIP-chip analysis of cell-cycle dynamics of condensin binding in relation to replication fork progression in both wild-type and origin-deleted strains.
Quimby BB, Yong-Gonzalez V, Anan T, Strunnikov AV, Dasso M. The promyelocytic leukemia protein stimulates SUMO conjugation in yeast. Oncogene 2006;25:2999-3005.
Strunnikov AV. SMC complexes in bacterial chromosome condensation and segregation. Plasmid 2006;55:135-44.
Yong-Gonzales V, Wang B, Butylin P, Ouspenski II, Strunnikov A. Condensin function at centromere chromatin facilitates proper kinetochore tension and ensures correct mitotic segregation of sister chromatids. Genes Cells 2007;12:1075-90.
1Graduate Partnerships Program
For further information, contact strunnik@mail.nih.gov.
