Alexander V. Strunnikov, PhD, Head, Unit on Chromosome Structure and Function
Vladimir Yong-Gonzales, PhD, Postdoctoral Fellow
Pavel Butylin, MSc, Visiting Fellow
Yoshimitsu Takahashi, PhD, Visiting Fellow
Bi-Dar Wang, PhD, Visiting Fellow
Stanimir Dulev, MSc, Predoctoral Fellow
Julia Panebianco, BSc, Postbaccalaureate Fellow

The eukaryotic ATPases of the SMC family constitute several essential eukaryotic protein complexes that determine higher-order chromosome structure and dynamics in eukaryotic cells. Condensin, one of these complexes, represents the main molecular machinery of chromosome condensation, a process indispensable for proper segregation of sister chromatids during anaphase. At present, the molecular mechanism of condensin activity in vivo is unknown. To understand the essence of condensin activity in chromatin, we focus on one aspect of this activity: the specificity of condensin targeting to the natural chromatin sites in budding yeast. We investigated the role of genetic and epigenetic factors in condensin's recognition of specific chromosomal domains within two research directions: the specificity mechanisms in condensin targeting to the nucleolar chromatin and condensin's role in the functionality of other chromosomal binding sites, which we recently identified by using a whole-genome analysis.

Molecular pathways governing condensin targeting to nucleolar organizer in mitosis

Wang, Panebianco, Butylin, Strunnikov

Our previous studies identified nucleolar chromatin (the rDNA genomic locus, or nucleolar organizer) as the major binding site for condensin in S. cerevisiae. Our subsequent genetic studies established that several pathways determine proper condensin localization to specific chromatin domains and thus facilitate the chromosome condensation process. Screening for molecular mechanisms determining specificity of mitotic condensin targeting to nucleolar chromatin focused on elucidating the role played by the nature of the rDNA locus in condensin affinity. The budding yeast system presents a unique opportunity to analyze the functional interface between the features of rDNA chromatin and condensin function, as it is possible to rearrange the tandemly repeated nucleolar organizer (NOR) locus so that all rRNA genes are episomal (diffuse nucleolus). In addition, it is possible to manipulate the copy number of tandem chromosomal rDNA repeats by genetic means. Thus, we initially used two strains of S. cerevisiae, one with a deletion of the natural NOR (with episomal rDNA) and the other with just 20 tandem repeats of the 9-kb rDNA. We assayed these cells for condensin binding to rDNA (using quantitative ChIP) in the course of cell cycle progression and compared them to wild type. Two important observations resulting from these experiments have led to a novel theory explaining the segregation mechanism of an actively transcribed nucleolus. First, four robust condensin binding sites identified within the rDNA in wild type changed their condensin enrichment in reverse correlation to the intensity of rDNA transcription. Second, condensin binding was excluded from the transcribed portion of the rDNA (encoding the 35S RNA precursor) in the diffuse nucleolus cells and the 20-rDNA repeat strain, both with hyperactivated Pol 1 transcription (per single rDNA unit) as a result of reduced rDNA copy number. In a series of experiments in which we repressed rDNA transcription by using rapamycin or a heterologous regulatable promoter, we further confirmed the reverse correlation between transcription and condensin binding. Thus, analysis of condensin binding to rDNA in the course of cell cycle and by means of modulating the nucleolar transcription levels has established that condensin occupancy of rDNA is negatively regulated by Pol I transcription.

These results allowed us to propose a model suggesting that the non-transcribed rDNA repeats, which are constitutively present in this tandemly repeated locus and constitute about half of all rDNA, are used for mitotic segregation of the nucleolus in its active/transcribed form, which is characteristic for yeast cells (see Figure 12.2). Moreover, it is likely that the requirement for segregation of the active nucleolus embodies the special role played by condensin in mitotic nucleolar segregation in budding yeast. We validated the model by direct time-lapse microscopic observations of nucleolar segregation in live budding yeast cells. In the strain with only 20 rDNA repeats, in which nearly all copies are transcribed throughout the cell cycle, segregation of the nucleolus (Sik1-RFP marker) was significantly delayed as compared with the rest of the genome (Scc3-GFP marker).

Figure 12.2

We found that another pathway controlling condensin binding to rDNA, particularly the replication fork-blocking region (RFB) in the non-transcribed spacer, is partially dependent on the sequence-specific RFB-binding protein Fob1. Such binding is likely involved in the interphase function of condensin; that is, it plays a role in maintaining the structural integrity of the nucleolar organizer. Nevertheless, the RFB-binding pathway is also activated in mitosis and thus is a likely regulator of an essential condensin function. The molecular components of this pathway are under investigation.

Analysis of condensin targeting to telomeres and centromeres

Yong-Gonzales, Wang, Takahashi, Dulev; in collaboration with Basrai, Lichten

As we showed recently, condensin has several critical sites located outside the nucleolar organizer. The sites correlate with landmarks of chromosome organization: DNA replication termination zones, subtelomeric regions, and centromeres. Studies on these sites with well-defined functions in the nucleus enable us to understand the role of condensin activity in the functioning of these chromosomal domains.

Our recent findings indicate that rearranging the NOR into multicopy rDNA plasmids (diffuse nucleolus) results in release of condensin from the transcribed portion of rDNA. At the same time, repression of rDNA transcription via a regulatable heterologous promoter attracts more condensin to rDNA, presumably from non-rDNA sites. Thus, the condensin binding pattern in the genome is at least partially regulated by the transcriptional state and physical organization of the rDNA locus. These findings allowed us to address two important questions in condensin biology: to what degree the condensin binding pattern across the genome is predetermined and the nature of condensin function in chromosomes when its recruitment for rDNA segregation is weakened. Thus, we employed chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip approach) to investigate the genomic distribution of condensin binding loci in the episomal rDNA (ErDNA) yeast strain. We established that condensin still has an essential role in segregating non-rDNA chromosomes in cells with a diffuse nucleolus. Moreover, we showed that, in ErDNA cells, condensin binding is increased preexisting condensing binding sites but that some is also redistributed to new locations, with a strong preference toward subtelomeric regions, thereby establishing a novel condensin-enriched domain.

We also characterized the scope of condensin involvement in kinetochore function and found that condensin mutants, mediated by centromere defects, elicit cell cycle arrest by activation of the spindle checkpoint pathway. A deficiency in the specialized centromere histone Cse4p (yeast CENP-A) may underlie the defects. Our finding opens an intriguing possibility that condensin plays a cis-function at the centromere, which is important for kinetochore assembly and/or function. Given that the recently discovered functional interactions of condensin with both telomeres and kinetochores open a new direction, we plan to focus on the molecular interface between the condensin function, telomere maintenance, and kinetochore activity.

COLLABORATORS

Munira A. Basrai, PhD, Genetics Branch, NCI, Bethesda, MD
Yoshiko Kikuchi, PhD, University of Tokyo, Tokyo, Japan
Michael J. Lichten, PhD, Laboratory of Biochemistry, NCI, Bethesda, MD

For further information, contact strunnik@mail.nih.gov.