Alexander Strunnikov

A Direct Link between Sister Chromatid Cohesion and Chromosome Condensation Revealed through the Analysis of MCD1 in S. cerevisiae

The S. cerevisiae MCD1 (mitotic chromosome determinant) gene was identified in genetic screens for genes important for chromosome structure. MCD1 is essential for viability and homologs are found from yeast to humans. Analysis of the mcd1 mutant and cell cycle-dependent expression pattern of Mcd1p suggest that this protein functions in chromosome morphogenesis from S phase through mitosis. The mcd1 mutant is defective in sister chromatid cohesion and chromosome condensation. The physical association between Mcd1p and Smc1p, one of the SMC family of chromosomal proteins, further suggests that Mcd1p functions directly on chromosomes. These data implicate Mcd1p as a nexus between cohesion and condensation. We present a model for mitotic chromosome structure that incorporates this previously unsuspected link.

Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein

Disruption of ypuG and ypuH open reading frames in Bacillus subtilis leads to temperature‐sensitive slow growth, a defect in chromosome structure and formation of anucleate cells. The genes, which were named scpA and scpB, were found to be epistatic to the smc gene. Fusions of ScpA and ScpB to the fluorescent proteins YFP or CFP showed that both proteins co‐localize to two or four discrete foci that were present at mid‐cell in young cells, and within both cell halves, generally adjacent to chromosomal origin regions, in older cells. ScpA and ScpB foci are associated with DNA and depend on the presence of SMC and both Scps. ScpA and ScpB are associated with each other and with SMC in vivo, as determined using the FRET technique and immunoprecipitation assays. Genes similar to scpA and scpB are present in many bacteria and archaea, which suggests that their gene products form a condensation complex with SMC in most prokaryotes. The observed foci could constitute condensation factories that pull DNA away from mid‐cell into both cell halves.

Characterization of the components of the putative mammalian sister chromatid cohesion complex.

Establishing and maintaining proper sister chromatid cohesion throughout the cell cycle are essential for maintaining genome integrity. To understand how sister chromatid cohesion occurs in mammals, we have cloned and characterized mouse orthologs of proteins known to be involved in sister chromatid cohesion in other organisms. The cDNAs for the mouse orthologs of SMC1S.c. and SMC3S.c. , mSMCB and mSMCD respectively, were cloned, and the corresponding transcripts and proteins were characterized. mSMCB and mSMCD are transcribed at similar levels in adult mouse tissues except in testis, which has an excess of mSMCD transcripts. The mSMCB and mSMCD proteins, as well as the PW29 protein, a mouse homolog of Mcd1pS.c./Rad21S.p., form a complex similar to cohesin in X. laevis. mSMCB, mSMCD and PW29 protein levels show no significant cell-cycle dependence. The bulk of the mSMCB, mSMCD and PW29 proteins undergo redistribution from the chromosome vicinity to the cytoplasm during prometaphase and back to the chromatin in telophase. This pattern of intracellular localization suggests a complex role for this group of SMC proteins in chromosome dynamics. The PW29 protein and PCNA, which have both been implicated in sister chromatid cohesion, do not colocalize, indicating that these proteins may not function in the same cohesion pathway. Overexpression of a PW29-GFP fusion protein in mouse fibroblasts leads to inhibition of proliferation, implicating this protein and its complex with SMC proteins in the control of mitotic cycle progression.

Condensin Binding at Distinct and Specific Chromosomal Sites in the Saccharomyces cerevisiae Genome

ABSTRACT Mitotic chromosome condensation is chiefly driven by the condensin complex. The specific recognition (targeting) of chromosomal sites by condensin is an important component of its in vivo activity. We previously identified the rRNA gene cluster in Saccharomyces cerevisiae as an important condensin-binding site, but both genetic and cell biology data suggested that condensin also acts elsewhere. In order to characterize the genomic distribution of condensin-binding sites and to assess the specificity of condensin targeting, we analyzed condensin-bound sites using chromatin immunoprecipitation and hybridization to whole-genome microarrays. The genomic condensin-binding map shows preferential binding sites over the length of every chromosome. This analysis and quantitative PCR validation confirmed condensin-occupied sites across the genome and in the specialized chromatin regions: near centromeres and telomeres and in heterochromatic regions. Condensin sites were also enriched in the zones of converging DNA replication. Comparison of condensin binding in cells arrested in G1 and mitosis revealed a cell cycle dependence of condensin binding at some sites. In mitotic cells, condensin was depleted at some sites while enriched at rRNA gene cluster, subtelomeric, and pericentromeric regions.

Cdc14p/FEAR Pathway Controls Segregation of Nucleolus in S. cerevisiae by Facilitating Condensin Targeting to rDNA Chromatin in Anaphase

The condensin complex is the chief molecular machine of mitotic chromosome condensation. Nucleolar concentration of condensin in mitosis was previously shown to correlate with proficiency of rDNA condensation and segregation. To uncover the mechanisms facilitating this targeting we conducted a screen for mutants that impair mitotic condensin congression to the nucleolus. Mutants in the cdc14, esp1 and cdc5 genes, which encode FEAR-network components, showed the most prominent defects in mitotic condensin localization. We established that Cdc14p activity released by the FEAR pathway was required for proper condensin-to-rDNA targeting in anaphase. The MEN pathway was dispensable for condensin-to-rDNA targeting, however MEN-mediated release of Cdc14p later in anaphase allowed for proper, albeit delayed, condensin targeting to rDNA and successful segregation of nucleolus in the slk19 FEAR mutant. Although condensin was physically dislodged from rDNA in the cdc14 mutant, it was properly assembled, phosphorylated and chromatin-bound, suggesting that condensin was mistargeted but active. This study identifies a novel pathway promoting condensin targeting to a specific chromosomal address, the rDNA locus.

SMC proteins and chromosome structure

The structure of chromosomes is largely determined by chromosome-associated proteins. Members of the SMC (structural maintenance of chromosomes) family play an important role in both prokaryotic and eukaryotic chromosome structure and dynamics. SMC proteins are involved in chromosome condensation, sister-chromatid cohesion, sex-chromosome dosage compensation, genetic recombination and DNA repair. There have been major advances recently in understanding the function of SMC proteins--including the identification of biochemical activities of SMC-containing protein complexes and the realization that individual SMC proteins might link seemingly unrelated aspects of chromosomal metabolism.

SIZ1/SIZ2 Control of Chromosome Transmission Fidelity Is Mediated by the Sumoylation of Topoisomerase II

The Smt3 (SUMO) protein is conjugated to substrate proteins through a cascade of E1, E2, and E3 enzymes. In budding yeast, the E3 step in sumoylation is largely controlled by Siz1p and Siz2p. Analysis of Siz− cells shows that SUMO E3 is required for minichromosome segregation and thus has a positive role in maintaining the fidelity of mitotic transmission of genetic information. Sumoylation of the carboxy-terminus of Top2p, a known SUMO target, is mediated by Siz1p and Siz2p both in vivo and in vitro. Sumoylation in vitro reveals that Top2p is an extremely potent substrate for Smt3p conjugation and that chromatin-bound Top2p can still be sumoylated, unlike many other SUMO substrates. By combining mutations in the TOP2 sumoylation sites and the SIZ1 and SIZ2 genes we demonstrate that the minichromosome segregation defect and dicentric minichromosome stabilization, both characteristic for Smt3p–E3-deficient cells, are mediated by the lack of Top2p sumoylation in these cells. A role for Smt3p-modification as a signal for Top2p targeting to pericentromeric regions was suggested by an analysis of Top2p–Smt3p fusion. We propose a model for the positive control of the centromeric pool of Top2p, required for high segregation fidelity, by Smt3p modification.

Human Condensin Function Is Essential for Centromeric Chromatin Assembly and Proper Sister Kinetochore Orientation

Condensins I and II in vertebrates are essential ATP-dependent complexes necessary for chromosome condensation in mitosis. Condensins depletion is known to perturb structure and function of centromeres, however the mechanism of this functional link remains elusive. Depletion of condensin activity is now shown to result in a significant loss of loading of CENP-A, the histone H3 variant found at active centromeres and the proposed epigenetic mark of centromere identity. Absence of condensins and/or CENP-A insufficiency produced a specific kinetochore defect, such that a functional mitotic checkpoint cannot prevent chromosome missegregation resulting from improper attachment of sister kinetochores to spindle microtubules. Spindle microtubule-dependent deformation of both inner kinetochores and the HEC1/Ndc80 microtubule-capturing module, then results in kinetochore separation from the Aurora B pool and ensuing reduced kinase activity at centromeres. Moreover, recovery from mitosis-inhibition by monastrol revealed a high incidence of merotelic attachment that was nearly identical with condensin depletion, Aurora B inactivation, or both, indicating that the Aurora B dysfunction is the key defect leading to chromosome missegregation in condensin-depleted cells. Thus, beyond a requirement for global chromosome condensation, condensins play a pivotal role in centromere assembly, proper spatial positioning of microtubule-capturing modules and positioning complexes of the inner centromere versus kinetochore plates.

Cooperation of sumoylated chromosomal proteins in rDNA maintenance.

SUMO is a posttranslational modifier that can modulate protein activities, interactions, and localizations. As the GFP-Smt3p fusion protein has a preference for subnucleolar localization, especially when deconjugation is impaired, the nucleolar role of SUMO can be the key to its biological functions. Using conditional triple SUMO E3 mutants, we show that defects in sumoylation impair rDNA maintenance, i.e., the rDNA segregation is defective and the rDNA copy number decreases in these mutants. Upon characterization of sumoylated proteins involved in rDNA maintenance, we established that Top1p and Top2p, which are sumoylated by Siz1p/Siz2p, most likely collaborate with substrates of Mms21p to maintain rDNA integrity. Cohesin and condensin subunits, which both play important roles in rDNA stability and structures, are potential substrates of Mms21, as their sumoylation depends on Mms21p, but not Siz1p and Siz2p. In addition, binding of cohesin and condensin to rDNA is altered in the mms21-CH E3-deficient mutant.

The promyelocytic leukemia protein stimulates SUMO conjugation in yeast

The promyelocytic leukemia gene was first identified through its fusion to the gene encoding the retinoic acid receptor alpha (RARα) in acute promyelocytic leukemia (APL) patients. The promyelocytic leukemia gene product (PML) becomes conjugated in vivo to the small ubiquitin-like protein SUMO-1, altering its behavior and capacity to recruit other proteins to PML nuclear bodies (PML-NBs). In the NB4 cell line, which was derived from an APL patient and expresses PML:RARα, we observed a retinoic acid-dependent change in the modification of specific proteins by SUMO-1. To dissect the interaction of PML with the SUMO-1 modification pathway, we used the budding yeast Saccharomyces cerevisiae as a model system through expression of PML and human SUMO-1 (hSUMO-1). We found that PML stimulated hSUMO-1 modification in yeast, in a manner that was dependent upon PMLs RING-finger domain. PML:RARα also stimulated hSUMO-1 conjugation in yeast. Interestingly, however, PML and PML:RARα differentially complemented yeast Smt3p conjugation pathway mutants. These findings point toward a potential function of PML and PML:RARα as SUMO E3 enzymes or E3 regulators, and suggest that fusion of RARα to PML may affect this activity.