Project description:Genome/chromosome organization is highly ordered and controls nuclear events. Here, we show that the TATA box-binding protein (TBP) interacts with the Cnd2 kleisin subunit of condensin to mediate interphase and mitotic chromosome organization in fission yeast. TBP recruits condensin onto RNA polymerase III-transcribed (Pol III) genes and highly transcribed Pol II genes; condensin in turn associates these genes with centromeres. Inhibition of the Cnd2-TBP interaction disrupts condensin localization across the genome and the proper assembly of mitotic chromosomes, leading to severe defects in chromosome segregation and eventually causing cellular lethality. We propose that the Cnd2-TBP interaction coordinates transcription with chromosomal architecture by linking dispersed gene loci with centromeres. This chromosome arrangement can contribute to the efficient transmission of physical force at the kinetochore to chromosomal arms, thereby supporting the fidelity of chromosome segregation. Genome-wide distributions of condensin and Pol III factors in fission yeast.
Project description:Genome/chromosome organization is highly ordered and controls nuclear events. Here, we show that the TATA box-binding protein (TBP) interacts with the Cnd2 kleisin subunit of condensin to mediate interphase and mitotic chromosome organization in fission yeast. TBP recruits condensin onto RNA polymerase III-transcribed (Pol III) genes and highly transcribed Pol II genes; condensin in turn associates these genes with centromeres. Inhibition of the Cnd2-TBP interaction disrupts condensin localization across the genome and the proper assembly of mitotic chromosomes, leading to severe defects in chromosome segregation and eventually causing cellular lethality. We propose that the Cnd2-TBP interaction coordinates transcription with chromosomal architecture by linking dispersed gene loci with centromeres. This chromosome arrangement can contribute to the efficient transmission of physical force at the kinetochore to chromosomal arms, thereby supporting the fidelity of chromosome segregation.
Project description:ABSTRACT: Condensin is a central regulator of mitotic genome structure, with mutants showing poorly condensed chromosomes and profound segregation defects. Here we identify the fission yeast NCT complex, comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), Casein Kinase II (CKII) and several TAFs, as a novel regulator of condensin function (where NCT mutants restore the formation of segregation-competent chromosomes in cells containing defective condensin). Synchronous ChIP-seq shows that NCT and condensin bind similar genomic regions, but only briefly co-localize during the periods of chromosome condensation and decondensation. These results are consistent with a model where NCT targets CKII to chromatin in a cell cycle-directed manner to modulate the activity of condensin during chromosome condensation and decondensation. DATA: Study includes ChIP-seq of fission yeast H3-K4Me3, H3-K36Me3, TBP, Taf7, Nrc1, Cka1 from aynchronous cells; Nrc1 and Cut3 (representing condensin) from four synchronized cell-cycle stages estimated as G2/M, Metaphase, Anaphase and G1/S.
Project description:Chromatin fibres dynamically change their organisation during cell cycle. In interphase nucleus, chromatin fibres are evenly distributed whereas their spatial occupancy are reorganised to form condensed chromosomes in mitosis. This process called chromosome condensation is necessary for an accomplishment of faithful chromosome segregation. One of the Structural Maintenance of Chromosomes complexes, Condensin, is indispensable for chromosome condensation. It remains, however, unknown how Condensin plays its role in shaping mitotic chromosome. Here we show that chromatin fibres change their interacting partners; short-range contacts in interphase nucleus are converted into long-range interactions to shape condensed chromosomes. This conversion of interactions among chromatin fibres results in the formation of larger domains within mitotic chromosomes. Condensin is solely in charge of the conversion and large domain formation in fission yeast mitosis. Our results show how fission yeast Condensin is involved in shaping mitotic chromosomes.
Project description:Chromatin fibres dynamically change their organisation during cell cycle. In interphase nucleus, chromatin fibres are evenly distributed whereas their spatial occupancy are reorganised to form condensed chromosomes in mitosis. This process called chromosome condensation is necessary for an accomplishment of faithful chromosome segregation. One of the Structural Maintenance of Chromosomes complexes, Condensin, is indispensable for chromosome condensation. It remains, however, unknown how Condensin plays its role in shaping mitotic chromosome. Here we show that chromatin fibres change their interacting partners; short-range contacts in interphase nucleus are converted into long-range interactions to shape condensed chromosomes. This conversion of interactions among chromatin fibres results in the formation of larger domains within mitotic chromosomes. Condensin is solely in charge of the conversion and large domain formation in fission yeast mitosis. Our results show how fission yeast Condensin is involved in shaping mitotic chromosomes.
Project description:Condensin protein complexes play central roles in the three-dimensional organization of chromosomes during mitotic and meiotic cell divisions. How condensin interacts with its chromatin substrates to promote sister chromatid decatenation and segregation is largely unknown. Previous work suggested that condensin, in addition to encircling chromatin fibers topologically within the large ring-shaped structure formed by its structural maintenance of chromosomes (SMC) and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA helices formed by condensinM-bM-^@M-^Ys HEAT-repeat subunits. Using detailed mapping data of the interfaces between the HEAT-repeat and the kleisin subunits, we generated mutant complexes that lack the Ycg1/CAP-G HEAT-repeat subunit. These tetrameric condensin complexes fail to associate stably with chromosomes in yeast and human cells. We suggest that condensin controls chromosome architecture by stabilizing chromatin loops of chromatin fibers through interaction with its unconventional HEAT-repeat DNA binding domain. Analysis of condensin binding genomewide in a wild type and a condensin mutant
Project description:The extreme length of chromosomal DNA requires organizing mechanisms to both promote functional genetic interactions and ensure faithful chromosome segregation when cells divide. Microscopy and genome wide contact frequency (Hi-C) analyses indicate that intra-chromosomal looping of DNA is a primary pathway of chromosomal organization during all stages of the cell cycle (Dekker, J. & Mirny, L. . Cell 164, 1110–1121 (2016). Although the enzymatic pathways required for DNA loop formation are yet to be fully characterized, the activity of the SMC family of proteins has been consistently associated with this process in interphase and mitosis. Here we use Hi-C to study the reorganization of budding yeast chromosome conformation in early mitosis and the role of SMCs in this process. Using polymer simulations, we find that the differences between interphase and mitotic Hi-C maps can be explained by the formation of intra-chromosomal (cis-) loops in mitotic chromosomes. We demonstrate that mitotic SMC cohesin activity is required for formation of cis-loops, independently of sister-chromatid cohesion. In contrast, SMC condensin is not required for loop formation in these early mitotic cells. Rather condensin activity promotes distinct higher order structures in the chromosomes at centromeres and in the rDNA proximal regions. Thus we demonstrate that cohesin-dependent cis-loops provide the primary higher order organization of budding yeast mitotic chromosomes, independently of condensin and sister chromatid cohesion.
Project description:The duplication and segregation of chromosomes involve the dynamic re-organization of their internal structure by conserved architectural proteins, such as structural maintenance of chromosomes complexes (i.e., cohesin and condensin). Although the roles of these factors is actively investigated, a genome-wide view of chromosome dynamic architecture at both small and large-scales during cell division remains elusive. Here we report the first comprehensive 4D analysis of the Saccharomyces cerevisiae genome higher-order organization during the cell cycle, and investigate the roles of SMC in the observed structural transitions. During replication, cohesion establishment promotes long-range intra-chromosomal contacts and correlates with the individualization of chromosomes, which culminates at metaphase. Mitotic chromosomes are then abruptly reorganized in anaphase by mechanical forces exerted by the mitotic spindle. The formation of a condensin-dependent loop, that bridges the centromere cluster with the rDNA loci, suggests that condensin-mediated forces may also directly facilitate segregation. This work provides a comprehensive overview of chromosome dynamics during the cell cycle of a unicellular eukaryote that recapitulates and unveils new features of highly conserved stages of the cell division.
Project description:Condensin protein complexes play central roles in the three-dimensional organization of chromosomes during mitotic and meiotic cell divisions. How condensin interacts with its chromatin substrates to promote sister chromatid decatenation and segregation is largely unknown. Previous work suggested that condensin, in addition to encircling chromatin fibers topologically within the large ring-shaped structure formed by its structural maintenance of chromosomes (SMC) and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA helices formed by condensin’s HEAT-repeat subunits. Using detailed mapping data of the interfaces between the HEAT-repeat and the kleisin subunits, we generated mutant complexes that lack the Ycg1/CAP-G HEAT-repeat subunit. These tetrameric condensin complexes fail to associate stably with chromosomes in yeast and human cells. We suggest that condensin controls chromosome architecture by stabilizing chromatin loops of chromatin fibers through interaction with its unconventional HEAT-repeat DNA binding domain.