Project description:The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C) has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.
Project description:DNA replication during S-phase is accompanied by establishment of sister chromatid cohesion to ensure faithful chromosome segregation. The Eco1 acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved interaction motif that Chl1 shares with GINS and polymerase α. We visualize recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1 helicase facilitates replication fork progression under conditions of nucleotide depletion, however this function does not require Ctf4 interaction. Conversely, Ctf4 interaction, but not helicase activity, is required for Chl1’s role in sister chromatid cohesion. A physical interaction between Chl1 and the cohesin complex during S-phase suggests that Chl1 contacts cohesin to facilitate its acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that coordinates replication fork progression and sister chromatid cohesion establishment.
Project description:The fidelity of chromosome duplication through cell divisions requires timely binding and release of the cohesin. Cohesin is a ring-shaped protein complex linking newly replicated sister chromatids to ensure their appropriate transmission through mitosis. Upon commencement of mitosis cohesin is removed from DNA in two steps: first, from chromosome arms resulting in sister chromatid resolution, and, second, from centromers leading to sister chromatid segregation. As DNA of eukaryotic chromosomes is assembled into chromatin, regulation of sister chromatid cohesion-segregation may involve chromatin modifying machinery, but this link is not well understood. Here we report that H2A-H2B histone chaperone NAP1, a factor, which is primarily implicated in chromatin assembly, is required for cohesin release from mitotic chromosome arms. NAP1 and cohesin protein complex interact directly and share multiple binding sites on chromatin. Depletion of the NAP1 hinders cohesin removal during mitosis resulting in accumulation of unresolved sister chromatids. Thus, in addition to its well established functions in chromatin dynamics, histone chaperone NAP1 coordinates cell cycle dependent cohesin release. These results reveal a novel molecular pathway for sister chromatid resolution and emphasizes a role for histone chaperones in control of eukaryotic genome replication and transmission.
Project description:As in Eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionaly towards the opposite terminus region. Here we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structuraly distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity and contacts are restricted to ~280 kb creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS that restricts short-range interactions. Combined, these results reveal the contributions of major, evolutionary conserved proteins in a bacterial chromosome organization.
Project description:Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome also may plays an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutant and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and change the chromosome organization at domain scales significantly. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulation (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (> 200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation. RNA-seq analysis to reveal the regulation process of Fur proteins.
Project description:The functions of cohesin are central to genome integrity, chromosome organization, and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2-Scc4 complex, however, little is known about how it stimulates the cohesin loading activity. Here we determine the large “hook” structure of Scc2 responsible for catalyzing cohesin loading. We identify key Scc2 surfaces that are crucial for DNA binding and for cohesin loading in vivo. Using previously determined structures and modeling, we derive a pseudo-atomic structure of the full-length Scc2-Scc4 complex. Finally, our crosslinking and electron microscopy analyses reveal that Scc2-Scc4 utilizes its modular structure to make multiple contacts with a folded cohesin at an interface created by the cohesin head-hinge interaction.
Project description:Three types of structurally related structural maintenance of chromosomes (SMC) complexes, referred to as condensins, have been identified in bacteria. Smc-ScpAB is present in most bacteria, whereas MukBEF is found in enterobacteria and MksBEF is scattered over the phylogenic tree. The contributions of these condensins to chromosome management were characterized in Pseudomonas aeruginosa, which carries both Smc-ScpAB and MksBEF. In this bacterium, SMC-ScpAB controls chromosome disposition by juxtaposing chromosome arms. In contrast, MksBEF is critical for chromosome segregation in the absence of the main segregation system, and it affects the higher-order architecture of the chromosome by promoting DNA contacts in the megabase range. Strikingly, our results reveal a prevalence of Smc-ScpAB over MksBEF involving a coordination of their activities with chromosome replication. They also show that E. coli MukBEF can substitute for MksBEF in P. aeruginosa while prevailing over Smc-ScpAB. Our results reveal a hierarchy between activities of bacterial condensins on the same chromosome.
Project description:The fidelity of chromosome duplication through cell divisions requires timely binding and release of the cohesin. Cohesin is a ring-shaped protein complex linking newly replicated sister chromatids to ensure their appropriate transmission through mitosis. Upon commencement of mitosis cohesin is removed from DNA in two steps: first, from chromosome arms resulting in sister chromatid resolution, and, second, from centromers leading to sister chromatid segregation. As DNA of eukaryotic chromosomes is assembled into chromatin, regulation of sister chromatid cohesion-segregation may involve chromatin modifying machinery, but this link is not well understood. Here we report that H2A-H2B histone chaperone NAP1, a factor, which is primarily implicated in chromatin assembly, is required for cohesin release from mitotic chromosome arms. NAP1 and cohesin protein complex interact directly and share multiple binding sites on chromatin. Depletion of the NAP1 hinders cohesin removal during mitosis resulting in accumulation of unresolved sister chromatids. Thus, in addition to its well established functions in chromatin dynamics, histone chaperone NAP1 coordinates cell cycle dependent cohesin release. These results reveal a novel molecular pathway for sister chromatid resolution and emphasizes a role for histone chaperones in control of eukaryotic genome replication and transmission. Genome-wide NAP1 and Cohesin ChIP-chip profiling in Drosophila S2 cells. The supplementary bed file S2_cohesin_sites.bed contains cohesin binding sites obtained by intersecting the sets of significant ChIP-chip peaks for SA (a cohesin subunit; stromalin) and SMC1.
Project description:Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome also may play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutant and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and change the chromosome organization at domain scales significantly. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulation (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (> 200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation. ChIP-seq analysis of Fur proteins binding in the genome of ZM532.
Project description:Meiosis produces gametes through a specialised, two-step cell division, which is highly error-prone in humans. Reductional meiosis I, where maternal and paternal chromosomes (homologs) segregate, is followed by equational meiosis II, where sister chromatids separate. Uniquely during meiosis I, sister kinetochores are monooriented and pericentromeric cohesin is protected. Here, we demonstrate that these key adaptations for reductional chromosome segregation are achieved through separable control of multiple kinases by the meiosis I-specific budding yeast Spo13 protein. Recruitment of Polo kinase to kinetochores directs monoorientation, while, independently, cohesin protection is achieved by controlling the effects of cohesin kinases. Therefore, reductional chromosome segregation, the defining feature of meiosis, is established by multifaceted kinase control by a master regulator. The recent identification of Spo13 orthologs, fission yeast Moa1 and mouse MEIKIN, suggests that kinase coordination by a master meiosis I regulator may be a general feature in the establishment of reductional chromosome segregation.