Title: The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning
Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres ( TEL s) and centromeres ( CEN s) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TEL s (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus ( STB ) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance. more »« less
Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locusSTB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STBbypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.
Dramatic change in chromosomal DNA morphology between interphase and mitosis is a defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin’s loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, accompanied by enhanced mixing of A and B chromatin compartments, and this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II’s NCAPG2 subunit. MCPH1’s ability to block condensin II’s association with chromatin is abrogated by the fusion of SMC2 with NCAPH2, hence may work by a mechanism similar to cohesin. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis.
Lariviere, Patrick J; Ashraf, A_H_M Zuberi; Navarro-Escalante, Lucio; Leonard, Sean P; Miller, Laurel G; Moran, Nancy A; Barrick, Jeffrey E
(, mBio)
Kaltenpoth, Martin
(Ed.)
ABSTRACT Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiontSnodgrassella alvi. We are also able to engineer the genomes of multiple strains ofS. alviand another species,Snodgrassella communis, which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont,Bartonella apis, which is an alphaproteobacterium. As expected, gene knockout inS. alviusing this approach isrecA-dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods. IMPORTANCEHoney bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health.
Abstract Dosage compensation inCaenorhabditis elegansequalizes X-linked gene expression between XX hermaphrodites and XO males. The process depends on a condensin- containing dosage compensation complex (DCC), which binds the X chromosomes in hermaphrodites to repress gene expression. Condensin IDCand an additional five DCC components must be present on the X during early embryogenesis in hermaphrodites to establish dosage compensation. However, whether the DCC’s continued presence is required to maintain the repressed state once established is unknown. Beyond the role of condensin IDCin X chromosome compaction, additional mechanisms contribute to X- linked gene repression. DPY-21, a non-condensin IDCDCC component, is an H4K20me2/3 demethylase whose activity enriches the repressive histone mark, H4 lysine 20 monomethylation, on the X chromosomes. In addition, CEC-4 tethers H3K9me3-rich chromosomal regions to the nuclear lamina, which also contributes to X- linked gene repression. To investigate the necessity of condensin IDCduring the larval and adult stages of hermaphrodites, we used the auxin-inducible degradation system to deplete the condensin IDCsubunit DPY-27. While DPY-27 depletion in the embryonic stages resulted in lethality, DPY-27 depleted larvae and adults survive. In these DPY-27 depleted strains, condensin IDCwas no longer associated with the X chromosome, the X became decondensed, and the H4K20me1 mark was gradually lost, leading to X-linked gene derepression. These results suggest that the stable maintenance of dosage compensation requires the continued presence of condensin IDC. A loss-of-function mutation incec-4, in addition to the depletion of DPY-27 or the genetic mutation ofdpy- 21, led to even more significant increases in X-linked gene expression, suggesting that tethering heterochromatic regions to the nuclear lamina helps stabilize repression mediated by condensin IDCand H4K20me1. Author SummaryIn some organisms, whether an individual becomes male, female, or hermaphrodite is determined by the number of their sex chromosomes. In the nematodeCaenorhabditis elegans, males have one X chromosome, whereas hermaphrodites have two X chromosomes. This difference in the number of X chromosomes is crucial for deciding whether an individual becomes a hermaphrodite or a male. However, having two X chromosomes can lead to problems because it results in different gene expression levels, resulting in hermaphrodite lethality. To solve this issue, many organisms undergo a process called dosage compensation. Dosage compensation inC. elegansis achieved by a group of proteins known as the dosage compensation complex (DCC), which includes a protein called DPY-27. The function of DPY-27 is essential during early embryonic development. This study shows that in contrast to early embryonic development, larvae and adults can still survive when DPY-27 is missing. In these worms, all known mechanisms involved in dosage compensation are disrupted and the X is no longer repressed. Our results suggest that the maintenance of dosage compensation in nematodes is an active process, and that it is essential for survival when the organism is developing, but once fully developed, the process becomes dispensable.
He, Yunyan; Lawrimore, Josh; Cook, Diana; Van Gorder, Elizabeth Erin; De Larimat, Solenn Claire; Adalsteinsson, David; Forest, M Gregory; Bloom, Kerry
(, Nucleic Acids Research)
null
(Ed.)
Abstract The revolution in understanding higher order chromosome dynamics and organization derives from treating the chromosome as a chain polymer and adapting appropriate polymer-based physical principles. Using basic principles, such as entropic fluctuations and timescales of relaxation of Rouse polymer chains, one can recapitulate the dominant features of chromatin motion observed in vivo. An emerging challenge is to relate the mechanical properties of chromatin to more nuanced organizational principles such as ubiquitous DNA loops. Toward this goal, we introduce a real-time numerical simulation model of a long chain polymer in the presence of histones and condensin, encoding physical principles of chromosome dynamics with coupled histone and condensin sources of transient loop generation. An exact experimental correlate of the model was obtained through analysis of a model-matching fluorescently labeled circular chromosome in live yeast cells. We show that experimentally observed chromosome compaction and variance in compaction are reproduced only with tandem interactions between histone and condensin, not from either individually. The hierarchical loop structures that emerge upon incorporation of histone and condensin activities significantly impact the dynamic and structural properties of chromatin. Moreover, simulations reveal that tandem condensin–histone activity is responsible for higher order chromosomal structures, including recently observed Z-loops.
Kumar, Deepanshu, Prajapati, Hemant Kumar, Mahilkar, Anjali, Ma, Chien-Hui, Mittal, Priyanka, Jayaram, Makkuni, and Ghosh, Santanu K.
"The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning". PLOS Genetics 17 (7). Country unknown/Code not available. https://doi.org/10.1371/journal.pgen.1009660.https://par.nsf.gov/biblio/10312443.
@article{osti_10312443,
place = {Country unknown/Code not available},
title = {The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning},
url = {https://par.nsf.gov/biblio/10312443},
DOI = {10.1371/journal.pgen.1009660},
abstractNote = {Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres ( TEL s) and centromeres ( CEN s) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TEL s (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus ( STB ) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.},
journal = {PLOS Genetics},
volume = {17},
number = {7},
author = {Kumar, Deepanshu and Prajapati, Hemant Kumar and Mahilkar, Anjali and Ma, Chien-Hui and Mittal, Priyanka and Jayaram, Makkuni and Ghosh, Santanu K.},
editor = {Malik, Harmit S.}
}
Warning: Leaving National Science Foundation Website
You are now leaving the National Science Foundation website to go to a non-government website.
Website:
NSF takes no responsibility for and exercises no control over the views expressed or the accuracy of
the information contained on this site. Also be aware that NSF's privacy policy does not apply to this site.