Histone H3K9 and H4 Acetylations and Transcription Facilitate the Initial CENP-AHCP−3 Deposition and De Novo Centromere Establishment in Caenorhabditis elegans Artificial Chromosomes

ABSTRACTForeign DNA microinjected into the Caenorhabditis elegans germline forms episomal extra-chromosomal arrays, or artificial chromosomes (ACs), in embryos. Injected linear, short DNA fragments concatemerize into high molecular weight (HMW)-DNA arrays that are visible as punctate DAPI-stained foci in oocytes, which undergo chromatinization and centromerization in embryos. The inner centromere, inner and outer kinetochore components, including AIR-2, CENP-AHCP-3, Mis18BP1KNL-2 and BUB-1, assemble onto the nascent ACs during the first mitosis. Yet, due to incomplete DNA replication of the nascent ACs, centromeric proteins are not oriented at the poleward faces of the nascent ACs in mitosis, resulting in lagging ACs. The DNA replication efficiency of ACs improves over several cell cycles. We found that a condensin subunit, SMC-4, but not the replicative helicase component, MCM-2, facilitates de novo CENP-AHCP-3 deposition on nascent ACs. Furthermore, H3K9ac, H4K5ac, and H4K12ac are highly enriched on newly chromatinized ACs. HAT-1 and RbAp46/48LIN-53, which are essential for de novo centromere formation and segregation competency of nascent ACs, also hyperacetylate histone H3 and H4. Different from centromere maintenance on endogenous chromosomes, where Mis18BP1KNL-2 functions upstream of RbAp46/48LIN-53, RbAp46/48LIN-53 depletion causes the loss of both CENP-AHCP-3 and Mis18BP1KNL-2 initial deposition at de novo centromeres on ACs.

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Construction and analysis of artificial chromosomes with de novo holocentromeres in Caenorhabditis elegans

Essays in Biochemistry ◽

10.1042/ebc20190067 ◽

2020 ◽

Vol 64 (2) ◽

pp. 233-249

Author(s):

Zhongyang Lin ◽

Karen Wing Yee Yuen

Keyword(s):

Caenorhabditis Elegans ◽

Dna Sequence ◽

De Novo ◽

Complex Mixture ◽

Chromosome Length ◽

Cis Acting ◽

Artificial Chromosomes ◽

C Elegans ◽

Successive Generations

Abstract Artificial chromosomes (ACs), generated in yeast (YACs) and human cells (HACs), have facilitated our understanding of the trans-acting proteins, cis-acting elements, such as the centromere, and epigenetic environments that are necessary to maintain chromosome stability. The centromere is the unique chromosomal region that assembles the kinetochore and connects to microtubules to orchestrate chromosome movement during cell division. While monocentromeres are the most commonly characterized centromere organization found in studied organisms, diffused holocentromeres along the chromosome length are observed in some plants, insects and nematodes. Based on the well-established DNA microinjection method in holocentric Caenorhabditis elegans, concatemerization of foreign DNA can efficiently generate megabase-sized extrachromosomal arrays (Exs), or worm ACs (WACs), for analyzing the mechanisms of WAC formation, de novo centromere formation, and segregation through mitosis and meiosis. This review summarizes the structural, size and stability characteristics of WACs. Incorporating LacO repeats in WACs and expressing LacI::GFP allows real-time tracking of newly formed WACs in vivo, whereas expressing LacI::GFP-chromatin modifier fusions can specifically adjust the chromatin environment of WACs. The WACs mature from passive transmission to autonomous segregation by establishing a holocentromere efficiently in a few cell cycles. Importantly, WAC formation does not require any C. elegans genomic DNA sequence. Thus, DNA substrates injected can be changed to evaluate the effects of DNA sequence and structure in WAC segregation. By injecting a complex mixture of DNA, a less repetitive WAC can be generated and propagated in successive generations for DNA sequencing and analysis of the established holocentromere on the WAC.

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DNA Sequence Preference for De Novo Centromere Formation on a Caenorhabditis elegans Artificial Chromosome

10.1101/2020.04.12.037994 ◽

2020 ◽

Cited By ~ 1

Author(s):

Zhongyang Lin ◽

Karen Wing Yee Yuen

Keyword(s):

Caenorhabditis Elegans ◽

Dna Sequence ◽

Dna Sequences ◽

De Novo ◽

Repetitive Sequences ◽

Model Organism ◽

Artificial Chromosome ◽

Centromeric Dna ◽

Artificial Chromosomes ◽

Non Homologous End Joining

ABSTRACTCentromeric DNA sequences vary in different species, but share common characteristics, like high AT-content, repetitiveness, and low, but not no, transcriptional activity. Yet, neocentromeres can be found on non-centromeric, ectopic sequences, suggesting that centromeres can be established and maintained epigenetically. In contrast, canonical centromeric DNA sequences are more competent in de novo centromere formation on artificial chromosomes (ACs). To determine if specific DNA sequence features are preferred for new centromere formation, we injected different DNA sequences into the gonad of a holocentric model organism, Caenorhabditis elegans, to form ACs in embryos, and monitored mitotic AC segregation. We demonstrated that AT-rich sequences, but not repetitive sequences, accelerated de novo centromere formation on ACs. We also injected fragmented Saccharomyces cerevisiae genomic DNA to construct a less repetitive, more complex AC that can propagate through generations. By whole-genome sequencing and de novo assembly of AC sequences, we deduced that this AC was formed through non-homologous end joining. By CENP-AHCP-3 chromatin immunoprecipitation followed by sequencing (ChIP-seq), we found that CENP-AHCP-3 domain width on both the AC and endogenous chromosomes is positively correlated with AT-content. Besides, CENP-AHCP-3 binds to unexpressed gene loci or non-genic regions on the AC, consistent with the organization of endogenous holocentromeres.

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Drosophila Ana2 is a conserved centriole duplication factor

The Journal of Cell Biology ◽

10.1083/jcb.200910016 ◽

2010 ◽

Vol 188 (3) ◽

pp. 313-323 ◽

Cited By ~ 102

Author(s):

Naomi R. Stevens ◽

Jeroen Dobbelaere ◽

Kathrin Brunk ◽

Anna Franz ◽

Jordan W. Raff

Keyword(s):

Drosophila Melanogaster ◽

Caenorhabditis Elegans ◽

De Novo ◽

Protein Family ◽

Centriole Duplication

In Caenorhabditis elegans, five proteins are required for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. Functional orthologues of all but SAS-5 have been found in other species. In Drosophila melanogaster and humans, Sak/Plk4, DSas-6/hSas-6, and DSas-4/CPAP—orthologues of ZYG-1, SAS-6, and SAS-4, respectively—are required for centriole duplication. Strikingly, all three fly proteins can induce the de novo formation of centriole-like structures when overexpressed in unfertilized eggs. Here, we find that of eight candidate duplication factors identified in cultured fly cells, only two, Ana2 and Asterless (Asl), share this ability. Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms. We show that Ana2 is the likely functional orthologue of SAS-5 and that it is also related to the vertebrate STIL/SIL protein family that has been linked to microcephaly in humans. We propose that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery.

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An Artificially Constructed De Novo Human Chromosome Behaves Almost Identically to Its Natural Counterpart during Metaphase and Anaphase in Living Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00355-06 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7682-7695 ◽

Cited By ~ 16

Author(s):

Tomohiro Tsuduki ◽

Megumi Nakano ◽

Nao Yasuoka ◽

Saeko Yamazaki ◽

Teruaki Okada ◽

...

Keyword(s):

Yeast Artificial Chromosome ◽

Fluorescent Protein ◽

De Novo ◽

Artificial Chromosome ◽

Time Lapse ◽

Living Cells ◽

Lac Repressor ◽

Host Chromosome ◽

Artificial Chromosomes ◽

Spindle Midzone

ABSTRACT Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing α-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input α-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.

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Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5563-5570.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5563-5570

Author(s):

S S Schneider ◽

J L Hiemstra ◽

B A Zehnbauer ◽

P Taillon-Miller ◽

D L Le Paslier ◽

...

Keyword(s):

Cell Line ◽

Gene Amplification ◽

De Novo ◽

Physical Map ◽

Neuroblastoma Cell ◽

Neuroblastoma Cell Line ◽

Chromosome 2 ◽

Human Neuroblastoma ◽

Artificial Chromosomes ◽

Yeast Artificial Chromosomes

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.

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Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Journal of Visualized Experiments ◽

10.3791/61170 ◽

2020 ◽

Author(s):

Margarita Elena Papandreou ◽

Konstantinos Palikaras ◽

Nektarios Tavernarakis

Keyword(s):

Protein Synthesis ◽

Caenorhabditis Elegans ◽

De Novo ◽

De Novo Protein Synthesis

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A conserved family of proteins facilitates nascent lipid droplet budding from the ER

The Journal of Cell Biology ◽

10.1083/jcb.201505067 ◽

2015 ◽

Vol 211 (2) ◽

pp. 261-271 ◽

Cited By ~ 137

Author(s):

Vineet Choudhary ◽

Namrata Ojha ◽

Andy Golden ◽

William A. Prinz

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Lipid Metabolism ◽

Caenorhabditis Elegans ◽

Lipid Droplet ◽

Lipid Droplets ◽

De Novo ◽

Fat Storage ◽

Higher Eukaryotes ◽

Er Membrane

Lipid droplets (LDs) are found in all cells and play critical roles in lipid metabolism. De novo LD biogenesis occurs in the endoplasmic reticulum (ER) but is not well understood. We imaged early stages of LD biogenesis using electron microscopy and found that nascent LDs form lens-like structures that are in the ER membrane, raising the question of how these nascent LDs bud from the ER as they grow. We found that a conserved family of proteins, fat storage-inducing transmembrane (FIT) proteins, is required for proper budding of LDs from the ER. Elimination or reduction of FIT proteins in yeast and higher eukaryotes causes LDs to remain in the ER membrane. Deletion of the single FIT protein in Caenorhabditis elegans is lethal, suggesting that LD budding is an essential process in this organism. Our findings indicated that FIT proteins are necessary to promote budding of nascent LDs from the ER.

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