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Abstract
Foreign DNA microinjected into the Caenorhabditis elegans syncytial gonad forms episomal extra-chromosomal arrays, or artificial chromosomes (ACs), in embryos. Short, linear DNA fragments injected concatemerize into high molecular weight (HMW) DNA arrays that are visible as punctate DAPI-stained foci in oocytes, and they undergo chromatinization and centromerization in embryos. The inner centromere, inner kinetochore and spindle checkpoint components, including AIR-2, CENP-AHCP-3, Mis18BP1KNL-2 and BUB-1, respectively, assemble onto the nascent ACs during the first mitosis. The DNA replication efficiency of ACs improves over several cell cycles, which correlates with the improvement of kinetochore bi-orientation and proper segregation of ACs. Depletion of condensin II subunits, like CAPG-2 and SMC-4, but not the replicative helicase component, MCM-2, reduces de novo CENP-AHCP-3 level on nascent ACs. Furthermore, H3K9ac, H4K5ac and H4K12ac are highly enriched on newly chromatinized ACs. RbAp46/48LIN-53 and HAT-1, which affect the acetylation of histone H3 and H4, are essential for chromatinization, de novo centromere formation and segregation competency of nascent ACs. RbAp46/48LIN-53 or HAT-1 depletion causes the loss of both CENP-AHCP-3 and Mis18BP1KNL-2 initial deposition at de novo centromeres on ACs. This phenomenon is different from centromere maintenance on endogenous chromosomes, where Mis18BP1KNL-2 functions upstream of RbAp46/48LIN-53.
Highlights
Histone H3 variant, CENP-A, replaces histone H3 in some of the centromeric nucleosomes and serves as the foundation for building the kinetochore [1], which connects the sister chromatids to opposite spindles and orchestrates chromosome movement in cell division
This is consistent with the finding that major sperm proteins trigger nuclear membrane breakdown [47] and release the nuclearlocalized histones and centromeric proteins to allow chromatinization and centromerization of the high molecular weight (HMW) DNA arrays, which are initially located in the cytoplasm
In wild-type, the average level of CENPAHCP-3 on nascent artificial chromosomes (ACs) formed from linear plasmid DNA is 1.3-fold higher than that on endogenous chromosomes in one-cell embryos (Supplementary Figure S1I)
Summary
Histone H3 variant, CENP-A, replaces histone H3 in some of the centromeric nucleosomes and serves as the foundation for building the kinetochore [1], which connects the sister chromatids to opposite spindles and orchestrates chromosome movement in cell division. New centromere formation has been observed in diverse species. Tethering CENP-A-specific chaperones to a euchromatin locus or overexpressing CENP-A could cause ectopic CENP-A localization and ectopic centromere formation in fission yeast and human cells [3,4]. Most artificial chromosome (AC) formation in these species often relies on the presence of their endogenous centromeric DNA sequences, which suggests endogenous centromeric DNA sequences are preferred for new centromere formation. The isolation of these ACs require long-term selection, and they have relatively low frequencies of de novo centromere formation, which has
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