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Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors.

ABSTRACT: The role of DNA sequence in determining replication timing (RT) and chromatin higher order organization remains elusive. To address this question, we have developed an extra-chromosomal replication system (E-BACs) consisting of ?200 kb human bacterial artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) stable segregation elements. E-BACs were stably maintained as autonomous mini-chromosomes in EBNA1-expressing HeLa or human induced pluripotent stem cells (hiPSCs) and established distinct RT patterns. An E-BAC harboring an early replicating chromosomal region replicated early during S phase, while E-BACs derived from RT transition regions (TTRs) and late replicating regions replicated in mid to late S phase. Analysis of E-BAC interactions with cellular chromatin (4C-seq) revealed that the early replicating E-BAC interacted broadly throughout the genome and preferentially with the early replicating compartment of the nucleus. In contrast, mid- to late-replicating E-BACs interacted with more specific late replicating chromosomal segments, some of which were shared between different E-BACs. Together, we describe a versatile system in which to study the structure and function of chromosomal segments that are stably maintained separately from the influence of cellular chromosome context.

SUBMITTER: Sima J

PROVIDER: S-EPMC5829748 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors.

Sima Jiao J Bartlett Daniel A DA Gordon Molly R MR Gilbert David M DM

Nucleic acids research 20180201 4

The role of DNA sequence in determining replication timing (RT) and chromatin higher order organization remains elusive. To address this question, we have developed an extra-chromosomal replication system (E-BACs) consisting of ∼200 kb human bacterial artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) stable segregation elements. E-BACs were stably maintained as autonomous mini-chromosomes in EBNA1-expressing HeLa or human induced pluripotent stem cells (hiPSCs) and established ...[more]

PMID: 29294101

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Project description:BACKGROUND: Human artificial chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of de novo HAC formation. Indeed, to date, only a small number of large (>100 kb) genomic loci have been reported to be successfully packaged into de novo HACs. RESULTS: We have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. We report here the creation of a novel, bimolecular system based on bacterial artificial chromosomes (BACs) for the construction of HACs incorporating any defined genomic region. We have utilized this vector system to rapidly design, construct and validate multiple de novo HACs containing large (100-200 kb) genomic loci including therapeutically significant genes for human growth hormone (HGH), polycystic kidney disease (PKD1) and beta-globin. We report significant differences in the ability of different genomic loci to support de novo HAC formation, suggesting possible effects of cis-acting genomic elements. Finally, as a proof of principle, we have observed sustained beta-globin gene expression from HACs incorporating the entire 200 kb beta-globin genomic locus for over 90 days in the absence of selection. CONCLUSION: Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus. We have evaluated the impact of different genomic loci on the frequency of HAC formation and identified segments of genomic DNA that appear to facilitate de novo HAC formation. These genomic loci may be useful for identifying discrete functional elements that may be incorporated into future generations of HAC vectors.

Dataset Information

Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors.

Publications

Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors.

Similar Datasets

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