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

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.