Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors [4C]

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 consisting of ~200kb human bacteria artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) replication origin elements (E-BACs). E-BACs were stably maintained as autonomous mini-chromosomes in both HeLa and human induced pluripotent stem cells (hiPSCs) and established their RT de novo. We applied 4C-seq to evaluate E-BACs'' sub-nuclear compartment. Overall design: 4C-seq profiles of E-BAC transfected K3-EBNA1 cells