Role of <i>cis</i>-regulatory element in controlling replication timing.

<p>(A) In wt DT40 cells, the selected site of insertion is located in a mid-late replicating region devoid of strong replication origin. One hypothesis is that this locus is naturally located more toward the nuclear envelope (NE). (B) Insertion of a strong replicator linked to two copies of the HS4 insulator or four binding sites for USF (FIV) does not induce a replication timing shift. The replication timing of the transgenic replicator follows that of its chromosomal environment. (C) Flanking the strong replicator with two copies of FIV induces a significant replication timing shift and recruitment of histone modifying enzymes. This could be accompanied by the formation of a small loop that relocates the replicator more toward the interior of the nucleus. The increased accessibility to S-phase kinases could be either due to local decondensation of the chromatin structure and/or to the nuclear repositioning of the modified chromosomal region. (D) The addition of a strong promoter nearby increases the replication timing shift and leads to the formation of an independent mid-early replicon.