Dynamic alterations of replication timing in mammalian cells.
BACKGROUND
The eukaryotic genome is divided into distinct replication timing domains, which are activated during S phase in a strictly conserved order. Cellular differentiation can alter replication timing in some loci, but recent experiments yielded conflicting data regarding the relationship between gene expression and replication timing. The genetic and epigenetic determinants of replication timing in mammalian cells have yet to be elucidated.


RESULTS
We developed a mammalian experimental system in which the timing of DNA replication can be altered in a controlled manner. This system utilizes sequences from the human beta-globin locus that exhibit orientation-dependent transcriptional silencing when inserted into the murine genome. We found that before insertion, the murine target site replicated late during S phase. After insertion, replication timing depended on the orientation of the transgene. In a transcription-permissive orientation, the transgene and flanking sequences replicated early. In the reverse (silencing-prone) orientation, these sequences replicated late. Early replication correlated with histone modifications of the transgene chromatin but could be observed in the absence of the beta-globin promoter. Importantly, the replication timing switch did not require a replication origin within the transgene.


CONCLUSIONS
Transgene insertions into mammalian heterochromatin can alter the timing of DNA replication at the insertion site. This differentiation-independent replication timing switch did not necessitate insertion of an active promoter or a replication origin. These observations suggest that the timing of DNA replication can be manipulated by changes in DNA sequence, but that the determinants of replication timing are distinct from the sequences that specify replication initiation sites.
Lin
Chii Mei
CM
Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Fu
Haiqing
H
Martinovsky
Maria
M
Bouhassira
Eric
E
Aladjem
Mirit I
MI
D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D004261:DNA Replication / Q000276:immunology*; D020868:Gene Silencing; D005914:Globins / Q000235:genetics; D006570:Heterochromatin / Q000235:genetics; D006801:Humans; D051379:Mice; D013997:Time Factors; D019076:Transgenes / Q000235:genetics / Q000502:physiology; D014407:Tumor Cells, Cultured
D006570:Heterochromatin; D005914:Globins