Dynamic alterations of replication timing in mammalian cells

Area of Science:

• Genomics
• Epigenetics
• Mammalian cell biology

Background:

• Eukaryotic genomes have replication timing domains active during S phase.
• Replication timing alterations during differentiation are known, but its link to gene expression is debated.
• Genetic and epigenetic factors influencing replication timing in mammals remain unclear.

Purpose of the Study:

• To investigate the genetic and epigenetic determinants of DNA replication timing.
• To develop a mammalian system for controlled alteration of replication timing.
• To explore the relationship between DNA sequence, chromatin modifications, and replication timing.

Main Methods:

• Developed a mammalian experimental system using human beta-globin locus sequences.
• Inserted transgenes into murine heterochromatin in orientation-dependent manner.
• Analyzed replication timing and histone modifications of transgene chromatin.

Main Results:

• Transgene insertion altered replication timing at the insertion site.
• Replication timing was orientation-dependent: permissive orientation led to early replication, silencing-prone orientation to late replication.
• Early replication correlated with histone modifications but did not require a promoter or replication origin.

Conclusions:

• Transgene insertions can manipulate DNA replication timing in mammalian heterochromatin.
• This replication timing switch is differentiation-independent and does not require an active promoter or replication origin.
• Replication timing determinants are distinct from sequences specifying replication initiation sites.