Genome organization of DNA replication timing and its link to chromatin and transcription

The replication of the genome is a highly organized process. Not every sequence
replicates at the same time, instead some genes replicate early, while others replicate
later during S phase. The timing of DNA replication is conserved within consecutive cell
divisions of a given cell type. The aim of this PhD thesis was a better understanding of
the regulation of DNA replication. In particular, I determined the genomic landscape of
the timing of DNA replication in the Drosophila genome, and defined the dynamics of
replication timing and its connection with chromatin and transcription.
Recent genome-wide studies of replication timing and transcription suggested a strong
relation between both processes since early replicating genes are more likely to be
expressed than genes replicating later during S phase. This correlation is not absolute,
therefore raising the question if replication timing is dynamic between different
epigenetic states, or if it is static and this correlation is driven mostly by a distinct set of
constitutively expressed genes. To create a defined replication timing program, initiation
of DNA replication needs to be controlled in space and time. The location and time of
firing of the closest origin of replication (ori) defines the replication timing of a certain
sequence. However, only few metazoan origins of replication have been identified, and
they lack a consensus sequence. Therefore it has been suggested that replication
initiation is defined epigenetically.
To address this problem I generated datasets for replication timing in two Drosophila cell
types representing different developmental states and gender, using high-resolution
tiling arrays. This detailed analysis permitted the identification of zones of replication
initiation throughout the whole genome. Surprisingly, I could identify a higher number of
initiation zones in early and late S phase than in mid S phase. This work also shows that
about 20% of the Drosophila genome replicates at different times in the two cell types.
These differences in replication timing correlate with differences in gene expression,
chromatin modifications and position in the nucleus relative to the nuclear periphery.
Interestingly, the dosage compensated male X chromosome replicates predominantly in
early S phase. This correlates with chromosome-wide hyperacetylation, often
independent of transcription differences. High levels of acetylation on Lysine 16 of
Histone H4 were also detected at initiation zones, supporting the model of epigenetically
defined replication initiation.
In addition, I addressed the potential role of chromatin-bound proteins in modulating
replication timing. Using RNA interference, I could show that the absence of Heterochromatin Protein 1 (HP1) has distinct effects on replication timing many of which
appear transcription independent.
Together, my results reveal organizational principles of DNA replication of the
Drosophila genome and indicate that replication timing is dynamic and chromatindependent.