Abstract
Author SummaryAll eukaryotic organisms must duplicate their genome precisely once before cell division. This occurs according to an established temporal program during S-phase (when DNA synthesis takes place) of the cell cycle. In vertebrates, this program is regulated at the level of large chromosomal domains ranging from 200 kb to 2 Mb, but the molecular mechanisms that control the temporal firing order of animal replication origins are not clearly understood. Using the genetically tractable chicken DT40 cell system, we identified a minimal combination of cis-regulatory DNA elements that is able to shift the timing of a naturally “mid-late” replicated region to “mid-early.” This critical group of elements is composed of one strong replication origin flanked by binding sequences for the upstream stimulatory factor (USF) protein. The additional presence of a strongly transcribed gene shifted the region towards an even earlier replication time, suggesting cooperation between cis-elements when establishing temporal programs of replication. We speculate that USF binding sequences cooperate with sites of replication initiation and transcribed genes to promote the establishment of early replicating domains along vertebrate genomes.
Highlights
The nuclear genome of higher eukaryotes is replicated according to an established temporal program
Using the genetically tractable chicken DT40 cell system, we identified a minimal combination of cisregulatory DNA elements that is able to shift the timing of a naturally ‘‘mid-late’’ replicated region to ‘‘mid-early.’’ This critical group of elements is composed of one strong replication origin flanked by binding sequences for the upstream stimulatory factor (USF) protein
We determined the genome-wide replication timing profiles of DT40 cells following pulse labeling with BrdU and cell sorting into three fractions
Summary
The nuclear genome of higher eukaryotes is replicated according to an established temporal program. Several studies suggest that post-translational histone modifications directly regulate replication timing, but the effects of altering particular modifications are relatively minor in vertebrates [7,8]. The cis tethering of histone acetyltransferase activity adjacent to the human b-globin origin of replication induced only a weak shift in replication timing from late to midlate S-phase in lymphocytes, suggesting that this signal is insufficient to organize the early domain of replication that exists in erythroid cells [7]. A minority (5/23) of 23 single copy loci displayed a weak shift of replication timing in a panel of mutant mouse ES cell lines that were disrupted for histone deacetylation; H3K4, H3K9, and H3K27 methylation; or DNA methylation activities [9]. A direct role for histone modifications in the regulation of early replication timing remains to be demonstrated in vertebrates.
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