Analysis Of Replication Timing Research Articles

Genome-scale analysis of replication timing: from bench to bioinformatics

Replication timing profiles are cell type-specific and reflect genome organization changes during differentiation. In this protocol, we describe how to analyze genome-wide replication timing (RT) in mammalian cells. Asynchronously cycling cells are pulse labeled with the nucleotide analog 5-bromo-2-deoxyuridine (BrdU) and sorted into S-phase fractions on the basis of DNA content using flow cytometry. BrdU-labeled DNA from each fraction is immunoprecipitated, amplified, differentially labeled and co-hybridized to a whole-genome comparative genomic hybridization microarray, which is currently more cost effective than high-throughput sequencing and equally capable of resolving features at the biologically relevant level of tens to hundreds of kilobases. We also present a guide to analyzing the resulting data sets based on methods we use routinely. Subjects include normalization, scaling and data quality measures, LOESS (local polynomial) smoothing of RT values, segmentation of data into domains and assignment of timing values to gene promoters. Finally, we cover clustering methods and means to relate changes in the replication program to gene expression and other genetic and epigenetic data sets. Some experience with R or similar programming languages is assumed. All together, the protocol takes ∼3 weeks per batch of samples.

A complete protocol for genome-scale analysis of replication timing in mammalian cells

A complete protocol for genome-scale analysis of replication timing in mammalian cells

Genome-wide studies highlight indirect links between human replication origins and gene regulation

To get insights into the regulation of replication initiation, we systematically mapped replication origins along 1% of the human genome in HeLa cells. We identified 283 origins, 10 times more than previously known. Origin density is strongly correlated with genomic landscapes, with clusters of closely spaced origins in GC-rich regions and no origins in large GC-poor regions. Origin sequences are evolutionarily conserved, and half of them map within or near CpG islands. Most of the origins overlap transcriptional regulatory elements, providing further evidence of a connection with gene regulation. Moreover, we identify c-JUN and c-FOS as important regulators of origin selection. Half of the identified replication initiation sites do not have an open chromatin configuration, showing the absence of a direct link with gene regulation. Replication timing analyses coupled with our origin mapping suggest that a relatively strict origin-timing program regulates the replication of the human genome.

Neural induction promotes large-scale chromatin reorganisation of theMash1locus

Determining how genes are epigenetically regulated to ensure their correct spatial and temporal expression during development is key to our understanding of cell lineage commitment. Here we examined epigenetic changes at an important proneural regulator gene Mash1 (Ascl1), as embryonic stem (ES) cells commit to the neural lineage. In ES cells where the Mash1 gene is transcriptionally repressed, the locus replicated late in S phase and was preferentially positioned at the nuclear periphery with other late-replicating genes (Neurod, Sprr2a). This peripheral location was coupled with low levels of histone H3K9 acetylation at the Mash1 promoter and enhanced H3K27 methylation but surprisingly location was not affected by removal of the Ezh2/Eed HMTase complex or several other chromatin-silencing candidates (G9a, SuV39h-1, Dnmt-1, Dnmt-3a and Dnmt-3b). Upon neural induction however, Mash1 transcription was upregulated (>100-fold), switched its time of replication from late to early in S phase and relocated towards the interior of the nucleus. This spatial repositioning was selective for neural commitment because Mash1 was peripheral in ES-derived mesoderm and other non-neural cell types. A bidirectional analysis of replication timing across a 2 Mb region flanking the Mash1 locus showed that chromatin changes were focused at Mash1. These results suggest that Mash1 is regulated by changes in chromatin structure and location and implicate the nuclear periphery as an important environment for maintaining the undifferentiated state of ES cells.

X chromosome choice occurs independently of asynchronous replication timing

In mammals, dosage compensation is achieved by X chromosome inactivation in female cells. Xist is required and sufficient for X inactivation, and Xist gene deletions result in completely skewed X inactivation. In this work, we analyzed skewing of X inactivation in mice with an Xist deletion encompassing sequence 5 KB upstream of the promoter through exon 3. We found that this mutation results in primary nonrandom X inactivation in which the wild-type X chromosome is always chosen for inactivation. To understand the molecular mechanisms that affect choice, we analyzed the role of replication timing in X inactivation choice. We found that the two Xist alleles and all regions tested on the X chromosome replicate asynchronously before the start of X inactivation. However, analysis of replication timing in cell lines with skewed X inactivation showed no preference for one of the two Xist alleles to replicate early in S-phase before the onset of X inactivation, indicating that asynchronous replication timing does not play a role in skewing of X inactivation.

Analysis of replication timing at the FRA10B and FRA16B fragile site loci.

The molecular basis for the cytogenetic appearance of chromosomal fragile sites is not yet understood. Late replication and further delay of replication at fragile sites expressing alleles has been observed for FRAXA, FRAXE and FRA3B fragile site loci. We analysed the timing of replication at the FRA10B and FRA16B loci to determine whether late replication is a feature which is shared by all fragile sites and, therefore, is a necessary condition for chromosomal fragile site expression. The FRA10B locus was located in a transitional region between early and late zones of replication. Fragile and non-fragile alleles exhibit a similar replication pattern proximal to the repeat but fragile alleles are delayed relative to non-fragile ones on the distal side. Although fragility at FRA10B appears to be caused by expansion of an AT-rich repeat in the region, replication time near the repeat was similar in fragile and non-fragile alleles. The FRA16B locus was late replicating and appeared to replicate even later on fragile chromosomes. While these observations are compatible with the hypothesis that delayed replication may play a role in fragile site expression, they suggest that replication delay may not need to occur at the expanded repeat region itself in order to be permissive for fragility.

Replication Timing Control Can Be Maintained in Extrachromosomally Amplified Genes

Extrachromosomal elements are common early intermediates of gene amplification in vivo and in cell culture. The time at which several extrachromosomal elements replicate was compared with that of the corresponding amplified or unamplified chromosomal sequences. The replication timing analysis employed a retroactive synchrony method in which fluorescence-activated cell sorting was used to obtain cells at different stages of the cell cycle. Extrachromosomally amplified Syrian hamster CAD genes (CAD is an acronym for the single gene which encodes the trifunctional protein which catalyzes the first three steps of uridine biosynthesis) replicated in a narrow window of early S-phase which was approximately the same as that of chromosomally amplified CAD genes. Similarly, extrachromosomally amplified mouse adenosine deaminase genes replicated at a discrete time in early S-phase which approximated the replication time of the unamplified adenosine deaminase gene. In contrast, the multicopy extrachromosomal Epstein-Barr virus genome replicated within a narrow window in late S-phase in latently infected human Rajii cells. The data indicate that localization within a chromosome is not required for the maintenance of replication timing control.

Replication timing control can be maintained in extrachromosomally amplified genes.

Extrachromosomal elements are common early intermediates of gene amplification in vivo and in cell culture. The time at which several extrachromosomal elements replicate was compared with that of the corresponding amplified or unamplified chromosomal sequences. The replication timing analysis employed a retroactive synchrony method in which fluorescence-activated cell sorting was used to obtain cells at different stages of the cell cycle. Extrachromosomally amplified Syrian hamster CAD genes (CAD is an acronym for the single gene which encodes the trifunctional protein which catalyzes the first three steps of uridine biosynthesis) replicated in a narrow window of early S-phase which was approximately the same as that of chromosomally amplified CAD genes. Similarly, extrachromosomally amplified mouse adenosine deaminase genes replicated at a discrete time in early S-phase which approximated the replication time of the unamplified adenosine deaminase gene. In contrast, the multicopy extrachromosomal Epstein-Barr virus genome replicated within a narrow window in late S-phase in latently infected human Rajii cells. The data indicate that localization within a chromosome is not required for the maintenance of replication timing control.