Figure 2. Relationship between cell culture models of mouse embryogenesis based on replication timing profiles.

Figure 2

Relationship between cell culture models of mouse embryogenesis based on replication timing profiles. Hierarchical clustering of 22 mouse cell lines based on replication timing profiles obtained by microarrays.8 The dendrogram reveals an epigenetic separation of cell types representing the late epiblast (EpiSCs) from the early epiblast (EPL and EBM3) as well as the ICM [ESCs (46C, D3, TT2) and fully reprogrammed iPSCs]. EpiSCs were more related to committed germ layer cell types of the early embryo [ectoderm (EBM6), neurectoderm (46CNPC, TT2NPC and EBM9), nascent mesoderm and endoderm]. Three partially reprogrammed iPSC (piPSC) lines were distinct from late embryonic cell types (MEFs and myoblasts), but were also distinct from ICM, epiblast or early germ layer cell types, forming an independent branch. The asterisk on the right indicates genomic segments that complete lineage-independent EtoL changes by the post-implantation epiblast stage (which roughly corresponds to 155 Mb total). Late replication of these segments was stably maintained in all downstream lineages and not reversed in piPSCs, which also exhibited difficulty in transcriptional reprogramming of genes within these segments. Figure was adapted from Hiratani et al with permission from Genome Research.8

Methods: Cells were pulse-labeled with BrdU, separated into early and late S-phase fractions by flow cytometry and BrdU-substituted DNA from each fraction was immunoprecipitated with an anti-BrdU antibody. The early and late replicating DNA samples were differentially labeled and cohybridized to whole-genome oligonucleotide microarrays. The ratio of the abundance of each probe in the early and late fraction ["replication timing ratio" = log2(Early/Late)] was then used to generate a replication timing profile for the entire genome at a density of one probe every 5.8 kb. Then, the whole genome was divided into 10,974 ~200-kb segments and their average replication timing ratios were compared between cell lines by hierarchical clustering. The heatmap shows the replication-timing ratios [= Log2(Early/Late)] of 10,974 ~200-kb segments, with red and green representing early and late replication, respectively. Segments framed in blue shows those with significant differential between any cell types, which represent 45% of the genome.

From: Autosomal Lyonization of Replication Domains During Early Mammalian Development

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