Reorganization of the DNA replication landscape during adipogenesis is closely linked with adipogenic gene expression.

The temporal order of DNA replication along the chromosomes is thought to reflect the transcriptional competence of the genome. During differentiation of mouse 3T3-L1 cells into adipocytes, cells undergo one or two rounds of cell division called mitotic clonal expansion (MCE). MCE is an essential step for adipogenesis; however, little is known about the regulation of DNA replication during this period. Here, we performed genome-wide mapping of replication timing (RT) in mouse 3T3-L1 cells before and during MCE, and identified a number of chromosomal regions shifting toward either earlier or later replication through two rounds of replication. These RT changes were confirmed in individual cells by single-cell DNA-replication sequencing. Coordinate changes between a shift toward earlier replication and transcriptional activation of adipogenesis-associated genes were observed. RT changes occurred before the full expression of these genes, indicating that RT reorganization might contribute to the mature adipocyte phenotype. To support this, cells undergoing two rounds of DNA replication during MCE had a higher potential to differentiate into lipid droplet-accumulating adipocytes, compared with cells undergoing a single round of DNA replication and non-replicating cells.

scRepli-Seq: A Powerful Tool to Study Replication Timing and Genome Instability.

Advances in "omics" technology have made it possible to study a wide range of cellular phenomena at the single-cell level. Recently, we developed single-cell DNA replication sequencing (scRepli-seq) that measures replication timing (RT) by copy number differences between replicated and unreplicated genomic DNA in replicating single mammalian cells. This method has been used to reveal previously unrecognized static and dynamic natures of several hundred kilobases to a few megabases-scale chromosomal units called RT domains. Because RT domains are highly correlated to A/B compartments detected by Hi-C, scRepli-seq data can be used to predict the 3D organization of the genome in the nuclear space. scRepli-seq, which essentially measures the copy number, can also be applied to study genome instability.