Single-Molecule Analysis Reveals Changes in the DNA Replication Program for the POU5F1 Locus upon Human Embryonic Stem Cell Differentiation

Abstract

Human embryonic stem cells (hESCs), due to their pluripotent nature, represent a particularly relevant model system to study the relationship between the replication program and differentiation state. Here, we define the basic properties of the replication program in hESCs and compare them to the programs of hESC-derived multipotent cells (neural rosette cells) and primary differentiated cells (microvascular endothelial cells [MECs]). We characterized three genomic loci: two pluripotency regulatory genes, POU5F1 (OCT4) and NANOG, and the IGH locus, a locus that is transcriptionally active specifically in B-lineage cells. We applied a high-resolution approach to capture images of individual replicated DNA molecules. We demonstrate that for the loci studied, several basic properties of replication, including the average speed of replication forks and the average density of initiation sites, were conserved among the cells analyzed. We also demonstrate, for the first time, the presence of initiation zones in hESCs. However, significant differences were evident in other aspects of replication for the DNA segment containing the POU5F1 gene. Specifically, the locations of centers of initiation zones and the direction of replication fork progression through the POU5F1 gene were conserved in two independent hESC lines but were different in hESC-derived multipotent cells and MECs. Thus, our data identify features of the replication program characteristic of hESCs and define specific changes in replication during hESC differentiation.Studies during the past few years suggest variability among different lines of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) with regard to differentiation and lineage specification (42). Thus, inconsistencies in the quality and purity of undifferentiated and differentiated cell populations from different passages are a serious concern for the development of translational applications in human disease (35). Current approaches to characterize the pluripotent behavior of hESCs are primarily limited to assays such as marker expression, in vitro differentiation, and in vivo teratoma formation. Therefore, it is critical for the field to develop additional methods for identifying characteristics that define the pluripotent state, particularly ones that could detect incompletely reprogrammed hiPSCs. One very important and defining epigenetic characteristic of ESCs is their DNA replication program.The DNA replication program specifies the sites along the DNA molecule at which replication initiates and when in the S phase these sites are activated. When tissue-specific gene loci are compared in different cell types, there are often differences in DNA replication timing, replication initiation sites, and the direction of replication fork progression (14,24,26,27,40). The replication program is implicated in many cellular functions, such as genome reprogramming, epigenetic modifications, gene expression, and development (reviewed in reference 20). In fact, small dif...

Global analysis reveals multiple pathways for unique regulation of mRNA decay in induced pluripotent stem cells

“…The vast majority of histone mRNA detected in an asynchronous cell population is therefore produced and degraded within S phase. We found that many histone mRNAs were stabilized in iPS cells, but cell cycle differences do not adequately account for the increase in histone mRNA stability, as S phase is of similar length in both cell types (Becker et al 2006;Schultz et al 2010). Overall abundance of histone mRNAs and proteins was also increased in iPS cells as compared with HFFs.…”

Regulation of DNA replication during development

“…In multiple ESC lines, the pattern of origin usage and replication fork progression is similar within the POU5F1 locus. By contrast, analysis of a differentiated cell line as well as a multipotent neuronal rosette cell line derived from one of the ESC lines used in this study revealed a change in origin usage and directionality of fork progression within the POU5F1 region (Schultz et al, 2010).…”

“…SMARD analysis of the replication program of the POU5F1 locus in human cells also revealed a change in both origin firing and replication fork progression as a function of differentiation (Schultz et al, 2010). In multiple ESC lines, the pattern of origin usage and replication fork progression is similar within the POU5F1 locus.…”

Genome-scale identification of active DNA replication origins

“…In addition, DNA combing is also limited by the DNA length that can be analyzed as it is hard to get molecules longer than 600-800 Kb. A related method called SMARD has been developed and applied to the Igh locus after its purification by pulsed field gel electrophoresis [11], and recently to Nanog and Oct4 gene environment [12].…”