Long-Lived Binding of Sox2 to DNA Predicts Cell Fate in the Four-Cell Mouse Embryo

Abstract

Transcription factor (TF) binding to DNA is fundamental for gene regulation. However, it remains unknown how the dynamics of TF-DNA interactions change during cell-fate determination in vivo. Here, we use photo-activatable FCS to quantify TF-DNA binding in single cells of developing mouse embryos. In blastocysts, the TFs Oct4 and Sox2, which control pluripotency, bind DNA more stably in pluripotent than in extraembryonic cells. By contrast, in the four-cell embryo, Sox2 engages in more long-lived interactions than does Oct4. Sox2 long-lived binding varies between blastomeres and is regulated by H3R26 methylation. Live-cell tracking demonstrates that those blastomeres with more long-lived binding contribute more pluripotent progeny, and reducing H3R26 methylation decreases long-lived binding, Sox2 target expression, and pluripotent cell numbers. Therefore, Sox2-DNA binding predicts mammalian cell fate as early as the four-cell stage. More generally, we reveal the dynamic repartitioning of TFs between DNA sites driven by physiological epigenetic changes. VIDEO ABSTRACT.

Coactivators and general transcription factors have two distinct dynamic populations dependent on transcription

“…FCS is particularly useful for the analysis of diffusion properties of very mobile factors and can be used to gain more detailed information than those obtained by photobleaching approaches. The single‐molecule sensitivity and the microsecond to a few seconds time resolution of FCS have for example allowed the dynamic repartitioning of DNA‐binding factors driven by physiological epigenetic changes, provided quantitative insights into chromatin‐binding dynamics of the Polycomb complex or participated to characterizing protein network dynamics in live cells (Steffen et al , 2013; Wachsmuth et al , 2015; White et al , 2016). Fluorescence intensity correlation analyses describe several physicochemical parameters of labelled factors, such as the total number of diffusing molecules ( N tot ), their apparent diffusion coefficient ( D ), and their apparent molecular weight (MW A ).…”

Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis

“…For example, recent experiments have shown that there is variable intercellular expression of proteins as early as the four-cell stage of a mouse embryo, even though the separation of fates into primitive endoderm, epiblast and trophectoderm takes place at the 16-to 32-cell transition (Goolam et al, 2016;White et al, 2016b). Furthermore, by measuring the binding dynamics of transcription factors to DNA, it was shown that Sox2, a transcription factor controlling pluripotency, exhibits much longer binding to DNA in specific blastomeres of the four-cell embryo; these blastomeres with longer-lived Sox2-DNA complexes later contributed to the pluripotent cells of the inner cell mass.…”

What have single-molecule studies taught us about gene expression?

“…For example, it has been observed that even before distinct lineages have been determined, Sox2 binding dynamics differ between four-cell blastocytes, and those cells with more long-lived Sox2 binding contribute more pluripotent progeny downstream (White et al 2016). Single-molecule tracking of fluorescently tagged p53 and glucocorticoid receptor (GR) show that, at transcriptionally active domains, TFs bind transiently (<1 sec dwell time on DNA), with only a small fraction (<8%) engaged in productive binding (∼5 sec dwell time on DNA) (Morisaki et al 2014).…”