Analysis of chromatin structure in mouse preimplantation embryos by fluorescent recovery after photobleaching

Abstract

Zygotes are totipotent cells that have the ability to differentiate into all cell types. It is believed that this ability is lost gradually and differentiation occurs along with the progression of preimplantation development. Here, we hypothesized that the loose chromatin structure is involved in the totipotency of one-cell stage embryos and that the change from loose to tight chromatin structure is associated with the loss of totipotency. To address this hypothesis, we investigated the mobility of eGFP-tagged histone H2B (eGFP-H2B), which is an index for the looseness of chromatin, during preimplantation development based on fluorescent recovery after photobleaching (FRAP) analysis. The highest mobility of eGFP-H2B was observed in pronuclei in 1-cell stage embryos and mobility gradually decreased during preimplantation development. The decrease in mobility between the 1- and 2-cell stages depended on DNA synthesis in 2-cell stage embryos. In nuclear transferred embryos, chromatin in the pseudopronuclei loosened to a level comparable to the pronuclei in 1-cell stage embryos. These results indicated that the mobility of eGFP-H2B is negatively correlated with the degree of differentiation of preimplantation embryos. Therefore, we suggest that highly loosened chromatin is involved in totipotency of 1-cell embryos and the loss of looseness is associated with differentiation during preimplantation development.

Minor zygotic gene activation is essential for mouse preimplantation development

“…Chromatin structure plays an important role in regulating transcription and it has been suggested that a loosened chromatin structure is involved in the promiscuous transcription that occurs during minor ZGA (5,25). We demonstrated by fluorescence recovery after photobleaching (FRAP) analysis that chromatin structure is extremely loosened in one-cell embryos and that it become tighter during the two-cell stage (26). FRAP analysis using an eGFP-tagged core histone estimates the mobility of core histones that reflects the looseness of chromatin structure (27,28).…”

“…1B), Pol I-mediated transcription would not be efficiently inhibited in our experimental conditions. Chromatin structure becomes "tighter" during MZT (26,39). The lack of a requirement for an enhancer-enhancers function by overcoming chromatin-mediated transcriptional repressionfor efficient expression of plasmid-borne reporter genes and promiscuous genome-wide transcription during minor ZGA likely reflect a loosened chromatin structure (5,25,40).…”

“…The lack of a requirement for an enhancer-enhancers function by overcoming chromatin-mediated transcriptional repressionfor efficient expression of plasmid-borne reporter genes and promiscuous genome-wide transcription during minor ZGA likely reflect a loosened chromatin structure (5,25,40). Reciprocally, the requirement for an enhancer for efficient expression of plasmid-borne reporter genes and a decrease in intergenic transcription during major ZGA presumably reflects chromatin structure becoming "tighter" (5,25,26,40). Indeed, the absence of minor ZGA caused the persistence of loosened state of chromatin structure at the two-cell stage (Fig.…”

“…FRAP. FRAP analysis was conducted as described previously with slight modification (26). Briefly, after photobleaching images were taken at 5-s intervals for 60 s. The mobile fraction (MF), an index of the rate of fluorescence recovery, was calculated using the following equation (38)(39)(40): MF = (F end − F post )/(F pre − F post ), where F end is the relative intensity of fluorescence at the endpoint, F post is immediately after photo-bleaching, and F pre is before photo-bleaching.…”

Specification of the First Mammalian Cell Lineages In Vivo and In Vitro

“…Chromatin structure is very relaxed after fertilization in both the mouse and human zygote (Wu et al 2018) and then slowly compacts throughout preimplantation development (Du et al 2017). The relaxed chromatin state is supported by the high mobility of histone proteins, which gradually decreases throughout preimplantation development, in line with the idea that increasing condensation of chromatin is associated with differentiation during development (Boskovic et al 2014;Ooga et al 2016).…”