Regulation And Function Of The Three-dimensional Chromatin Structure During The Pluripotent To Totipotent State Transition In Embryonic Stem Cells

After fertilization,epigenetically different parental genomes are reprogrammed toward totipotency and zygote genome activation(ZGA)is initiated.In mice,ZGA peaks at the 2-cell(2C)stage and is characterized by the activation of transcripts of 2-cell specific genes such as the Zscan4 cluster and the endogenous retrovirus MERVL.After the ZGA event,the differentiation potential of cells is gradually restricted.The molecular characteristics of totipotency were once thought to be unique to zygotes and the earliest cleavage embryos.Mouse embryonic stem cells(ESCs)are derived from the inner cell mass of the blastocyst stage,and are considered as pluripotent cells because they have the ability to develop into all three germ cells,but they rarely contribute to the formation of extraembryonic tissues.However,a very small percentage(0.2%-5%)of mouse ESCs cultured in vitro have totipotency,or expanded pluripotency.They have the ability to differentiate into both embryonic tissues and extra-embryonic tissues.Such cells are known as 2C-like cells(2CLCs).The discoveries of 2CLC provide a favorable experimental tool for studying totipotency outside of early embryos and the molecular regulation of ZGA.In order to explore the role of three-dimensional(3D)genome architecture in regulating cell potential,we used the pluripotency-to-totipotency transition in mouse ESCs as a model and defined the role of relaxed 3D genome conformation in establishing totipotency.By using low-input technologies,we presented genome-wide chromatin contact maps(Hi-C)of totipotent 2CLCs and pluripotent ESCs,together with their transcriptome(RNA-seq),chromatin accessibility(ATAC-seq),and histone modification profiles(ChIP-seq).This rich dataset has allowed us to investigate the dynamics of chromatin state and gene expression during the pluripotent to totipotent state transition.We revealed that chromatin architecture of ESCs became more relaxed during the spontaneous 2C-like transition,with globally weakened chromatin loops.This included not only topologically associated domain(TAD)boundary loops but also chromatin loops between ESC enhancers particularly super-enhancers and their neighboring gene promoters.The loss of the two types of chromatin loops leads to a decrease in the insulation of the TAD boundaries and the activity of the ESC-specific enhancers/super enhancers,which results in the transcriptional down-regulation of pluripotent genes(such as Oct4,Sox2,and Nanog).We also observed formation of 2C enhancers during ESC to 2CLC transition.Up-regulated genes(such as Nelfa)during the 2C-like transition tend to have ESC TAD boundaries separating them from neighboring 2C enhancers.Therefore,disruption of TAD boundary loops in ESCs may facilitate the contacts between 2C enhancers that are likely sporadically activated in ESCs and the promoters of nearby 2C genes,thus promoting their expression,which in turn fully activates the 2C-like transcriptional program.We further demonstrated a causal relationship between relaxed chromatin architecture and totipotency acquisition.The results showed that disruption of the chromatin architecture regulator CTCF or cohesin in ESCs to disrupt the two types of chromatin loops could facilitate 2C-like transition.In summary,the three-dimensional chromatin structure plays a key role in regulating cell potential.