Alterations in chromatin architecture promote totipotent-like features in a DUX-dependent manner

2-cell like cells (2CLC) are a transiently cycling population of cells with totipotent-associated features. Interestingly, CTCF depletion leads to 2CLC conversion in mouse ESC. However, the mechanism underlying this reprogramming or whether totipotent-like conversion depends on chromatin structural alterations or to CTCF-specific effects is unclear. We demonstrate that 2CLC conversion mediated by CTCF loss depends on the transcription factor DUX and correlates with nucleolar integrity. We show that depletion of the cohesin release factor WAPL in ESC also promotes 2CLC reprogramming which is increased by CTCF co-depletion. Single-cell RNAseq/ATACseq analyses in CTCF/WAPL-depleted ESC revealed that chromatin accessibility precedes 2C-associated gene expression. Finally, although distinct transcriptional changes were associated to WAPL or CTCF depletion in human ESC, we did not detect any similarity in their transcriptome with those of early stages of human embryo development suggesting a lack of conservation. Our work revealed an intertwined link between higher-order chromatin organization and totipotency in mice.