During the first lineage segregation, a mammalian totipotent embryo differentiates into the inner cell mass (ICM) and trophectoderm (TE). However, how transcription factors (TFs) regulate this earliest cell-fate decision in vivo remains elusive, with their regulomes primarily inferred from cultured cells. Here, we investigated the TF regulomes during the first lineage specification in early mouse embryos, spanning the pre-initiation, initiation, commitment, and maintenance phases. Unexpectedly, we found that TFAP2C, a trophoblast regulator, bound and activated both early TE and inner cell mass (ICM) genes at the totipotent (two- to eight-cell) stages ('bipotency activation'). Tfap2c deficiency caused downregulation of early ICM genes, including Nanog, Nr5a2, and Tdgf1, and early TE genes, including Tfeb and Itgb5, in eight-cell embryos. Transcription defects in both ICM and TE lineages were also found in blastocysts, accompanied by increased apoptosis and reduced cell numbers in ICMs. Upon trophoblast commitment, TFAP2C left early ICM genes but acquired binding to late TE genes in blastocysts, where it co-bound with CDX2, and later to extra-embryonic ectoderm (ExE) genes, where it cooperatively co-occupied with the former ICM regulator SOX2. Finally, 'bipotency activation' in totipotent embryos also applied to a pluripotency regulator NR5A2, which similarly bound and activated both ICM and TE lineage genes at the eight-cell stage. These data reveal a unique transcription circuity of totipotency underpinned by highly adaptable lineage regulators.

Zygotic genome activation (ZGA) marks the beginning of the embryonic program for a totipotent embryo, which gives rise to the inner cell mass (ICM) where pluripotent epiblast arises, and extraembryonic trophectoderm. However, how ZGA is connected to the first lineage segregation in mammalian embryos remains elusive. Here, we investigated the role of nuclear receptor (NR) transcription factors (TFs), whose motifs are highly enriched and accessible from the 2-cell (2C) to 8-cell (8C) stages in mouse embryos. We found that NR5A2, an NR TF strongly induced upon ZGA, was required for this connection. Upon Nr5a2 knockdown or knockout, embryos developed beyond 2C normally with the zygotic genome largely activated. However, 4-8C-specific gene activation was substantially impaired and Nr5a2-deficient embryos subsequently arrested at the morula stage. Genome-wide chromatin binding analysis showed that NR5A2-bound cis-regulatory elements in both 2C and 8C embryos are strongly enriched for B1 elements where its binding motif is embedded. NR5A2 was not required for the global opening of its binding sites in 2C embryos but was essential to the opening of its 8C-specific binding sites. These 8C-specific, but not 2C-specific, binding sites are enriched near genes involved in blastocyst and stem cell regulation, and are often bound by master pluripotency TFs in blastocysts and embryonic stem cells (ESCs). Importantly, NR5A2 regulated key pluripotency genes Nanog and Pou5f1/Oct4, and primitive endoderm regulatory genes including Gata6 among many early ICM genes, as well as key trophectoderm regulatory genes including Tead4 and Gata3 at the 8C stage. By contrast, master pluripotency TFs NANOG, SOX2, and OCT4 targeted both early and late ICM genes in mouse ESCs. Taken together, these data identify NR5A2 as a key regulator in totipotent embryos that bridges ZGA to the first lineage segregation during mouse early development.

Somatic cell nuclear transfer (SCNT) can reprogram a somatic nucleus to a totipotent state. However, the re-organization of 3D chromatin structure in this process remains poorly understood. Using low-input Hi-C, we revealed that, during SCNT, the transferred nucleus first enters a mitotic-like state (premature chromatin condensation). Unlike fertilized embryos, SCNT embryos show stronger topologically associating domains (TADs) at the 1-cell stage. TADs become weaker at the 2-cell stage, followed by gradual consolidation. Compartments A/B are markedly weak in 1-cell SCNT embryos and become increasingly strengthened afterward. By the 8-cell stage, somatic chromatin architecture is largely reset to embryonic patterns. Unexpectedly, we found cohesin represses minor zygotic genome activation (ZGA) genes (2-cell-specific genes) in pluripotent and differentiated cells, and pre-depleting cohesin in donor cells facilitates minor ZGA and SCNT. These data reveal multi-step reprogramming of 3D chromatin architecture during SCNT and support dual roles of cohesin in TAD formation and minor ZGA repression.