The N-terminus of Stag1 is required to repress the 2C program by maintaining rRNA expression and nucleolar integrity.

Our understanding of how STAG proteins contribute to cell identity and disease have largely been studied from the perspective of chromosome topology and protein-coding gene expression. Here, we show that STAG1 is the dominant paralog in mouse embryonic stem cells (mESCs) and is required for pluripotency. mESCs express a wide diversity of naturally occurring Stag1 isoforms, resulting in complex regulation of both the levels of STAG paralogs and the proportion of their unique terminal ends. Skewing the balance of these isoforms impacts cell identity. We define a novel role for STAG1, in particular its N-terminus, in regulating repeat expression, nucleolar integrity, and repression of the two-cell (2C) state to maintain mESC identity. Our results move beyond protein-coding gene regulation via chromatin loops to new roles for STAG1 in nucleolar structure and function, and offer fresh perspectives on how STAG proteins, known to be cancer targets, contribute to cell identity and disease.