Investigating the effect of Daxx deletion on gene and repeat expression in embryonic stem cells and their differentiated neural derivatives

Genomes comprise a large fraction of repetitive sequences folded into constitutive heterochromatin to protect genome integrity and cell identity. De novo formation of heterochromatin during preimplantation development is essential to preserve the ground-state of pluripotency and the self-renewal capacity of embryonic stem cells (ESCs). In this study we find that DAXX, an H3.3 chaperone, is essential for ESCs maintenance in the ground-state of pluripotency. DAXX accumulates at pericentromeric regions, and recruits PML and SETDB1, thereby promoting heterochromatin formation. In the absence of DAXX, the 3D-architecture and physical properties of pericentric and peripheral heterochromatin are disrupted, resulting in derepression of major satellite DNA, transposable elements and genes associated with the nuclear lamina. Our data reveal that DAXX is crucial for the maintenance and 3D-organization of the heterochromatin compartment and protects ESCs viability. Overall design: To investigate the function of DAXX we generated a transgenic mouse ESC line lacking a functional copy of the Daxx gene (Daxx-/-). We cultured wildtype parental (WT; E14Tg2a) and Daxx-/- ESCs in three conditions: 1). Serum + LIF (ESCs), 2). 2i + Vitamin C (ground state pluripotency) and 3). Retinoic Acid -LIF (Neural differentiated). We isolated total RNA from each of these samples across three independent replicates and, following rRNA depletion, performed RNA-seq on each sample. We performed comparative gene and repeat expression analysis between WT and Daxx-/- cells cultured under each of these three conditions.