The ZBTB24-CDCA7-HELLS axis suppresses the totipotent 2C-like reprogramming by maintaining Dux methylation and repression

Two-cell-like cells (2CLCs), a rare population (~0.5%) in murine embryonic stem cell (mESC) cultures, are in a transient totipotent-like state resembling that of 2C-stage embryos, and their discovery and characterization have greatly facilitated the study of early developmental events, such as zygotic genome activation. However, the molecular determinants governing 2C-like reprogramming remains to be elucidated. Here, we show that ZBTB24, CDCA7 and HELLS, components of a molecular pathway that is involved in the pathogenesis of immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, function as negative regulators of 2C-like reprogramming by maintaining DNA methylation of the Dux cluster, a master inducer of the 2C-like state. Disruption of the ZBTB24-CDCA7-HELLS axis results in Dux hypomethylation and derepression, leading to dramatic upregulation of 2C-specific genes, which can be reversed by site-specific re-methylation in the Dux promoter. We also provide evidence that CDCA7 is enriched at the Dux cluster and recruits the CDCA7-HELLS chromatin remodeling complex to constitutive heterochromatin. Our study uncovers a key role for the ZBTB24-CDCA7-HELLS axis in safeguarding the mESC state by suppressing the 2C-like reprogramming. To assess the impact of the deficiency of Zbtb24, Cdca7 or Hells on H3K9me3 occupancy at Dux cluster and 2C-specific genes, we conducted CUT&Tag sequencing (CUT&Tag-Seq) with H3K9me3 antibody (ab8898, abcam) in WT, two to three independent Zbtb24-, Cdca7-, or Hells-KO mESC lines.To assess the impact of the deficiency of Zbtb24, Cdca7 or Hells on H3K9me3 occupancy at Dux cluster and 2C-specific genes, we conducted CUT&Tag sequencing (CUT&Tag-Seq) with H3K9me3 antibody (ab8898, abcam) in WT, two to three independent Zbtb24-, Cdca7-, or Hells-KO mESC lines.