The ZBTB24-CDCA7-HELLS axis suppresses the totipotent 2C-like cell 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 investigate the inhibitory effects of ICF-related genes on 2C-like reprogramming, we generated stable cell lines with Dnmt3b-, Zbtb24-, Cdca7- and Hells-knockouts by CRISPR-Cas9 technology. RNA-seq was conducted on WT, Dnmt3b-KO, Zbtb24-KO, Cdca7-KO and Hells-KO cells to assess the transcriptional profile. To explore whether the facilitation of 2C-like reprogramming in the deficiency of ZBTB24-CDCA7-HELLS axis is Dux dependent, we employed CRISPR-Cas9 technology to knockout Dux cluster in Zbtb24-KO, Cdca7-KO and Hells-KO cells individually to generate Zbtb24/Dux double knockout (DKO), Cdca7/Dux-DKO and Hells/Dux-DKO cell lines. We performed gene expression profiling analysis using data obtained from RNA-seq of WT cells, single-KO and double-knockout cells. Additionally, we treated WT mESCs with DMSO control or 2 μM DNMT1 protein degrader GSK-3484862 for three days for inducing global DNA demethylation.