Proper telomere length is essential for indefinite self-renewal of embryonic stem (ES) cells and cancer cells. Telomerase-deficient late generation mouse ES cells and human ALT cancer cells are able to propagate for numerous passages, suggesting telomerase-independent mechanisms responding for telomere maintenance. However, the underlying mechanisms ensuring the telomere length maintenance are unclear. Here, using late generation telomerase KO (G4 Terc^-/-) ESCs as a model, we show that Zscan4, highly upregulated in G4 Terc^-/- ESCs, is responsible for the prolonged culture of these cells with stably short telomeres. Mechanistically, G4 Terc^-/- ESCs showed reduced levels of DNA methylation and H3K9me3 at Zscan4 promoter and subtelomeres, which relieved the expression of Zscan4. Similarly, human ZSCAN4 was also derepressed by reduced H3K9me3 at its promoter in ALT U2 OS cells, and depletion of ZSCAN4 significantly shortened telomeres. Our results define a similar conserved pathway contributing to the telomere maintenance in telomerase-deficient late generation mESCs and human ALT U2OS cancer cells.

CDCA7 , encoding a protein with a carboxyl-terminal cysteine-rich domain (CRD), is mutated in immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a disease related to hypomethylation of juxtacentromeric satellite DNA. How CDCA7 directs DNA methylation to juxtacentromeric regions is unknown. Here, we show that the CDCA7 CRD adopts a unique zinc-binding structure that recognizes a CpG dyad in a non-B DNA formed by two sequence motifs. CDCA7, but not ICF mutants, preferentially binds the non-B DNA with strand-specific CpG hemi-methylation. The unmethylated sequence motif is highly enriched at centromeres of human chromosomes, whereas the methylated motif is distributed throughout the genome. At S phase, CDCA7, but not ICF mutants, is concentrated in constitutive heterochromatin foci, and the formation of such foci can be inhibited by exogenous hemi-methylated non-B DNA bound by the CRD. Binding of the non-B DNA formed in juxtacentromeric regions during DNA replication provides a mechanism by which CDCA7 controls the specificity of DNA methylation.

Zygotic genome activation occurs in two-cell (2C) embryos, and a 2C-like state is also activated in sporadic (~1%) naïve embryonic stem cells in mice. Elevated chromatin accessibility is critical for the 2C-like state to occur, yet the underlying molecular mechanisms remain elusive. Zscan4 exhibits burst expression in 2C embryos and 2C-like cells. Here, we show that Zscan4 mediates chromatin remodeling to promote the chromatin accessibility for achieving the 2C-like state. Through coimmunoprecipitation/mass spectrometry, we identified that Zscan4 interacts with the corepressors Kap1/Trim28, Lsd1, and Hdac1, also with H3K9me3 modifiers Suv39h1/2, to transiently form a repressive chromatin complex. Then, Zscan4 mediates the degradation of these chromatin repressors by recruiting Trim25 as an E3 ligase, enabling the ubiquitination of Lsd1, Hdac1, and Suv39h1/2. Degradation of the chromatin repressors promotes the chromatin accessibility for activation of the 2C-like state. These findings reveal the molecular insights into the roles of Zscan4 in promoting full activation of the 2C-like state.

Two-cell-like cells (2CLCs), a rare population (∼0.5%) in mouse 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 remain 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.