5‐Hydroxymethylcytosine Dynamics Reveals Coordinated Reprogramming of Parental Genomes and X Chromosome Dosage Balance in Mouse SCNT Embryos

Abstract Somatic cell nuclear transfer (SCNT) embryos exhibit widespread epigenetic defects, particularly aberrant DNA methylation. DNA 5‐hydroxymethylcytosine (5hmC) is involved in methylation reprogramming during early embryonic development, yet its role in SCNT embryos remains largely unknown. Here, the genome‐wide 5hmC landscapes in mouse SCNT embryos are systematically profiled with parental allele specificity. It is revealed that both maternal and paternal genomes of donor somatic cells acquire a transient, sperm‐like but attenuated and allele symmetric distribution of 5hmC at the 2‐cell stage, distinct from the parental asymmetric pattern observed in naturally fertilized eggs. This is characterized by insufficient DNA hydroxymethylation of the X chromosome in female SCNT embryos, as well as resistance to 5hmC‐associated DNA demethylation at germline imprinting control regions (gICRs). While de novo 5hmC generation is closely associated with initial DNA demethylation during somatic‐to‐zygotic transition, it later becomes uncoupled from ongoing methylation changes. Importantly, global elevation of 5hmC via Tet3 overexpression leads to premature activation of developmental genes at the 2‐cell stage and severely impairs SCNT embryo development. These findings reveal unique dynamics and functional consequences of abnormal 5hmC remodeling in SCNT embryos, highlighting the precise regulation of 5hmC generation as a key epigenetic event for successful mammalian cloning.