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

## TL;DR

This study shows that mouse embryos created through cloning have unusual patterns of a DNA modification called 5hmC, which affects proper development and gene activation.

## Contribution

The study reveals novel insights into 5hmC dynamics and its role in epigenetic reprogramming during mouse SCNT embryogenesis.

## Key findings

- SCNT embryos show a transient, symmetric 5hmC pattern distinct from natural embryos.
- X chromosomes and imprinting control regions resist proper 5hmC remodeling in SCNT embryos.
- Elevated 5hmC leads to premature gene activation and impaired embryonic development.

## 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.

Genome‐wide, high‐resolution profiling of hydroxymethylation in mouse SCNT embryos reveals a transient, allele‐symmetric 5hmC reprogramming pattern distinct from natural embryos, with X‐chromosomes and imprinting control regions resistant to proper remodeling. Ectopic generation of 5hmC prematurely activates developmental genes and impairs embryogenesis, highlighting the necessity of both precise levels and correct distribution of hydroxymethylation for successful mammalian cloning.