Divergent 3D genome architecture of male germ cells across vertebrates

The evolutionary plasticity of the three-dimensional (3D) genome organization in vertebrates, and its transmission through the germ line, is central to understanding genome function and evolution. Yet, the mechanisms regulating these processes remain poorly characterized across lineages. Here, we integrate fluorescence-activated cell sorting, in situ chromosome capture conformation (Hi-C), and single-cell RNA sequencing to investigate germ line genome architecture in eutherians, marsupials, and reptiles, lineages that last shared a common ancestor ~350 million years ago. We uncover lineage-specific chromatin folding patterns in germ cells, shaped by chromosome morphology and genome size, which constrain DNA loop formation and inter-chromosomal interactions during meiosis. We also explore the relationship between 3D genome remodeling and gene regulation in the context of meiotic sex chromosome inactivation (MSCI). In the tammar wallaby, we identify regions of the X that escape MSCI checkpoint, suggesting incomplete silencing in marsupials. These findings provide high-resolution insights into the evolution of germ line chromatin architecture and the co-evolution of genome structure and function across vertebrates. The study reveals how the 3D genome changes during germ cell formation across vertebrates that split over 350 million years ago, uncovering shared and species‑specific patterns and showing that genome size and chromosome shape drive DNA folding.