Limb cell-fate commissioning is guided by widespread coordinated genome detachment from the nuclear lamina [Multiome]

Diverse forms of heterochromatin block inappropriate transcription and safeguard differentiation and cell identity. Yet, how and when heterochromatin is reconfigured to facilitate changes in cell-fate remains a key open question. Here, we address this by mapping a prevalent heterochromatic feature - genome-lamina interactions - relative to transcription in single-cells during mouse embryogenesis. We find that lamina-genome interactions remain relatively uniform following gastrulation but are extensively reconfigured during organogenesis in diverse tissues. Focusing on limb development, we demonstrate that genome-lamina interactions are selectively released at key developmental genes and their surrounding regulatory domains in early multipotent progenitors. Strikingly, this “lamina-release” often precedes later gene expression, suggesting it primes regulatory domains for future potential activation. Lamina-release also coincides with the putative binding of crucial limb transcription factors and so is closely intertwined with the regulatory machinery that drives limb formation. Conversely, we show that CTCF-defined boundaries of topologically-associated domains (TADs) constrains the spread of lamina-release at a limb gene locus. This ensures independent heterochromatin dynamics between neighbouring domains. Together, this suggests a previously unrecognised process where genome-lamina interactions are selectively dismantled at regulatory domains to transition loci toward more permissive chromatin states, thereby potentiating cell-type specific activation. Our work thus reveals how systematic heterochromatin reorganization links to developmental multipotency, providing mechanistic insight into how progenitors traverse diverse cell-fates in vivo.