Single-cell dynamics of genome-nucleolus interactions captured by nucleolar laser microdissection (NoLMseq).

Gene positioning in nuclear space is central to regulation, with repressive chromatin domains often contacting the nuclear lamina or nucleoli. The nucleolus undergoes structural changes in different cellular states, potentially altering genome organization. Yet, how nucleolar states influence 3D-genome architecture remains underexplored, largely due to the lack of methods able to map nucleolar-associated domains (NADs) in single cells and stressed nucleoli. We developed NoLMseq, a technique combining laser-capture microdissection and DNA sequencing to map NADs in single cells. NoLMseq uncovered unexplored features of chromosome organization around nucleoli, including NAD heterogeneity in mouse embryonic stem cells, yielding two major populations with distinct chromatin and developmental states. NADs predominantly contact nucleoli monoallelically, with contact frequency correlating with gene expression and chromatin states. Under nucleolar stress, NoLMseq revealed extensive chromosome reorganization, highlighting the importance of nucleolus integrity in genome organization. Thus, NoLMseq provides a critical tool to study 3D-genome responses to nucleolar states in health and disease.