Chromatin folding motifs define the basic grammar of TAD architecture in single alleles

Chromatin structure varies dramatically among single cells, yet the specific motifs underpinning this organization remain poorly understood. Using imaging-based spatial genomics, we visualized 3D chromatin organization across seven mouse tissues. We demonstrate that single chromatin traces can be decomposed into loop and domain Chromatin Folding Motifs (CFMs). Polymer simulations revealed that loop-extrusion reproduces loop CFMs, while cis-regulatory interactions are primarily responsible for domain CFMs. Notably, a unique set of CFMs describes single-cell chromatin architecture across multiple cell types, underscoring the universality of CFMs in organizing chromatin. In the pancreas, cells with different transcriptional programs exhibited distinct CFM usage, highlighting the functional relevance of CFMs. Perturbation of transcription and CTCF levels in a type 2 diabetes model altered CFM usage, demonstrating that disease induction affects both architectural and cis-regulatory interactions. Our results uncover how chromatin is organized in single cells and reveal the alterations that occur during disease.