Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation [Hi-C]

Precise Hox gene expression is crucial for embryonic patterning. Local transcription factor binding and distal enhancer elements have emerged as the major regulatory modes controlling the initiation and maintenance of Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce ‘synthetic regulatory reconstitution’, a novel conceptual framework for studying gene regulation and apply it to the mammalian HoxA cluster. To measure the HoxA intrinsic regulatory potential and distal enhancer contributions, we de novo synthesized and delivered variant rat HoxA clusters (130-170 kilobases each) to an ectopic location in the mouse genome. We find that a minimal HoxA cluster lacking distal enhancers induces the appropriate gene set and establishes distinct chromatin domains in response to patterning signals, while distal enhancers are required for full transcriptional output. These results suggest that the genomically compact Hox clusters contain all the information to decode patterning signals and maintain positional information Hi-C was used to investigate topological organization in cells carrying synthetic Hox cluster variants during ESC-to-MN differentiation