Synthetic genomic reconstitution reveals principles of mammalianHoxcluster regulation

Precise Hox gene expression is crucial for embryonic patterning. Intra- Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modes controlling 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 HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130-170 kilobases each) to an ectopic location in the mouse genome. We find that a HoxA cluster lacking distal enhancers recapitulates correct patterns of chromatin remodeling and transcription in response to patterning signals, while distal enhancers are required for full transcriptional output. Synthetic regulatory reconstitution is a generalizable strategy to decipher the regulatory logic of gene expression in complex genomes. One-Sentence Summary Reconstitution of gene regulation using large DNA constructs unravels the regulatory logic of a developmental gene locus.