Synthetic regulatory reconstitution reveals principles of mammalian <i>Hox</i> cluster regulation

Abstract

Precise <i>Hox</i> gene expression is crucial for embryonic patterning. Intra-<i>Hox</i> transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling <i>Hox</i> gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the <i>HoxA</i> cluster. We synthesized and delivered variant rat <i>HoxA</i> clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal <i>HoxA</i> cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.