Range extender mediates long-distance enhancer activity.

Area of Science:

• Developmental Biology
• Genetics
• Molecular Biology

Background:

• Mammalian transcriptional enhancers typically regulate gene promoters over tens of kilobases.
• Some enhancers, however, function over megabase distances, but the underlying sequence features are unknown.

Purpose of the Study:

• To identify sequence features enabling long-distance enhancer-promoter interactions.
• To investigate the mechanism of long-range gene regulation in mammalian development.

Main Methods:

• In vivo enhancer-replacement experiments at the mouse Shh locus.
• Identification and characterization of the range extender (REX) element.
• Mutation analysis of conserved motifs within the REX element and the ZRS enhancer.
• Generation of knock-in mice to assess limb development.

Main Results:

• Short- and medium-range limb enhancers cannot activate gene expression at long distances.
• A novel cis-acting element, REX, confers long-distance regulatory activity.
• Addition of REX to other enhancers increased their interaction range by up to an order of magnitude.
• Conserved [C/T]AATTA homeodomain motifs within REX are critical for its activity and are enriched in long-range limb enhancers.
• Mutation of these motifs in the ZRS enhancer abolished long-range activity, causing severe limb reduction.

Conclusions:

• The REX element is necessary and sufficient to confer long-distance activation by remote enhancers.
• A specific sequence signature, including [C/T]AATTA motifs, is associated with long-range enhancer-promoter interactions.
• This discovery provides critical insights into the mechanisms of long-range gene regulation during mammalian development.