Developmentally regulated Shh expression is robust to TAD perturbations.

Area of Science:

• Genomics
• Developmental Biology
• Epigenetics

Background:

• Topologically associating domains (TADs) are crucial for regulating gene expression by organizing chromatin.
• The Sonic hedgehog (Shh) gene is located within a TAD containing all its enhancers, making it a model for studying TAD function.

Purpose of the Study:

• To investigate the role of chromatin conformation and TAD integrity in developmental gene regulation.
• To determine the impact of Shh TAD perturbations on Shh expression and organismal development.

Main Methods:

• Genetic manipulation of the Shh TAD, including internal deletions and boundary alterations.
• Chromosome conformation capture (3C) assays to analyze chromatin structure.
• Fluorescence in situ hybridization (FISH) to assess gene expression patterns.

Main Results:

• Substantial alterations to the Shh TAD structure did not affect Shh expression levels or patterns during development, except when enhancers were deleted.
• Most TAD perturbations did not result in detectable phenotypes.
• Ectopic enhancer activity from the Shh limb enhancer was observed on the Mnx1 gene in a neighboring TAD only after a large deletion at one TAD boundary.

Conclusions:

• Developmental gene regulation, specifically for Shh, is remarkably robust to significant perturbations in TAD structure.
• TADs may play a less critical role in constraining enhancer activity than previously assumed, particularly for genes with established regulatory elements within their domain.
• While TAD integrity is important, developmental gene regulation exhibits resilience to structural changes, highlighting complex regulatory networks.