A multi-enhancer RET regulatory code is disrupted in Hirschsprung disease

Area of Science:

• Genetics
• Developmental Biology
• Molecular Biology

Background:

• Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of enteric ganglia in the distal bowel.
• Major genetic risk factors involve polymorphisms in cis-regulatory elements (CREs) of the RET gene, crucial for enteric nervous system (ENS) development.
• These variants reduce RET expression by affecting transcription factor binding and dysregulating the RET-EDNRB gene regulatory network (GRN).

Purpose of the Study:

• To identify additional HSCR-associated risk variants within RET CREs that influence gene expression.
• To investigate the functional impact of these variants on RET enhancer activity and transcription factor binding.
• To understand the cumulative and synergistic effects of multiple variant-containing enhancers on RET gene expression.

Main Methods:

• Utilized siRNA, ChIP, and CRISPR-Cas9 deletion analyses in the SK-N-SH cell line.
• Identified and screened 22 HSCR-associated variants in candidate RET CREs.
• Assessed in vitro enhancer activity and transcription factor binding (PAX3) for variant-containing enhancers.

Main Results:

• Identified 22 HSCR-associated variants in candidate RET CREs.
• Seven variants demonstrated differential allele-specific in vitro enhancer activity.
• Four of these seven variants affected RET gene expression, with two being bound by PAX3.
• Deletion of multiple variant-containing enhancers showed synergistic effects on RET gene expression.

Conclusions:

• Common sequence variants in at least 10 RET enhancers contribute to HSCR risk.
• Seven of these enhancers experimentally show altered RET gene expression, extending the known RET-EDNRB GRN.
• This reveals an extensive regulatory code modulating HSCR risk through common sequence variants in RET enhancers.