A multi-enhancer RET regulatory code is disrupted in Hirschsprung disease
- Sumantra Chatterjee1,2,
- Kameko M. Karasaki3,
- Lauren E. Fries1,
- Ashish Kapoor4 and
- Aravinda Chakravarti1,2
- 1Center for Human Genetics and Genomics, New York University Grossman School of Medicine, New York, New York 10016, USA;
- 2Department of Neuroscience and Physiology, New York University Grossman School of Medicine, New York, New York 10016, USA;
- 3Department of Biology, Johns Hopkins University, Baltimore, Maryland 21205, USA;
- 4Institute of Molecular Medicine, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA
Abstract
The major genetic risk factors for Hirschsprung disease (HSCR) are three common polymorphisms within cis-regulatory elements (CREs) of the receptor tyrosine kinase gene RET, which reduce its expression during enteric nervous system (ENS) development. These risk variants attenuate binding of the transcription factors RARB, GATA2, and SOX10 to their cognate CREs, reduce RET gene expression, and dysregulate other ENS and HSCR genes in the RET–EDNRB gene regulatory network (GRN). Here, we use siRNA, ChIP, and CRISPR-Cas9 deletion analyses in the SK-N-SH cell line to ask how many additional HSCR-associated risk variants reside in RET CREs that affect its gene expression. We identify 22 HSCR-associated variants in candidate RET CREs, of which seven have differential allele-specific in vitro enhancer activity, and four of these seven affect RET gene expression; of these, two enhancers are bound by the transcription factor PAX3. We also show that deleting multiple variant-containing enhancers leads to synergistic effects on RET gene expression. These, coupled with our prior results, show that common sequence variants in at least 10 RET enhancers affect HSCR risk, seven with experimental evidence of affecting RET gene expression, extending the known RET–EDNRB GRN to reveal an extensive regulatory code modulating disease risk at a single gene.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.275667.121.
- Received April 20, 2021.
- Accepted October 5, 2021.
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