HDAC9 structural variants disrupting TWIST1 transcriptional regulation lead to craniofacial and limb malformations
- Naama Hirsch1,2,
- Idit Dahan1,2,
- Eva D'haene3,
- Matan Avni1,2,
- Sarah Vergult3,
- Marta Vidal-García4,
- Pamela Magini5,
- Claudio Graziano5,
- Giulia Severi5,
- Elena Bonora5,6,
- Anna Maria Nardone7,
- Francesco Brancati8,9,
- Alberto Fernández-Jaén10,
- Olson J. Rory11,
- Benedikt Hallgrímsson4 and
- Ramon Y. Birnbaum1,2
- 1Department of Life Sciences, Faculty of Natural Sciences, The Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel;
- 2Center of Evolutionary Genomics and Medicine, The Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel;
- 3Center for Medical Genetics, Ghent University, 9000, Ghent, Belgium;
- 4Department of Cell Biology and Anatomy, Alberta Children's Hospital Research Institute, University of Calgary, T2N 1N4, Calgary, Alberta, Canada;
- 5U.O. Genetica Medica, IRCCS Azienda Ospedaliero-Universitaria di Bologna, 40138, Bologna, Italy;
- 6Department of Medical and Surgical Sciences, University of Bologna, 40126, Bologna, Italy;
- 7Medical Genetics Unit, Policlinico Tor Vergata University Hospital, 00133, Rome, Italy;
- 8Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100, L'Aquila, Italy;
- 9Human Functional Genomics Laboratory, San Raffaele Pisana, 00167, Rome, Italy;
- 10Department of Pediatrics and Neurology, Hospital Universitario Quirónsalud, School of Medicine, Universidad Europea de Madrid, 28223, Madrid, Spain;
- 11Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA
Abstract
Structural variants (SVs) can affect protein-coding sequences as well as gene regulatory elements. However, SVs disrupting protein-coding sequences that also function as cis-regulatory elements remain largely uncharacterized. Here, we show that craniosynostosis patients with SVs containing the histone deacetylase 9 (HDAC9) protein-coding sequence are associated with disruption of TWIST1 regulatory elements that reside within the HDAC9 sequence. Based on SVs within the HDAC9‐TWIST1 locus, we defined the 3′-HDAC9 sequence as a critical TWIST1 regulatory region, encompassing craniofacial TWIST1 enhancers and CTCF sites. Deletions of either Twist1 enhancers (eTw5-7Δ/Δ) or CTCF site (CTCF-5Δ/Δ) within the Hdac9 protein-coding sequence led to decreased Twist1 expression and altered anterior/posterior limb expression patterns of SHH pathway genes. This decreased Twist1 expression results in a smaller sized and asymmetric skull and polydactyly that resembles Twist1+/− mouse phenotype. Chromatin conformation analysis revealed that the Twist1 promoter interacts with Hdac9 sequences that encompass Twist1 enhancers and a CTCF site, and that interactions depended on the presence of both regulatory regions. Finally, a large inversion of the entire Hdac9 sequence (Hdac9INV/+) in mice that does not disrupt Hdac9 expression but repositions Twist1 regulatory elements showed decreased Twist1 expression and led to a craniosynostosis-like phenotype and polydactyly. Thus, our study elucidates essential components of TWIST1 transcriptional machinery that reside within the HDAC9 sequence. It suggests that SVs encompassing protein-coding sequences could lead to a phenotype that is not attributed to its protein function but rather to a disruption of the transcriptional regulation of a nearby 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.276196.121.
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Freely available online through the Genome Research Open Access option.
- Received September 12, 2021.
- Accepted June 2, 2022.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
