HDAC9 structural variants disrupting TWIST1 transcriptional regulation lead to craniofacial and limb malformations

• Published in: bioRxiv
• Main Authors: Hirsch, Naama, Dahan, Idit, Dhaene, Eva, Avni, Matan, Vergult, Sarah, Marta Vidal Garcia, Magini, Pamela, Graziano, Claudio, Severi, Giulia, Bonora, Elena, Nardone, Anna Maria, Brancati, Francesco, Fernandez-Jaen, Alberto, Olson, Rory J, Hallgrimsson, Benedikt, Birnbaum, Ramon Y
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 11.08.2021
• Subjects: Amino acid sequence; Chromatin; Cranial sutures; Craniosynostosis; Enhancers; Gene regulation; Genotype & phenotype; Hedgehog protein; Histone deacetylase; Limb malformations; Phenotypes; Polydactyly; Proteins; Regulatory sequences; Transcription
• DOI: 10.1101/2021.08.10.455254

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 HDAC9 sequence. Based on SVs within the HDAC9-TWIST1 locus, we defined the 3' HDAC9 sequence (~500Kb) as a critical TWIST1 regulatory region, encompassing craniofacial TWIST1 enhancers and CTCF sites. Deletions of either Twist1 enhancers (eTw5-7Δ/Δ) or Ctcf site (CtcfΔ/Δ) within the Hdac9 protein-coding sequence in mice 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 region 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 elucidated essential components of TWIST1 transcriptional machinery that reside within the HDAC9 sequence, suggesting that SVs, encompassing protein-coding sequence, such as HDAC9, 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, such as TWIST1. Competing Interest Statement The authors have declared no competing interest.