Published on Wed Aug 11 2021

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

Hirsch, N., Dahan, I., Dhaene, E., Avni, M., Vergult, S., Vidal Garcia, M., Magini, P., Graziano, C., Severi, G., Bonora, E., Nardone, A. M., Brancati, F., Fernandez-Jaen, A., Olson, R. J., Hallgrimsson, B., Birnbaum, R. Y.

Structural variants (SVs) can affect protein-coding sequences as well as gene regulatory elements. 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.

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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 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 (eTw5-7{Delta}/{Delta}) or Ctcf site (Ctcf{Delta}/{Delta}) 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.