RT Journal
A1 Hirsch, Naama
A1 Dahan, Idit
A1 D'haene, Eva
A1 Avni, Matan
A1 Vergult, Sarah
A1 Vidal-García, Marta
A1 Magini, Pamela
A1 Graziano, Claudio
A1 Severi, Giulia
A1 Bonora, Elena
A1 Nardone, Anna Maria
A1 Brancati, Francesco
A1 Fernández-Jaén, Alberto
A1 Rory, Olson J.
A1 Hallgrímsson, Benedikt
A1 Birnbaum, Ramon Y.
T1 HDAC9 structural variants disrupting TWIST1 transcriptional regulation lead to craniofacial and limb malformations
JF Genome Research 
JO Genome Research 
YR 2022 
FD July 01 
VO 32 
IS 7 
SP 1242 
OP 1253 
DO 10.1101/gr.276196.121 
UL http://genome.cshlp.org/content/32/7/1242.abstract 
AB 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.