An ENU-induced mutation in Twist1 transactivation domain causes hindlimb polydactyly with complete penetrance and dominant-negatively impairs E2A-dependent transcription

Abstract

Twist1 encodes a basic helix-loop-helix transcription factor (TF), which forms homodimer or heterodimer with other TFs, like E2A, to regulate target genes' expression. Mutations in TWIST1 are associated with Saethre-Chotzen syndrome (SCS), a rare congenital disorder characterized with osteogenesis abnormalities. However, how dysfunction of TWIST1 leads to SCS is still largely unknown. Here, using an unbiased ENU-induced mutagenesis screening, we identified a novel Twist1 mutation and the mutant mouse phenocopies some features of SCS in a dominant manner. Physically, our mutation p.F191S lies at the edge of a predicted α-helix in Twist1 transactivation (TA) domain. Adjacent to F191, a consecutive three-residue (AFS) has been hit by 3 human and 2 mouse disease-associated mutations, including ours. Unlike previously reported mouse null and p.S192P alleles that lead to hindlimb polydactyly with incomplete penetrance but a severe craniofacial malformation, our p.F191S causes the polydactyly (84.2% bilateral and 15.8% unilateral) with complete penetrance but a mild craniofacial malformation. Consistent with the higher penetrance, p.F191S has stronger impairment on E2A-dependent transcription than p.S192P. Although human p.A186T and mouse p.S192P disease mutations are adjacent to ours, these three mutations function differently to impair the E2A-dependent transcription. Unlike p.A186T and p.S192S that disturb local protein conformation and unstabilize the mutant proteins, p.F191S keeps the mutant protein stable and its interaction with E2A entire. Therefore, we argue that p.F191S we identified acts in a dominant-negative manner to impair E2Adependent transcription and to cause the biological consequences. In addition, the mutant mouse we provided here could be an additional and valuable model for better understanding the disease mechanisms underlying SCS caused by TWIST1 dysfunction.The function of TWIST1 in osteogenesis has been reflected by the identification of the TWIST1 mutations in Saethre-Chotzen syndrome (SCS), a rare congenital disorder often associated with cone-shaped head, asymmetrical face, hand and foot malformation, and even mental retardation 1-3 . Further studies demonstrated that Twist1 is also involved in epithelial-mesenchymal transition and cell migration during embryonic development and contributes to the invasion of carcinoma cells and tumor metastasis 4-7 .Twist1 encodes a basic helix-loop-helix (bHLH) transcription factor and Twist1 DNA-binding capability essential for its functions has been intensively studied previously [8][9][10][11][12][13] . Twist1 either functions as homodimer or DiscussionIdentification of a novel Twist1 mutation responsible for hindlimb polydactyly. In ENU-induced mutagenesis, mutations that generate stop codon (nonsense), disrupt splicing site, or change amino acid (missense) are most likely disease-causing 22,23 . Among our 160 mutations identified by our exome-sequencing, no stop-gain and splicing site mutation was found. Instead, we identified 16 nonsynonymous mutati...

A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration

“…For ENU-induced mutagenesis, the NMF291 −/+ males were i.p. injected with ENU (80–110 mg/kg Body Weight (B. W.)) for three consecutive weeks, as previously reported ( Salinger and Justice, 2008 ; Chen et al, 2020 ). After an infertility test, the ENU-treated males were crossed to untreated NMF291 −/+ females for G 1 .…”

A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration

“…For ENU-induced mutagenesis, the NMF291 -/+ males were i.p. injected with ENU (80-110 mg/kg B. W.) for three consecutive weeks, as previously reported (83,84). After an infertility test, the ENU-treated males were crossed to untreated NMF291 -/+ females for G1.…”

TCF7L2 acts as a molecular switch in midbrain to control mammal vocalization through a transcriptional repression mechanism

“…Male C57BL/6J mice were treated with ENU (80-110 mg/kg B. W.) for three consecutive weeks by intraperitoneal injection at the age of 12 weeks according previously reported (Chen et al, 2020). The ENU-treated males were crossed with C57BL/6J females and the resulting G1 pups were subjected to USV measurement at P5 with maternal deprivation in a 5-minute interval by a commercial USV detector (Med Associates Inc).…”

“…The mutant candidates were selected by previously described criteria (Chen et al, 2020). ΔEx5', Lysates were centrifuged at 12000 x g for 10 minutes at 4°C.…”