Mutations in the basic domain and the loop–helix II junction of TWIST abolish DNA binding in Saethre–Chotzen syndrome

Abstract

Saethre^Chotzen syndrome is an autosomal dominant skull disorder resulting from premature fusion of coronal sutures (craniosynostosis). It is caused by mutations in the TWIST gene encoding a basic Helix^Loop^Helix transcription factor. Here we report on the identification of a novel mutation affecting a highly conserved residue of the basic domain. Unlike nonsense and missense mutations lying within helices, this mutation does not affect protein stability or heterodimerisation of TWIST with its partner E12. However, it does abolish TWIST binding capacity to a target E-box as efficiently as two missense mutations in the loop^helix II junction. By contrast, elongation of the loop through a 7 amino acid insertion appears not to hamper binding to the DNA target. We conclude that loss of TWIST protein function in Saethre^Chotzen patients can occur at three different levels, namely protein stability, dimerisation, and DNA binding and that the loop^helix II junction is essential for effective protein^DNA interaction. ß

Mechanism of Transcriptional Activation by the Proto-oncogene Twist1

“…The in vitro DNA binding of Twist1 has, on several occasions and for different E-boxes, been shown to require the presence of E-proteins (10,11,75), yet transactivation of FGFR2 and periostin and binding of Twist1 to the muscle creatine kinase E-box has recently been reported also in the absence of E-proteins (57). It remains to be established whether these observations reflect differences in the specific experimental settings or in the physiological state of the Twist1 protein.…”

Down-Regulation of Ubiquitin Ligase Cbl Induced by Twist Haploinsufficiency in Saethre-Chotzen Syndrome Results in Increased PI3K/Akt Signaling and Osteoblast Proliferation

“…10 The mutations studied cause Twist haploinsufficiency by inducing either degradation of truncated Twist protein (Y103X, Q109X) or loss of Twist DNA binding capacities (R118C). 6,7 Coronal sutures from patients and controls were fixed in 10% formaldehyde and embedded in paraffin, deparaffinized in xylene, and rehydrated through a graded series of ethanol. Sections were digested with 20 g/ml proteinase K for 15 minutes at 37°C.…”

“…4,5 Twist mutations in SCS cause Twist protein degradation, resulting in Twist haploinsufficiency, loss of dimerization with E proteins, and reduced binding to DNA canonical sequences in the promoter of target genes. 6,7 Despite the important implication of Twist mutations in craniosynostosis in SCS, our knowledge of the molecular mechanisms by which Twist alters the osteoblast phenotype in SCS remains incomplete. Our previous studies showed that Twist haploinsufficiency induced by deletion of the bHLH domain in SCS alters the osteoblast phenotype by affecting signaling molecules that control cell differentiation and apoptosis.…”

PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage

“…bHLH proteins form active dimers with E-box proteins and bind to a core sequence (CANNTG, referred to as E-box) in the regulatory elements of many lineage-specific genes in muscle, cartilage and osteogenic cells. Germ-line mutations of the Twist-1 gene that result in haploinsufficiency lead to the development of one of the most commonly inherited craniosynostosis conditions, the Saethre-Chotzen syndrome, which is characterized by premature fusion of cranial sutures and limb abnormalities (Gripp et al, 2000;Ghouzzi et al, 2001;Yang et al, 2004;Cai and Jabs, 2005). Expression of Twist-1 has also been implicated in the inhibition of differentiation of various cell lineages including osteoblasts and myoblasts (Spicer et al, 1996;Bialek et al, 2004;Hayashi et al, 2007).…”