Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities

Abstract

Autosomal dominant mutations in the bHLH transcription factor TWIST1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome (SCS). The molecular mechanism underlying these phenotypes is poorly understood. We show that the ectopic expression of the related bHLH factor Hand2 phenocopies Twist1 loss-of-function phenotypes in the limb, and that they display a gene dosage-dependent antagonistic interaction. Twist1 and Hand2 dimerization partner choice can be modulated by PKA and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Interestingly, multiple TWIST1 mutations associated with SCS alter PKA-mediated Twist1 phosphorylation, suggesting that misregulation of Twist1 dimerization via either stoichiometric or posttranslational mechanisms underlies SCS phenotypes.Studies of developing vertebrate limbs have yielded many insights into the process of embryonic pattern formation. Prominent among these are the identification of a growing catalog of transcription factors that orchestrate limb patterning. While the genetic and biochemical interactions of these transcription factors are clearly important for integrating patterning information, these interactions are poorly understood. Twist1 and Hand2 are basic helix-loop-helix (bHLH) transcription factors within the Twist family, and are attractive candidates for investigating such interactions. Each is required for distinct yet subtly related aspects of limb development, and biochemical studies have revealed a complex regulation of their protein-protein interactions 1-3 .Early limb bud expression of Twist1 is observed primarily in the peripheral mesenchyme, and Twist1 is required for maintenance of the overlying apical ectodermal ridge (AER) 4-7 . Twist1 haploinsufficiency in mice and humans is associated with a range of limb abnormalities. Twist1 heterozygous null mice display a partially penetrant preaxial polydactyly 8,9 . Human Correspondence should be addressed to A.B.F. tfirulli@iupui.edu (317) 278-5814 and E.L. elaufer@columbia.edu (212) Here we investigate the biochemical and genetic interactions between Twist1 and Hand2 both in vitro and during limb development. We show that PKA and B56δ-containing PP2A can regulate Twist1 and Hand2 phosphorylation at the conserved helix I residues, that hypophosphorylation and phosphorylation mimics of these residues alter bHLH dimerization affinities, and that a population of TWIST1 mutations that cause SCS in humans exhibit disregulation of this phosphoregulatory circuit. We also show that ectopic Hand2 expression phenocopies multiple SCS-like limb phenotypes, that the appropriate genetic dosage of Hand2 and Twist1 is critical for proper limb development, and that these interactions require the phosphoregulated helix I residues. These findings support a mechanism where the Twist family dimerization partner choices are modulated by both the relative levels of gene expression and the phosphorylation state of key helix I residues, thereby dictating changes i...

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

“…Protein kinase A phosphorylates two conserved PKB-mediated Twist-1 phosphorylation A Vichalkovski et al residues within the HLH domain of both Twist-1 and Hand2 (T125/S127 and T112/S114, respectively, in mice and T121/S123 and T112/S114 in human) bringing about their dimerization, which is necessary for the regulation of target genes during limb development. A group of Twist-1 mutations identified in patients with Saethre-Chotzen syndrome was reported to disrupt protein kinase A-mediated phosphorylation, emphasizing the importance of Twist-1 in development (Firulli et al, 2005). In contrast to the S42A mutation, most mutations within the bHLH domain of Twist-1 negatively affect its transcriptional repressor function (Sosic et al, 2003).…”

Hand1 phosphoregulation within the distal arch neural crest is essential for craniofacial morphogenesis

“…7). Twist1 lossof-function mutations are associated with craniofacial defects during embryogenesis (Chen and Behringer, 1995) and the disease SCS (Barnes and Firulli, 2009;Firulli et al, 2005;Jabs, 2004). At E10.5, Twist1 is robustly expressed within the NCC of the medial and lateral nasal processes (lnp), the maxillary process (mp), as well as the first (I) and second (II) PAs (Fig.…”

“…Compelling evidence shows that Twist family bHLH factors mediate biological function by dimer choice (Castanon et al, 2001;Firulli et al, 2003Firulli et al, , 2005Firulli et al, , 2007. Dimer choice is regulated, in part, by a threonine and serine pair that is evolutionarily conserved among all Twist family members.…”

“…Indeed, changes in bHLH dimer choices affect craniofacial development (Connerney et al, 2006). Dysregulation of Twist1 phosphorylation at these threonine and serine residues causes SCS (Cai and Jabs, 2005;Firulli et al, 2005). Dimerization of Twistfamily bHLH factors is governed by their co-expression within a cell, their colocalization within the nucleus, their level of relative expression and the phosphorylation of the conserved threonine and serine residues within Helix I of the bHLH domain Firulli et al, 2003Firulli et al, , 2005Firulli et al, , 2007.…”

Expression analysis of limb element markers during mouse embryonic development

“…Here, we show nerve innervation patterns throughout limb outgrowth. Similarly, cartilage and bone analysis is usually assessed in mouse limb studies at specific time points to study specific gene function and action (for example, Amarilio et al, 2007; Collinson et al, 2018; Firulli et al, 2005; Nowlan et al, 2010; Ray et al, 2015; Tavella et al, 2004). Here, we show in detail the progression of cartilage and bone development throughout fore‐ and hindlimb patterning.…”