Mutations within Helix I of Twist1 Result in Distinct Limb Defects and Variation of DNA Binding Affinities

Abstract

Twist1 is a basic helix-loop-helix (bHLH) factor that plays an important role in limb development. Haploinsufficiency of Twist1 results in polydactyly via the inability of Twist1 to antagonistically regulate the related factor Hand2. The mechanism modulating Twist1-Hand2 antagonism is via phosphoregulation of conserved threonine and serine residues in helix I of the bHLH domain. Phosphoregulation alters the dimerization affinities for both proteins. Here we show that the expression of Twist1 and Twist1 phosphoregulation mutants results in distinct limb phenotypes in mice. In addition to dimer regulation, Twist1 phosphoregulation affects the DNA binding affinities of Twist1 in a partner-dependent and cis-element-dependent manner. In order to gain a better understanding of the specific Twist1 transcriptional complexes that function during limb morphogensis, we employ a series of Twist1-tethered dimers that include the known Twist1 partners, E12 and Hand2, as well as a tethered Twist1 homodimer. We show that these dimers behave in a manner similar to monomerically expressed bHLH factors and result in distinct limb phenotypes that correlate well with those observed from the limb expression of Twist1 and Twist1 phosphoregulation mutants. Taken together, this study shows that the Twist1 dimer affinity for a given partner can modulate the DNA binding affinity and that Twist1 dimer choice determines phenotypic outcome during limb development.Members of the Twist family of bHLH 2 proteins are evolutionarily conserved transcription factors that play fundamental roles in the normal development of a number of tissues, including extraembryonic membranes, teeth, jaw, heart, and limbs (1-3). Although bHLH/helix-loop-helix proteins can be organized into five groups, bHLH factors can be generally classified into two groups: the ubiquitously expressed E-proteins (class A) and the tissue-specific/restricted bHLH factors (class B) (for a review, see Ref. 4). The classic model for bHLH function is that a heterodimer containing a member from class A and a member from class B interacts via the amphipathic ␣-helices. This interaction juxtaposes the basic domains of both proteins, such that a DNA binding domain is formed that recognizes a DNA cis-element termed an E-box (CANNTG) (4). The requirement for class A/class B heterodimerization for many bHLH factors is well established, and in the case of the skeletal myogenic bHLH factors (MyoD1, Myogenin, Myf5, and Mrf4), heterodimerization is critical for proper biological function (4, 5). Other class B factors, such as members of the Twist family of bHLH proteins, not only function as heterodimers with E-proteins but can also form homodimers and heterodimers with other class B bHLH proteins (1-3, 6). It is thought that the control of bHLH dimerization is a mechanism that could control transcriptional response.Recently, we have shown that members of the Twist family of bHLH proteins contain an evolutionarily conserved threonine and serine within helix 1 that can be phosphoregulated by the a...

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

“…The complexity of regulating the bHLH pool lies in determining all combinations of Hand bHLH dimer complexes within the extremely narrow temporal window when this dysfunction is occurring. Additionally, we have to consider that altering the Twistfamily dimer regulation also affects DNA-binding affinities (Firulli et al, 2007). Therefore, it is also possible that Hand1 mutant proteins could target promoters inappropriately and/or facilitate other bHLH factors to do likewise, thus further effecting gene expression.…”

“…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.…”

“…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. Overexpression studies alter the stoichiometry of this bHLH pool.…”

GSK3 Temporally Regulates Neurogenin 2 Proneural Activity in the Neocortex

“…7F ). A similar approach was used previously to dissect apart the roles of Twist homodimers and heterodimers in Drosophila mesoderm (Castanon et al, 2001) and mouse limb development (Neuhold and Wold, 1993;Firulli et al, 2007). In protein degradation assays, Neurog2ϳNeurog2 homodimers had half-lives of Ͻ30 min (Fig.…”

TWIST1 and TWIST2 regulate glycogen storage and inflammatory genes in skeletal muscle

“…Thus overexpression and ablation have similar effects, which may initially appear contradictory. However, as TWIST is a bHLH transcription factor, overexpression could have similar effects to silencing where homodimers have opposing effects to those mediated by heterodimers (Firulli et al 2007, Connerney et al 2008. Thus, the effects of TWIST proteins on cytokine expression appear to have a U-shaped curve with both extremes triggering a pro-inflammatory response.…”