Phosphoregulation of Twist1 provides a mechanism of cell fate control

Abstract

Basic Helix-loop-Helix (bHLH) factors play a significant role in both development and disease. bHLH factors function as protein dimers where two bHLH factors compose an active transcriptional complex. In various species, the bHLH factor Twist has been shown to play critical roles in diverse developmental systems such as mesoderm formation, neurogenesis, myogenesis, and neural crest cell migration and differentiation. Pathologically, Twist1 is a master regulator of epithelial-to-mesenchymal transition (EMT) and is causative of the autosomal-dominant human disease Saethre Chotzen Syndrome (SCS). Given the wide spectrum of Twist1 expression in the developing embryo and the diverse roles it plays within these forming tissues, the question of how Twist1 fills some of these specific roles has been largely unanswered. Recent work has shown that Twist's biological function can be regulated by its partner choice within a given cell. Our work has identified a phosphoregulatory circuit where phosphorylation of key residues within the bHLH domain alters partner affinities for Twist1; and more recently, we show that the DNA binding affinity of the complexes that do form is affected in a cis-element dependent manner. Such perturbations are complex as they not only affect direct transcriptional programs of Twist1, but they indirectly affect the transcriptional outcomes of any bHLH factor that can dimerize with Twist1. Thus, the resulting lineage-restricted cell fate defects are a combination of loss-of-function and gain-of-function events. Relating the observed phenotypes of defective Twist function with this complex regulatory mechanism will add insight into our understanding of the critical functions of this complex transcription factor.

Fig. (1)

Regulatory conservation of Twist-family bHLH factors . Top shows amino acid alignment of human TWIST1 with murine protein family members Twist2, Hand1 and 2, Paraxis and Scleraxis. The conservation of the phosphoregulated threonine (T) and serine (S) is noted by black shading. Conservation is maintained back to invertebrates [25]. Red-bolded residues shown in the human sequence identify specific point mutations found within SCS patients. Middle panels show a wildtype and Twist1 null embryo at time of death E11.5. Note the pronounced exencephaly (white arrowhead), hypoplastic limb buds (lb), and reduced lateral mesoderm (lm). Bottom shows the phosphoregulatory circuit that governs Twist-family dimer control and DNA binding. PKA is capable of phosphorylation Twist1 whereas only PP2A complexes containing B56δ can specifically dephosphorylate the helix I resides.

Fig. (2)

Model of Twist–family bHLH protein dimer regulation. Twist-family proteins have been shown to exhibit promiscuous dimerization characteristics that allow for multiple functional partners. In addition to expression levels of bHLH proteins within a cell as well as E-protein titration via Id factors, the phosphorylation state modulates Twist-family protein dimer affinities for its available partners thereby driving biological function. Expression of hypophosphorylation or phosphorylation mimic forms of the protein conveys distinct phenotypes in vivo. (Fig. (2) adapted from [24] PKA, PKC, and the Protein Phosphatase 2A Influence HAND Factor Function: A Mechanism for Tissue-Specific Transcriptional Regulation © 2003 with permission from Elsevier).

Fig. (3)

Gene balance model between Twist1 and Hand2 in the developing limb. Left shows genotypes that convey Twist1 haploinsufficiency resulting in polydactyly where as genotypes to the right convey normal limb development. Of note, point mutations that disrupt phosphorylation (Twist1T125;S127A: TW1AA) of Twist1 result in phenotypes indistinguishable from a genetic imbalance with Hand2. Below is an E17.5 day transgenic mouse embryo expressing Hand2 via the Prx1-limb-specific promoter. Obvious is right forepaw polydactyly with left forepaw showing normal digit formation. Given that Prx1-expression via this promoter fragment is not asymmetric [31], this example shows the critical balance of Twist-Hand2 gene dosage as subtle differences in expression between left and right limbs within the same animal can result in different phenotypes.