In this study, we describe two mechanisms by which the activities of Twist1 and Hand2 can be regulated, either by changes in their relative levels of expression or through phosphorylation. Specific threonine and serine residues in helix I shared by Twist1 and Hand2 are conserved among a majority of Twist family members. We find that these residues can be post-translationally modified via the actions of PKA and B56δ containing PP2A, and provide evidence that dimerization affinity can be altered by such modifications. We also find that ectopic Hand2 expression phenocopies limb phenotypes resulting from mutations in Twist1, including those presented in patients with SCS. We furthermore demonstrate a gene dose-dependent interaction between Twist1 and Hand2 in both combination gain-of-function and combination loss-of-function experiments and show that a mutation that alters Twist1 dimerization and phosphorylation in vitro also alters its genetic interactions with Hand2 in vivo. These data strongly support models in which Twist family bHLH protein dimerization partner choice is crucial for normal development.
Early models of bHLH function posited that competition between transcriptionally active E proteins and transcriptionally inactive HLH proteins for dimerization with tissue specific bHLH proteins was the primary mechanism regulating the formation of bHLH complexes18,29
. However, it is now clear that regulation by both phosphorylation and interactions with a wider spectrum of dimerization partners are also significant additions to this model. We previously showed that PKA and B56δ-containing PP2A regulate Hand1 dimerization affinities for other bHLH proteins through phosphorylation of helix I (ref. 20
). The current demonstration that Twist1 and Hand2 are similarly modified suggests that other Twist-family proteins that share the phosphorylated helix I residues also share this regulatory mechanism. The embryological significance of the Twist1 post-translational modifications is reflected in the phenotypes observed in SCS patients12
, in C. elegans
models and in the data presented here () in which Twist1 point mutations that disrupt the PKA/PP2A circuit alter the developmental activity of Twist1 (ref. 24
). These observations provide a potential mechanistic explanation for several mutations found in SCS patients. These include five basic domain mutations and mutation of the conserved phosphorylation-regulated helix I serine10,12,24
The use of phosphorylation to regulate Twist family dimerization may extend beyond interactions with the Twist family and E proteins. For example Twist1 can bind the myogenic factor MyoD, and negatively regulate its activity30,31
. Interestingly, a Twist1 R120A;R122A;R124A triple mutation was reported to disrupt interactions between MyoD and Twist1 (ref. 30
). Our results show that mutating these residues individually also disrupts PKA-mediated phosphorylation of Twist1 helix I (). These data are consistent with the idea that the MyoD and Twist1 interaction may be regulated in part by phosphorylation of Twist1.
Although the Twist1 basic domain mutations can affect the ability of PKA to phosphorylate the helix I residues, it is possible that the developmental effects may result solely from changes in DNA binding specificity. We feel this is unlikely for several reasons. First, Twist carrying several of the SCS mutations tested here remains capable of DNA binding as C. elegans
. Nevertheless, DNA binding is not required for all Twist1 activity, as a Twist1 mutant with a non-DNA binding basic domain can still inhibit transcriptional activation by MyoD and Mef2 (ref. 31
). Similarly, Hand2 that lacks the basic domain remains competent to induce polydactyly when misexpressed in mouse limbs32
. Thus multiple lines of evidence point toward Twist1 having DNA binding-independent activities that may be influenced by properties of its basic domain.
While proper Twist1
gene dosage has long been appreciated as critical for normal development, the molecular basis for this is less clear8,9,11,12
. Our results indicate that at least in the limb, reduced Twist1
or increased Hand2
dosage disrupts a critical antagonistic balance between Twist1
and Hand2. Hand2
are expressed in the limb in overlapping domains, and these proteins can form heterodimers with each other, form homodimers, or form heterodimers with other partners. Changing the Twist1:Hand2
dosage probably alters the relative amounts of the various possible protein combinations, leading to changes in target gene expression and consequent developmental defects. While we currently do not know which complexes are most critical, there is precedent for specific Twist dimer complexes differentially regulating transcription of target genes21
and thus each unique dimer likely exhibits unique biological activities. Studies in Drosophila using forced dimers show that Twist homodimers specify mesoderm and the somatic myogenic lineage while Twist-daughterless heterodimers repress the transcription of genes required for somatic myogenesis21
. A similar experimental strategy may help define the functions of the various Twist1 and Hand2 dimer combinations in the limb.
Some of the ectopic Hand2
limb phenotypes reported here may reflect a normal developmental mechanism where high levels of Hand2 are required to antagonize Twist1 activity. The AER disruption associated with Hand2 misexpression is one example. AER disruption can be observed adjacently to ectopic outgrowths that correspond to sites of Hand2 virus infection, and thus presumably is due to high levels of Hand2 expression (, data not shown). As Twist1 is normally required in the mesenchyme for the function of an FGF signaling loop between mesenchyme and ectoderm that maintains the AER4,5,33
, the Hand2
virus phenotype may be caused by interference with this Twist1 activity. Interestingly, Hand2
expression is normally highest in the posterior limb, adjacent to ectoderm just proximal of the AER, and from which the AER has regressed in concert with distal limb outgrowth. Thus, one normal function of high level Hand2
expression may be to limit the posterior-proximal extent of the AER.
Bialek et al.34
recently showed that a novel carboxy-terminal ‘Twist domain’ conserved in Twist1 and Twist2 binds Runx proteins and inhibits Runx transcriptional activity. As Runx2 promotes ossification of cartilaginous and membranous bone, Twist proteins thereby act to delay the onset of ossification. It is proposed that in SCS patients, either mutation of the Runx binding domain or reducing Twist1 levels generates excess free Runx2 protein that promotes premature ossification, resulting in the craniofacial and skeletal defects of SCS. How Hand2 activity fits into this paradigm is unclear. Hand2 levels in the limb may modulate the availability of Twist1 for interaction with Runx proteins. Or, as Runx mutants with polydactyly have not been described35–38
, interactions between Hand2 and Twist1 in the early limb may operate independently of Runx pathways. Whether changes in Hand2
gene dosage can alter osteogenic SCS phenotypes is an interesting question. Similarly, the Twist1 double alanine mutation tested here does not suppress the Hand2-induced polydactyly phenotype and also does not cause gross cartilage defects. Whether the loss of this latter activity is due to defective Hand2 interactions or reflects a requirement for these residues for broader aspects of Twist1 functionality remains to be determined. These and future studies will expand our understanding of how combinatorial interactions among broadly expressed transcription factors drive highly context-specific developmental programs.