Autosomal dominant mutations in the gene encoding the basic helix-loop-helix transcription factor Twist1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome. The molecular mechanism underlying these phenotypes is poorly understood. We show that ectopic expression of the related basic helix-loop-helix factor Hand2 phenocopies Twist1 loss of function in the limb and that the two factors have a gene dosage–dependent antagonistic interaction. Dimerization partner choice by Twist1 and Hand2 can be modulated by protein kinase A– and protein phosphatase 2A–regulated phosphorylation of conserved helix I residues. Notably, multiple Twist1 mutations associated with Saethre-Chotzen syndrome alter protein kinase A–mediated phosphorylation of Twist1, suggesting that misregulation of Twist1 dimerization through either stoichiometric or post-translational mechanisms underlies phenotypes of individuals with Saethre-Chotzen syndrome.

Members of the Twist-family of bHLH proteins play a pivotal role in a number of essential developmental programs. Twist-family bHLH proteins function by dimerizing with other bHLH members and binding to cis- regulatory elements, called E-boxes. While Twist-family members may simply exhibit a preference in terms of high-affinity binding partners, a complex, multilevel cascade of regulation creates a dynamic role for these bHLH proteins. We summarize in this review information on each Twist-family member concerning expression pattern, function, regulation, downstream targets, and interactions with other bHLH proteins. Additionally, we focus on the phospho-regulatory mechanisms that tightly control posttranslational modification of Twist-family member bHLH proteins.

The basic Helix‐Loop‐Helix (bHLH) transcription factors Hand1 and Hand2 play critical roles in the development of multiple organ systems during embryogenesis. The dynamic expression patterns of these two factors within developing tissues obfuscate their respective unique and redundant organogenic functions. To define cell lineages potentially dependent upon Hand gene expression, we generated a mutant allele in which the coding region of Hand1 is replaced by Cre recombinase. Subsequent Cre‐mediated activation of β‐galactosidase or eYFP reporter alleles enabled lineage trace analyses that clearly define the fate of Hand1‐expressing cells. Hand1‐driven Cre marks specific lineages within the extra embryonic tissues, placenta, sympathetic nervous system, limbs, jaw, and several cell types within the cardiovascular system. Comparisons between Hand1 expression and Hand1‐lineage greatly refine our understanding of its dynamic spatial‐temporal expression domains and raise the possibility of novel Hand1 functions in structures not thought to be Hand1‐dependent. Developmental Dynamics 239:3086–3097, 2010.

The interactions of diverse transcription factors mediate the molecular programs that regulate mammalian heart development. Among these, Nkx2.5 and the Mef2c regulate common downstream targets and exhibit striking phenotypic similarities when disrupted, suggesting a potential interaction during heart development. Co‐immunoprecipitation and mammalian two‐hybrid experiments revealed a direct molecular interaction between Nkx2.5 and Mef2c. Assessment of mRNA expression verified spatiotemporal cardiac coexpression. Finally, genetic interaction studies employing histological and molecular analyses showed that, although Nkx2.5^−/− and Mef2c^−/− individual mutants both have identifiable ventricles, Nkx2.5^−/−;Mef2c^−/− double mutants do not, and that mutant cardiomyocytes express only atrial and second heart field markers. Molecular marker and cell death and proliferation analyses provide evidence that ventricular hypoplasia is the result of defective ventricular cell differentiation. Collectively, these data support a hypothesis where physical, functional, and genetic interactions between Nkx2.5 and Mef2c are necessary for ventricle formation. Developmental Dynamics 237:3809–3819, 2008.

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.