Anthony B. Firulli, PhD

The biological question that holds my research interest is gaining an understanding of the transcriptional mechanisms that control the cell specification and differentiation of multi-potential cells. When I was a graduate student, the myogenic basic loop-helix (bHLH) transcription factors were identified and shown to program fibroblast cell lines such as 10T1/2 to the skeletal muscle lineage. The finding that a single family of transcription factors could define cell identity led me to look for novel transcription factors that played similar roles in the specification of tissues such as the heart. These efforts resulted in my study of the Twist class of bHLH factors Hand1, Hand2, and Twist1. Unlike the myogenic bHLH transcription factors, Twist-family bHLH factors are expressed in a wide range of tissues including heart, cardiac neural crest, lateral mesoderm, the developing sympathetic nervous system and other mesenchymal cell populations during development as well as in pathologies such as cancer. In our study of the biological properties of Twist-family proteins, we discovered that unlike the myogenic bHLH factors, these bHLH factors exhibit promiscuous dimerization characteristics such that Hand1, Hand2 and Twist1 can form homo and hetero dimers in addition to heterodimers with E-proteins. This expanded dimerization profile sets up a model whereby changes in the biological function of these factors is dependent on the various bHLH proteins expressed within a given cell and the mechanisms that dictate dimer partner choice. If in fact Twist-family biological function is regulated by dimer partner choice then an obvious question: How is dimer choice regulated? Must be asked. Our first insights into the mechanisms that control Twist-family dimerization choices came from our discovery of a phosphoregulatory circuit composed of the kinases PKC and PKA and the trimeric phosphatase, PP2A, containing the B56d regulatory subunit. These enzymes modulate the phosphorylation state of Hand and Twist proteins on 2 evolutionarily conserved residues within Helix I of the bHLH domain. Florescence Resonance Energy Transfer (FRET) and in vivo expression analysis show that phosphorylation of Hand1, Hand2 and Twist1 on these conserved residues modulates dimer partner choice, and in the case of Twist1, human mutations that hobble normal phosphoregulation result in the autosomal dominant human disease Saethre Chotzen Syndrome (SCS; OMIM 101400.) Furthermore, loss of normal Twist1 phosphorylation in SCS effects Twist1's ability to antagonize the functions of Hand2 and that by reducing the gene dosage of Hand2 one can rescue the SCS phenotypes in the Twist1 haploinsufficient mouse model validating the hypothesis that Twist-family dimer choice is critical for normal development to proceed. To pursue our research goals we employ both conditional gain-of-function and loss-of-function mouse models, standard molecular and biochemical techniques as well as transcriptional analysis using in vivo and in vitro systems. We have gone on to show that dimer regulation affects craniofacial morphogenesis, that Twist1 is a master repressor or the neuronal program and have identified the regulatory control domains of Hand1. Our future efforts will further characterize the posttranslational modifications within Twist family of proteins and to explore how phosphoregulation and partner choice drive tissue-specific development programs using gene targeted mouse models that allow for the conditional activation of mutant protein expression in the wide spectrum of mesenchymal tissues that require Twist-class bHLH factors for proper development. By using large sequence data that has revealed mutations of Hand and Twist proteins found in human patients presenting with disease, we will model these mutations in vivo and deduce their contributions to congenital defects that account for 1% of all live births.