Research:
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 fibroblasts 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 HAND class of bHLH factors HAND1 and HAND2. Unlike the myogenic bHLH factors HAND1 and 2 are expressed in a wide range of tissues including heart, cardiac neural crest, lateral mesoderm and the developing sympathetic nervous system. In addition, HAND1 is expressed in extraembryonic structures and HAND2 is expressed in the maternally derived deciduum. As the molecular cascades controlling cell specification and differentiation of non-skeletal muscle tissues were investigated several observations were made. First, as novel transcription factors were cloned and studied none were found to exhibit the tissue specific fidelity of the myogenic bHLH factors. Secondly, as gene-targeting strategies were employed to knock out factors in the heart and other organs it was clear that although defects in development were identified, tissue specification was never compromised. For example, the HAND1 and HAND 2 null mice exhibit cardiac and extraembryonic abnormalities, but do not show a compromise to the cell commitment decision of these tissues. These findings support a hypothesis that the majority of tissue-specific gene expression is facilitated by multiple transcription factors, which are more broadly expressed and that via protein-protein interactions these broadly expFebruary 6, 2008xes and driving tissue specific transcription.

In our study of the biological properties of HAND1 and HAND2, we discovered that unlike the myogenic bHLH factors, HAND factors exhibit promiscuous dimerization characteristics in that HAND1 and HAND2 can form homo and hetero HAND dimers as well as heterodimers with class A (E-proteins) other class B bHLH factors. This broad dimerization profile fits our hypothesis nicely as the broad affects of HAND factors within the tissues that they are expressed can be explained by the formation of unique tissue-specific dimers that drive the desired genetic program. Implicit in this model is that for HAND1 and HAND2 to dimerize with numerous bHLH partners; the dimerization choices of HAND factors must be a regulated. Our most recent work has shown some of the first insight into the mechanisms that control HAND dimerization choices. We have discovered that phosphorylation of HAND1 is elevated during the differentiation of RCHOI trophoblast stem cells. HAND1 has been shown to be required for the differentiation of this cell type and we have shown that the lack of HAND1 in mice results in a incomplete of differentiation of giant cell trophoblasts.  Using in vitro and in vivo methods we have determined that 3-amino acid residues conserved between HAND1 and HAND2 account for the increased phosphorylation and they can be modified by the kinases PKC and PKA. Moreover we discovered that a specific form of the trimeric phosphatase PP2A containing the B56d regulatory subunit can specifically dephosphorylate HAND1 on two of these modified residues. Efforts to deduce the biological significance of these modifications and interactions using Florescence Resonance Energy Transfer (FRET) and in vivo expression analysis show that phosphorylation of HAND1 by PKC and PKA facilitates an increase Alters HAND dimerization affinities and these alterations effect biological function, allowing for the formation of unique dimer pools within a cell which we feel allows for the activation of specific genetic programs within specific tissue lineages.

Our future goals are to further characterize the posttranslational modifications within HAND1 and HAND2 as well as the modifications that occur within E-proteins and coexpressed class B bHLH factors such as the HES related transcription factors HRT1, 2 and 3. By studying the phosphorylation of these factors during cardiac and trophoblast development and deducing the effect of these modifications on transcriptional activity/dimerization/DNA binding our hope is to define the specific dimers (and the mechanisms controlling there formation) that are responsible for HAND function in heart and extraembryonic development. In addition, by employing engineered HAND proteins that are predisposed to form certain dimers vs. others and using gene targeting technologies to knock these mutants into the mouse the genetic consequences of HAND dimerization choices can be deduced and add valuable insight into the molecular programs that orchestrate tissue-specific.

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