REGULATION OF THE TRANSCRIPTION FACTOR SLUG IN THE EMBRYONIC HEART

During embryonic heart development the transcription factor slug is required for the initial step in the process of heart valve formation, epithelial-mesenchymal cell transformation.  This transcription factor plays a similar critical role in a variety of cell transformations in the embryo and in metastasis.  The Runyan laboratory has shown that slug mRNA expression is regulated in endothelial cells by Transforming Growth Factor ß2 (TGFß2) but not by its closely related isoform, TGFß3.  This argues that the slug promoter should have a TGFß2 responsive element.  Clearly the differing pattern of expression of the TGFß isoforms in the embryo argues for some specificity of function but to date there has been no observation of a TGFß isoform-specific signaling pathway.  Thus the embryonic heart provides a potentially important system where we can explore isoform specific gene regulation.

The proximal 1.185 kb of upstream sequence from the slug gene was cloned and ligated into a reporter construct.  This reporter construct expresses a secretable alkaline phosphatase (SEAP) under the control of the inserted putative promoter and contains a SV40 enhancer downstream of the enzyme.  This construct was transfected into embryonic heart AV canal explants along with a second plasmid expressing green fluorescent protein (GFP).  Explants were seen to be transfected by the expression of GFP.   Initial data suggest that there is some promoter activity with the inserted slug sequence.  The expression of the alkaline phosphatase reporter under the influence of TGFß2 is currently being examined.  If the present upstream sequence does not contain a TGFß2 responsive domain, additional upstream sequence will be obtained.  A TGFß2 responsive promoter will be characterized by comparison to exogenous TGFß3 and by the response of the promoter to isoform-specific blocking antibodies.

Identification of the TGFß2 responsive element will be performed by sequential deletion analysis of the promoter and continued testing with exogenous growth factor or blocking antibody.  Once identified the minimal response element will be screened for known transcription factor binding.  Single base alterations will be performed to test the specificity of the recognition.  Complete identification of the TGFß2 response element will be completed by inserting the sequence into a promoter of a non-TGFß2 responsive gene.  If the new construct becomes TGFß2 responsive, the response element is sufficient to mediate an isoform-specific signaling pathway and its components.

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