Robert Benezra - US grants

The long term objective of the research being proposed is to provide a molecular description of primary events leading to the onset of differentiation in mammalian cells. Specifically, this research will focus on providing a detailed description of the mechanism of action of the protein Id, a molecule shown to inhibit the differentiation process in multiple cell lineages. The main model system that will be sued to study Id activity is the muscle differentiation pathway in which Id has been shown to antagonize the action of the muscle determination gene MyoD. 1) The regions of the Id protein molecule required for its activity will be determined by site directed mutagenesis followed by functional analysis in transfection assays. Both the ability of Id to inhibit myogenesis and its ability to overcome the growth suppressive properties of MyoD will be investigated in these studies. 2) The Id gene will be isolated from a mouse genomic library and the enhancer elements responsible for its regulated expression during differentiation will be identified by deletion analysis, transient transfections and the use of transgenic mice. 3) Inducible versions of the Id gene will be constructed to determine the phenotype of timed over expression of Id in cultured cells and transgenic mice.

Benezra 97-23522 The main objective of the research being proposed is to determine the function of the Id gene family during mammalian development using a genetic approach. The Id genes (Id 14) have been demonstrated both biochemically and in tissue culture models to be negative regulators of the basic helix-loop-helix (bHLH) family of transcription factors controlling cell fate decisions in such diverse processes as hematopoiesis, adipogenesis, myogenesis, osteogenesis and others. By analyzing the phenotypes of mice that harbor deletions in two of the Id genes (Idl and Id3) which are known to be expressed in similar patterns during development, the role of these proteins in controlling differentiation in vivo will be determined. In addition, it will be determined if disruption of the Id genes can compensate for the loss of the E protein family of bHLH proteins known to be targets for Id mediated repression biochemically. In this way, a direct genetic link between the Id and E protein families can be established genetically. Preliminary experiments indicate that indeed Idl and Id3 are required for post natal viability since double homozygous null mice developed in our laboratory usually die within one day of birth. These mice display evidence of proliferative and/or differentiation disorders in multiple tissue types and also a dramatic sex ratio distortion. These observations will be expanded by detailed molecular analyses both on tissue samples and fibroblasts derived from the mutant mice in order to define more clearly the role of the Id gene family during mammalian development. In addition, it has been observed that disruption of the Id genes can compensate for some of the defects observed in the E protein knockout mice. In particular, whereas homozygous disruption of the E proteins leads to postnatal lethality in a majority of mice, loss of both an E protein and Idl restores the postnatal viability of these mice. This observation will be extended to determine the cause of the lethali ty in the E protein knockout mice as well as the mechanism of rescue by the loss of Idl. These experiments will thus increase our understanding of the mechanism of action of the Id gene family and define more precisely the nature of the Id/ E protein interaction in viva.