The purpose of this grant is to analyze the function of the human TAN-1 gene, now referred to as NOTCH1, which encodes a transmembrane receptor implicated in a number of normal developmental processes and pathologic states. Previous work by this laboratory indicates that the gene is expressed as a 350 kD precursor polypeptide that is cleaved to produce a heterodimer in which an extracellular 230 kD subunit remains bound to a 120 kD transmembrane subunit by non-covalent forces. Engagement of ligand seems to activate NOTCH1 by dissociating the 230 kD subunit, which otherwise represses the intrinsic activity of the transmembrane subunit. Experiments are planned to investigate a number of issues related to changes in the receptor during the process of activation, including whether the release of the 230 kD subunit involves a proteolytic cleavage of the 120 kD subunit to remove the site at which the 230 kD subunit is bound. The portion the 230 kD subunit responsible for the repression of the 120kD subunit will be investigated by generating mutations within the 230 kD subunit. The fate of the 230 kD subunit after ligand-induced dissociation from the receptor complex will be studied to resolve apparent inconsistencies in experimental observations about the destination of free subunit after its release. To gain a more accurate assessment about the anatomic sites and developmental timing of NOTCH1 activation, transgenic mice will be generated with reporter transgenes encoding the jelly fish green fluorescent protein under control of a NOTCH1-sensitive promoter. To address questions related to the differential regulation of the NOTCH1-3 genes, particularly with regard to feedback regulation of transcription, the promoters for these three genes will be analyzed and used to investigate regulatory proteins that bind to them. To better understand the role of NOTCH1 in thymocyte development, experiments will be performed in which NOTCH1 signaling is disrupted or modified in T cell precursors as they differentiate in a thymic culture system derived from Rhesus macaques. Finally, alterations of gene expression produced by activation of NOTCH1 will be studied by hybridization of probes for total cellular RNA to cDNA microarrays. Besides providing insights into genes regulated directly and indirectly by NOTCH1, studies with microarrays may reveal the identity of genes which collaborate with truncated NOTCH1 in the malignant transformation of cells having chromosomal translocations that divide the NOTCH1 gene.
| Callahan, J; Aster, J; Sklar, J et al. (2000) Intracellular forms of human NOTCH1 interact at distinctly different levels with RBP-jkappa in human B and T cells. Leukemia 14:84-92 |
| Rand, M D; Grimm, L M; Artavanis-Tsakonas, S et al. (2000) Calcium depletion dissociates and activates heterodimeric notch receptors. Mol Cell Biol 20:1825-35 |
| Luo, B; Aster, J C; Hasserjian, R P et al. (1997) Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor. Mol Cell Biol 17:6057-67 |
| Aster, J C; Robertson, E S; Hasserjian, R P et al. (1997) Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription. J Biol Chem 272:11336-43 |
