This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Notch proteins are conserved surface receptors that regulate development through cell-cell contact in multicellular organisms. Notch signaling triggers cell growth, cell differentiation, or cell death, in a context dependent manner. Upon activation by ligand engagement, Notch is released from the cell membrane by two proteolytic events, allowing the intracellular portion (ICN) to move to the nucleus. ICN associates with two other proteins, CSL and Mastermind, in the nucleus to form the Notch transcription complex (NTC), which activates gene transcription. Aberrant Notch signals have been implicated in cancer and a number of human diseases. More than half of T-cell acute lymphoblastic leukemia (T-ALL) patients have mutated forms of Notch that are constitutively active, resulting in aberrant transcription of Notch targets. Trimer NTCs can self-associate on specialized DNA elements with two CSL-binding sites oriented head-to-head with a defined spacer between the two sites, referred to as an SPS (for Sequence Paired Site or Supressor of Hairless Paired Site). A mutant form of ICN (R1985A) is able to assemble into NTC complexes and perform some known Notch functions, including promotion of T cell differentiation. However this mutant form of ICN is not able to form dimers and does not promote leukemia in a mouse model for T-ALL in which wild-type ICN is leukemogenic. The goal of this work is to determine the structural and energetic foundations underlying Notch mediated dimerization and explore its role in gene regulation using biophysical, biochemical and cellular techniques.
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