The objective of this proposal is to define the regulation and deregulation of a large proto- oncogene, BCL-2, within its genomic context by developing approaches which utilize yeast artificial chromosomes (YACs). A number of disease associated genes, especially oncogenes, span hundreds of kilobases. Major questions exist concerning the purpose of this design and the mechanism of their regulation in normal and disease states. The most common translocation, the t(14;18) of follicular lymphoma, results in a BCL-2-IG fusion gene that is transcriptionally deregulated. However, the mechanism is uncertain since the IG heavy chain locus is introduced 225 kb downstream of the BCL-2 promoters. This proposal aims to retrofit YAC clones of the entire BCL-2 gene by inserting portions of Ig to recreate and dissect the t(14;18). Modified YACs are transfected into mammalian cells by protoplast fusion. Full length stable integrants will be identified and species specific RT PCR, S1 protection, Western, and immunohistochemical assays used to confirm expression of YAC origin BCL2. YAC transfection conditions will be tailored for preB and mature B lymphoid cell lines and the utility of microinjection will be assessed. Since prior minigenes failed to express, cells bearing an integrated BCL2- YAC will be assessed for stage specific expression.This should resolve whether the IG locus is sufficient to deregulate BCL2 and the effects of the IG enhancer extend across the 225 kb intron. The efficiency and precision of modifying YACs provides the capability to compare alternative rearrangement sites and address other mechanisms.BCL2-YACs provide reagents to determine if Intron II (225 kb) is continuously transcribed and cis spliced or whether BCL2 can be trans spliced. Finally, the normal BCL2-YAC provides the substrate to identify the cis regulatory sequences responsible for its differential expression. The use of YAC fragmenting vectors and interstitial deletion vectors is an attractive avenue to define positive acting enhancer(s). A YAC approach to BCL2 provides a model system for addressing other oncogenes.
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| Walensky, Loren D (2013) Protein-protein interactions: A PUMA mechanism unfolds. Nat Chem Biol 9:141-3 |
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