The hallmark of Burkitt Lymphoma and murine plasmacytoma cells is the translocation of the c-myc oncogene to one of the immunoglobulin gene loci that harbor B-cell specific enhancer elements. The rearranged c-myc allele is maintained in a highly active state even though the non-translocated allele is transcriptionally silent, as it is in mature B cells. Previous experimental data from this laboratory demonstrate that the normal expression of the mammalian c-myc gene requires an insulator element that protects the c-myc gene locus from the negative influence of surrounding heterochromatin, and efficiently inhibits enhancer-promoter interaction. Despite its function as a chromatin boundary, this insulator element appears to be ineffective in Burkitt Lymphoma cells in which the translocation breakpoint occurred upstream of the barrier/boundary element at the c-myc gene. This suggests that additional epigenetic changes inactivate the enhancer-blocking activity. The long-term objectives of our study are to define the molecular mechanisms by which insulator regions and chromatin organizing elements protect the normal expression of genes, and to determine the functional consequences of their reorganization through chromosomal translocations. In the first specific aim, we will focus on the identification of essential sequence elements that mediate the enhancer blocking and barrier activity of the c-myc insulator at the endogenous c-myc gene. The analysis of the insulator region will be performed in its normal chromosomal """"""""neighborhood"""""""" of the human c-myc gene locus. Additional experiments that utilize a novel approach based on quantitative mass spectrometry will determine the set of structural and regulatory proteins that is required for insulator function. In the second specific aim, the role of this element in the context of chromosomal translocations in Burkitt Lymphoma will be studied. A comparative analysis of chromatin structure at the translocated and non-translocated allele will define the role of chromatin components and histone modifications in the expression of c-myc alleles. The results will broaden our understanding of the role of chromatin elements in the specific regulation and nuclear organization of mammalian oncogenes in normal and malignant cells.
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