MMTV strains that cause T-cell lymphomas contain a large deletion in the LTR that includes two negative regulatory elements, the promoter proximal NRE and promoter distal NRE. Loss of one or both NREs increases transcription from the MMTV LTR in a tissue-specific manner. Specific mutations in the promoter proximal NRE increase MMTV expression in transient transfection assays and in lymphoid tissues of transgenic mice. Two nuclear protein complexes bind to the NREs. Those complexes contain the nuclear matrix-associated proteins SATB1 and Cux/CDP. The objective of this application is to determine the role of SATB1 and Cux/CDP in tissue-specific MMTV expression, and to determine whether the loss of transcriptional suppression in lymphoid tissues is required for T-cell lymphomagenesis. First, SATB1 will be overexpressed in cell lines that lack it, and in transgenic mice. The effect of SATB1 overexpression on MMTV expression will be analyzed using MMTV LTR-reporter genes and by measuring endogenous MMTV RNA. The effect of SATB1 overexpression on MMTV replication will be measured in SATB1-transgenic mice. Mutations of SATB1 will be analyzed for their ability to bind DNA, to bind to the nuclear matrix, and to localize in discrete cellular compartments. Second, Cux/CDP will be overexpressed in differentiated mouse mammary cells and in transgenic mice to determine if it will repress MMTV-LTR-directed expression. Antisense Cux RNA expression and Cux knockout mice will be used to determine if MMTV transcription is increased in the absence of Cux. Finally, mutations of Cux and SATB1 binding sites and MMTV Sag will be introduced into a full-length infectious MMTV provirus in order to evaluate the role of these elements in the induction of T-cell lymphoma.
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