The goal of our work is to determine how TALl and LYL1 oncoproteins are connected to the core cell cycle machinery in human T-cell acute lymphoblastic leukemias (T-ALL). Our hypothesis is that TALl impacts the cell cycle machinery by transcriptionally upregulating cyclin D3, while LYL1 signals via cyclin D2. TALl and LYL1 are basic helix-loop helix (bHLH) transcription factors implicated in the pathogenesis of T-ALL. The transcriptional targets for these oncoproteins are currently unknown. D-type cyclins (D1, D2 and D3) are key components of the core cell cycle machinery in mammalian cells. The expression of D-cyclins is controlled by the mitogenic and oncogenic signal transduction pathways. In our preliminary studies, we determined that T-ALL cases with activated TALl express high levels of cyclin D3 mRNA and protein, and virtually no cyclin D2. On the other hand, we found that T-ALL cases with activated LYL1 express high levels of cyclin D2. We propose three mechanistic models to explain how these bHLH might control the expression of D-cyclins: (1) """"""""gain of function"""""""": over expressed TALl or LYL1 directly transactivate cyclin D promoters; (2) """"""""loss of function"""""""": Class I bHLH proteins, such as E2A, normally function as homodimers to repress cyclin D promoters. Over expression of TALl or LYL1 drives formation of inactive TALI-E2A or LYL1-E2A heterodimers, leading to de-repression of cyclin D promoters; (3) """"""""indirect action"""""""": TALl and LYL1 influence the expression of D-cyclins indirectly, by transcriptionally controlling a gene encoding an activator or repressor of D-cyclins. In the studies described in the Specific Aims 1 and 2, we will address these possibilities at a mechanistic level, by analyzing the molecular connection between TALl or LYL1 and the core cell cycle machinery in human T-ALL cells. We will identify transcriptional complexes that bind to cyclin D promoters in TALl- and LYL1 -positive T-ALL cells using EMSA and chromatin immunoprecipitation analyses. In the Specific Aim 3, we will use mice lacking cyclin D3, or D2, that we generated, to probe the requirement for TALl and LYL1 in malignant transformation using genetic means.
The Specific Aims are: 1. To determine how TALl oncogene is connected to the cell cycle machinery in human T-ALL; 2. To determine how LYL1 oncogene is connected to the cell cycle machinery in human T-ALL; 3. To study the wiring of bHLH proteins to the cell cycle machinery using genetically altered mice.
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