Mechanism of HTLV-1 Tax-mediated activation of CDK6 transcription.

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DESCRIPTION (provided by applicant): The complex retrovirus, human T-cell leukemia virus type 1 (HTLV-1), is the etiological agent of a fatal T-cell malignancy termed adult T-cell leukemia and a neurodegenerative disorder defined as tropical spastic paraparesis/HTLV-1-associated myelopathy. The HTLV-1-encoded Tax protein plays an essential role in viral propagation by activating transcription of the chromosomally integrated HTLV-1 genome and by stimulating cell proliferation, which both serve to replicate the viral genome. Stimulation of cell proliferation is also paramount to the persistence of HTLV-1 infection and is believed to underlie the pathological effects caused by the virus. Among several cellular processes affected by Tax, its capacity to deregulate the expression of numerous cellular genes is a key factor leading to cell proliferation. Although the current paradigm suggests that Tax lacks DNA-binding activity, this protein is able to form complexes with the transcription factor CREB that then associate with enhancer elements within the viral promoter. These complexes nucleate the formation of a preinitiation complex that notably includes the coactivator p300, which is recruited directly by Tax. It is believed that Tax utilizes similar mechanisms to activate transcription from certain cellular promoters. To test this hypothesis, we used a ChIP-on-CHIP approach to identify regions within the human genome where Tax is specifically associated. Several sites were found to be potential targets for Tax-recruitment, among which a region of the CDK6 promoter consistently exhibited a high-level of Tax- enrichment. Cdk6 is a positive regulator of the G1- to S-phase transition and maintains cells in a state of proliferation. These functions parallel the effects of Tax in infected cells, and indeed, we have confirmed results from a previous study demonstrating that CDK6 expression is activated by Tax. Unexpectedly, further analysis of the association of Tax upstream of CDK6 revealed that Tax alone binds directly to the DNA, independently of a requirement for an ancillary factor. We also found that p300 is not recruited to this Tax-DNA complex. These findings are unprecedented and challenge some of the current models for Tax-mediated activation of transcription. Our objectives in this proposal are to, first, characterize the novel Tax-DNA complex and, second, define the Tax-dependent events involved in the activation of CDK6 expression. We will utilize a series of biochemical approaches to characterize the DNA element that is recognized by Tax, domains of the viral protein required for DNA-binding, and the stoichiometry of Tax when bound to DNA. We will employ complementary in vitro and in vivo approaches to identify cellular transcriptional regulators that are recruited to the CDK6 promoter by Tax, and define their roles during Tax-mediated transcriptional activation. These studies will yield a more refined understanding of events involved in the chronic proliferation of HTLV-1-infected cells and, ultimately, the pathological effects caused by the virus. In addition, they will provide insight into a novel mechanism through which Tax deregulates cellular gene expression. PUBLIC HEALTH RELEVANCE: Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with an aggressive leukemia and a neurodegenerative disease. This virus encodes a viral protein, Tax, involved in regulating viral replication and in activating cell cycle progression. This proposal investigates a novel mechanism of how Tax activates the transcription of a cellular gene important for cell cycle progression that, when overexpressed, potentially contributes to the proliferation of HTLV-1 infected cells.