? PROJECT 3 Human T-cell leukemia virus type 1 (HTLV-1) is the cause of an aggressive, refractory T cell malignancy, adult T-cell leukemia / lymphoma (ATL). The integrated proviral genome has a single binding site (BS) for the chromatin insulator protein CTCF (CTCF-BS). The HTLV-1 CTCF-BS is located within the 3?portion of the genome, downstream of 5? proviral DNA that is generally methylated and bound by histones with markers of inactive chromatin, and upstream of 3? proviral DNA with generally unmethylated DNA and bound by histone with markers of active chromatin. More than 50,000 CTCF-BS are also present in the human genome, which can mediate DNA looping with the HTLV-1 CTCF-BS. In addition to, or as a result of its looping activity, CTCF regulates epigenetic chromatin modifications and transcription. CTCF-BS in several DNA viruses are known to be important for latency, reactivation from latency, and/or viral transcription. However, very little is known about the role of CTCF in retroviruses, and there is only limited information about the epigenetic regulation of HTLV- 1. Our premise is that CTCF regulates establishment of HTLV-1 latency, and the rate of virus replication and transformation in humanized mice.
In Aim 1, we will use a Jurkat cell line model of HTLV-1 latency to examine if the effect of CTCF on establishment of latency is mediated by preference for certain integration site characteristics, and whether this affects the pattern of DNA methylation and H3K4me3 and H3K36me3 modifications of histones bound to the 5? and 3? proviral DNA.
Aim 2 will examine the effect of mutation of the CTCF-BS on HTLV-1 replication and transformation in humanized mice. For this purpose, we will use two different humanized mouse models, one of which is a model of HTLV-induced lymphoma and the other a model of HTLV-induced leukemia. Once again, we will examine whether specificity of proviral integration sites affects virus replication and transformation, and epigenetic modifications.
Aim 3 will examine the effect of CRISPR/Cas9 editing of the CTCF-BS in ATL cell lines on DNA looping and their ability to proliferate in immunodeficient mice. We will also determine if reconstitution of DNA looping independent of CTCF, through use of defective Cas9 fused to looping protein Lim domain-binding protein 1 (LDB1) affects ATL cell proliferation in immunodeficient mice. In summary, these studies will provide fundamental new information about the mechanism whereby the CTCF insulator protein regulates HTLV-1 transcription, splicing, latency, integration preference, immortalization, and tumorigenesis.
- PROJECT 3 This project investigates the role of a cellular protein (CTCF) that binds to a specific site (CTCF-BS) in the integrated HTLV-1 DNA as well as many sites in the lymphocyte DNA; this interaction is known to introduce loops into DNA and affect expression of genes. We will investigate whether the CTCF-BS affects the ability of HTLV-1 to replicate, establish latency, a state in which the viral DNA is present in the cell but it is not copied into RNA and proteins, and cause leukemia and lymphoma in immunodeficient mice. These studies will elucidate the mechanism of action of CTCF in regulating HTLV-1 replication and transformation.
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