|dc.description.abstract||A key requirement of the retroviral lifecycle is integration of the proviral genome into the host cell genome. This makes retroviral vectors uniquely suited for gene therapy applications. While murine leukemia virus (MLV) and human immunodeficiency virus-1 (HIV-1) based vectors are commonly used, we propose that avian leukosis virus (ALV) may be a safer vector. Previous gene therapy trials, while successful, had issues with integration into and activation of oncogenes leading to the formation of cancer. In this thesis, we show that ALV integration is relatively random with only slight integration site preferences in the chicken genome, potentially making it less prone to insertional mutagenesis.
To better understand how this random integration pattern is achieved, we also determine host cell factors that regulate ALV integration. Host proteins have previously been shown to target integration of MLV and HIV-1 to transcription start sites and active genes respectively. We show here that the cellular FACT (facilitates chromatin transcription) complex proteins regulate ALV integration efficiency in vitro and in vivo by binding directly to the ALV integrase protein. We show that the integration pattern of ALV in vivo changes significantly with varying expression levels of FACT complex. We hypothesize based on the observed direct binding of the FACT complex to the integrase protein and the observed effect in vivo, that the FACT complex may be acting as a bimodal tether to recruit the ALV pre-integration complex to specific genomic locations, thereby influencing both efficiency and pattern of integration.
We also explore other host cell protein candidates that selectively bound the ALV integrase protein. Interestingly, we discovered that the BET family of proteins, which are known to regulate MLV integration, also seem to have an effect on ALV integration efficiency in vivo. We also see subtle effects of BET protein inhibition on integration targeting. Interestingly, BET protein inhibition in a FACT knockdown background has the largest effect on integration targeting suggesting a potential collaborative effect of the cellular host factors. Other factors explored, such as nucleolin and UBTF (upstream binding transcription factor) were found to regulate the ALV lifecycle but not directly at the level of integration.
In addition to analyzing the regulation of ALV integration, this thesis also documents how the subsequent selection of integration sites in vivo can be used to identify novel genes involved in tumorigenesis. Because retroviruses contain strong promoter and enhancer elements, the insertion of the proviral genome into the host cell genome can have profound effects on host gene expression. Depending on the site of integration, this can activate gene expression or promote the expression of altered gene products. In vivo, integration sites into genes that contribute to the regulation of proliferation, immortalization or apoptosis are selected for over time and can lead to the formation of tumors. We identified selected, or expanded, integration sites in B-cell lymphomas. This led to the identification of novel oncogenes CTDSPL and CTDSPL2 as well as the putative noncoding TAPAS RNA.
CTDSPL and CTDSPL2 have been previously shown to regulate the phosphorylation status of the C-terminal domain of RNA polymerase II. They also play a role in regulating pRb phosphorylation and thus have been previously characterized as tumor suppressor genes. To the contrary, in our system we observe that CTDSPL and CTDSPL2 while having slight negative effects on cell proliferation, promote cell migration and protect cells against apoptosis, attributes more characteristic of an oncogene. Truncated proteins, like those generated by ALV integration within CTDSPL and CTDSPL2 in tumors, also promote immortalization in primary cell culture. Thus, we hypothesize that integrations into these genes were selected for in tumors due to the immortalization role of the truncated protein products.
The most common expanded integration site identified in B-cell lymphomas was in the TERT (telomerase reverse transcriptase) promoter region. We found that these integrations were predominantly in the opposite transcriptional orientation to TERT and were in fact promoting the expression of a truncated form of a novel antisense long noncoding RNA, which we have named TAPAS (TERT antisense promoter associated) RNA. TAPAS RNA is conserved in most birds and we find evidence for a similar transcript in humans. We provide evidence here for a role of TAPAS RNA in regulating TERT expression in cis in both chickens and humans.||