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Retrovirus-like promoters in the human genome

University of British Columbia

Several families of repetitive sequences related to integrated retroviruses (proviruses) have been identified in the human genome. The largest of these families, the RTVL-H family, has close to 1000 members, in addition to several hundred solitary long terminal repeats (LTRs). The similarity of these LTRs in structure and organization to the LTRs of proviruses suggest that they may act as transcriptional regulators of gene expression. To test this hypothesis, I initially examined the ability of different RTVL-H LTRs to drive expression of the reporter gene chloramphenicol acetyltransferase (CAT) in a variety of human and murine cell lines. These studies revealed that RTVL-H LTRs are heterogeneous in their ability to regulate the expression of linked genes. Although all of five LTRs tested could promote expression of the CAT gene, their relative promoter activities as well as range of activities varied widely. One LTR, H6, displayed strong promoter activity in human (NTera2Dl, 293, Hep2), monkey (COS-1), and mouse (3T3) cells. RNA mapping studies localized the transcription start site to the expected location in the H6 LTR. RTVL-H LTRs were also shown to contain sequences that could increase transcription from the human i globin promoter and be influenced by SV4O enhancer sequences. Over the course of these studies, I observed a difference in RTVL-H promoter activity in CV-1 and COS-1 cells (COS-1 cells are African green monkey kidney cells transformed by SV4O and CV-1 cells are their “untransformed” parent cell line). To examine the possibility that this effect was mediated by SV4O encoded proteins, LTR-CAT constructs were cotransfected with expression vectors encoding SV4O proteins. The results of these studies showed that SV4O T antigen could activate expression from certain RTVL-H LTRs 5-30 fold. In addition, this transactivation effect was observed in two CV- 1 cells lines containing stably integrated LTR-CAT constructs. These results demonstrate that a known transforming protein can alter the transcriptional capabilities of RTVL-H LTRs. The results of the above studies suggested that RTVL-H LTRs may have the ability to influence the expression of unrelated cellular genes. Thus, a differential screening strategy was used to identify five cDNA clones that appear to have been promoted from RTVL-H LTRs. In one case, clone AF-4, the LTR has been shown to be normally linked to a CpG island. Another clone, AF-3, appears to be a new insertion of an RTVL-H element into a large gene with a region of homology to yeast CDC4. The third clone , AF-5, is the result of a splicing event between an RTVL-H element and a novel gene with two regions of homology with phospholipase A2. Taken together, these results suggest a general evolutionary role for RTVL-H LTRs in the regulation of gene expression and raise the possibility that activation or rearrangements involving these sequences may alter the normal regulation of cellular genes and thus contribute to human disease.

Thesis/Dissertation

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2008-12-19

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http://hdl.handle.net/2429/3211

Genetics

University of British Columbia

UBCV

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