Characterization of the molecular mechanisms regulating human reduced folate carrier gene expression in human tissues.

摘要

Reduced folates are responsible for one carbon transfer reactions that result in the synthesis of DNA, RNA, and protein precursors. The lack of these cofactors is strongly associated with the development of fetal abnormalities, cardiovascular disease, and chromosomal instability. Since mammalian cells lack the ability to synthesize reduced folates de novo, a sophisticated active facilitative transporter called the reduced folate carrier (RFC) has evolved. RFC is the primary route of entry for reduced folate cofactors, but also transports antifolate cancer chemotherapeutics [e.g., Methotrexate (Mtx)]. For this reason, membrane transport is a critical determinant of antitumor activity. Clinical studies have demonstrated that altered human RFC (hRFC) gene expression is strongly associated with Mtx-related drug resistance. Therefore, my dissertation focused on characterizing the molecular mechanisms regulating hRFC in human tissues. This study demonstrated that hRFC is differentially but ubiquitously expressed in normal human tissues. At least eighteen unique hRFC transcripts were identified in tissues differentially expressing hRFC. These transcripts result from seven non-coding exons (A1, A2, A, B, C, D, and E), of which, exons A, B, C, and D alternatively splice. Non-coding exon usage was heterogeneous; however, malignant tissues predominantly expressed non-coding exons B and A. For this reason, the factors influencing the transcriptional control of each promoter were determined. Oncogenic transcription factors (e.g., cMyc, Ikaros, and AP2) were involved in regulating the expression of promoters B and A. Since many of the factors binding promoter B were able to alter chromatin structure through histone acetylation, the acetylation status of promoter B was assayed by transfections and chromatin immunoprecipitations. By inhibiting class I histone deacetylases, promoter B activity increased, but more importantly, histone 3 acetylation increased, as well. In addition, a functional polymorphism that upregulated promoter A activity was identified. The data in this dissertation demonstrate that altered transcription factor composition, chromatin remodeling, and promoter structure can regulate promoter/exon usage, which provides a molecular mechanism for tissue-specific expression and/or altered promoter/exon usage in normal and malignant tissues.