Mechanisms of SSX gene expression regulation at the promoter level

Abstract

Cancer Testis (CT) Antigen Genes are not transcribed in any of the adult tissues except spermatogonia, oogonia and trophoblasts. This tight regulation of expression is reversed resulting in the reactivation of CT transcription in a wide variety of cancers. CT genes are coordinately expressed and known to be regulated epigenetically. CT genes are reactivated in cancers by a mechanism that leads to the specific hypomethylation of their promoter-proximal sequences. The mechanisms leading to this phenomenon are unknown. The main objective of this thesis was to further unravel the mechanisms regulating CT gene expression at the promoter level. For this purpose, SSX4 ,a typical CT-X gene known to be under the control of a bidirectional promoter, was chosen as a model. We characterized the minimal critical sequences controlling the sense and antisense promoter and discovered a bidirectional promoter with overlapping promoter activities within a 40 bp region. To study how the antisense promoter could mediate sense promoter repression and vice versa, we used two different reporter genes for each of the promoters in a single construct and found that measurable antisense promoter activity was dramatically reduced upon the introduction of a reporter for the sense promoter. The SSX4 antisense promoter is capable of producing a noncoding transcript from the neighboring ornitine aminotransferase-like pseudogene in vivo. This, however, wasn’t confirmed in this study. The possibility of transcriptional interference or the production of a small dsRNA that could affect the regulation of SSX4 gene expression is discussed in the context of the data. Results from experiments where the down-regulation of DICER was studied as a mechanism that could influence CT gene expression are also discussed