Transcription factor involvement in the rat PNS-derived stem cell line family, RT4
Reinhart, Adam J
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The long-term objectives of this project were twofold: (1) to identify transcription factors (both novel and previously identified) which may influence cell fate and/or maturation in the nervous system, and (2) to investigate the role(s) that these transcription factors may play in either the cell-fate decisions and/or maturation of an in vitro model of peripheral neurogenesis called RT4. In the first phase of this project, five POU-domain genes (Oct-1, Oct-2, Bm-2, Bm- 5 and Tst-1/SCIP), as well as REST/NRSF (a repressor of neuronal-specific gene expression) were shown to be expressed, and their expression levels characterized in the RT4 cell line family. We also reported that 4 candidate transcription factors (Bm-1, Bm-3, Bm-4 and MASH-1) were not expressed in the RT4 cell line family. The second phase of this project involved the elucidation of the possible role(s) of Tst-1/SCIP and REST/NRSF in the RT4 cell line family. We examined whether Tst-1/SCIP influenced the conversion of RT4 stem cells to derivative cell types. The conversion frequency was essentially unchanged when transfected with wild-type Tst-1/SCIP, or a dominant-negative version of Tst-1/SCIP, or pcDNA3 vector alone. Although the conversion frequency among transfection groups (i.e., Tst-1/SCIP; Tst-1/SCIP AN; pcDNA3 vector alone) groups was approximately the same to all of the derivative cell types, the total number of stably transfected colonies within the Tst-1/SCIP transfection group was considerably less than the other transfection groups. The transcription factor REST/NRSF was examined in terms of its possible role(s) in regulating maturation-specific gene expression. Although we were able to demonstrate that the REST/NRSF-mediated repression of an RE 1-CAT reporter was at least partially relieved, we were unable to detect the de novo expression of a set of maturation-specific genes in RT4-B8, which would expect to be expressed if the cell line had been stimulated to mature.