Related Experiment Videos
Direct and indirect mechanisms of repression participate in suppression of T-cell-specific gene expression in T x
L Shurman1, R Laskov, Y Bergman
1Hubert H. Humphrey Center for Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Abstract:
Expression of tissue-specific genes can be altered upon fusion of mammalian cells of different types. To resolve the genetic basis of this phenomenon and to identify components of the regulatory circuits that are involved, we have established a series of somatic cell hybrids between mouse T cells and L cells. These hybrids have an unusual and interesting phenotype. Unlike many hybrid cells studied, in which the expression of an entire set of tissue-specific genes was coordinately extinguished, in our T x L-cell hybrids only two out of seven T-cell-restricted genes were completely extinguished, whereas the other genes were repressed to various degrees. These hybrids extinguish the production of TCR beta and Thy-1 mRNA, repress the expression of TCR alpha, GATA-3, TCF-1, and LEF-1 genes to different extents, exhibit small changes in the level of CD3-epsilon mRNA, and continue to express the fibroblast-specific fibronectin gene, and the ets-1 gene. In this study we have evaluated for the first time the molecular mechanisms that underlie the repression of TCR alpha and TCR beta chain genes in T x L-cell hybrids. We have shown that multiple repression mechanisms, both direct and indirect, contribute to TCR alpha and TCR beta suppression. Repression of the expression of these genes correlated not only with the downregulation of GATA-3, TCF-1, and LEF-1 transcription factor expression, and with a change in the chromatin structure, but more importantly, with the activation of the silencer activity. Our study provides evidence for the existence of at least two negatively regulating elements, located at the TCR alpha enhancer-containing fragment and at the silencer region, which are active in our hybrid cells. We have shown that there was no correlation between the levels of GATA-3, TCF-1, and LEF-1 expression versus the level of TCR alpha mRNA in the independent hybrids. In contrast, both the silencer activity and the ability of the TCR alpha enhancer to downregulate thymidine kinase (TK) promoter activity were found to be in an inverse correlation with the ability of the different hybrid cells to express TCR alpha mRNA.
Insights
Somatic cell hybrids reveal complex gene repression mechanisms. T-cell specific genes like TCR alpha and TCR beta are suppressed through multiple pathways, including silencer activity, not just transcription factor levels.
Area of Science:
- Mammalian cell fusion and gene regulation
- Molecular mechanisms of gene silencing
- T-cell specific gene expression
Background:
- Cell fusion can alter tissue-specific gene expression.
- Previous studies often showed coordinate extinction of gene sets.
- T-cell and L-cell hybrids present a unique gene expression profile.
Purpose of the Study:
- Investigate the genetic basis of altered gene expression in somatic cell hybrids.
- Identify regulatory circuit components involved in gene repression.
- Elucidate molecular mechanisms underlying TCR alpha and TCR beta gene repression.
Main Methods:
- Established somatic cell hybrids between mouse T cells and L cells.
- Analyzed expression levels of various T-cell restricted genes (TCR beta, Thy-1, TCR alpha, GATA-3, TCF-1, LEF-1, CD3-epsilon).
- Evaluated molecular mechanisms including transcription factor levels, chromatin structure, and silencer activity.
Main Results:
- T x L-cell hybrids showed differential repression of T-cell genes, with TCR beta and Thy-1 fully extinguished.
- TCR alpha, GATA-3, TCF-1, and LEF-1 were repressed to varying degrees.
- Gene repression correlated with altered chromatin, downregulated transcription factors, and importantly, activated silencer elements.
Conclusions:
- Multiple direct and indirect repression mechanisms contribute to TCR alpha and TCR beta suppression in hybrids.
- Silencer activity and TCR alpha enhancer function inversely correlate with TCR alpha mRNA expression.
- Gene repression is not solely dependent on GATA-3, TCF-1, and LEF-1 levels.