a weakened transcriptional enhancer yields variegated gene expression一个被削弱了的转录增强收益组合基因表达.pdfVIP

A Weakened Transcriptional Enhancer Yields Variegated Gene Expression Cathy Collins, Peter Azmi, Maribel Berru, Xiaofu Zhu, Marc J. Shulman* Department of Immunology, University of Toronto, Toronto, Canada Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, ,1.4 kb 39 of the promoter, but inactive ,6 kb 39, indicating that the activity of the core enhancer was distan