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The strength of reporter gene expression increased with multiple enhancer cassettes, plateauing with the use of four enhancer elements (4 × Grm6).
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Lastly, genes are frequently regulated by multiple enhancer elements or clusters of enhancer elements.
The constructs tested carried 2, 4, 8, or 16 copies of the 200-bp enhancer cassettes in tandem.
Further studies have identified multiple enhancer elements involved in this regulation [ 8- 11].
We speculate that in those instances, the GFP expression cassette may have fallen under the control of multiple enhancers – some tissue-specific, some ubiquitous.
And studies in Drosophila suggest, too, that interactions of multiple enhancers are commonplace. .
Data on long-distance enhancer-mediated activation of gene promoters and complex regulation of gene expression by multiple enhancers have prompted the hypothesis that the action of enhancers is restricted by insulators.
One such interaction overlaps the ß-catenin/TCF4 enhancer elements A through E. It is therefore possible that through ternary interactions, multiple enhancers are concurrently juxtaposed to MYC.
Some genes have multiple enhancers with overlapping activities, typically including a primary enhancer and secondary or 'shadow' enhancer that is even more distant.
Genes under high regulatory load from multiple enhancers or super-enhancers are known key genes for disease and cell identity.
Second, most genes are regulated by multiple enhancers, but only one enhancer can be tested in a reporter assay.
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