Question

8. To analyze whether a putative transcriptional enhancer activates transcription, one could use which of the following approaches?

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Answer 1

Ans. -

A variety of methods can be used to identify targets that are likely to be regulated directly by a transcription factor. Timing is one criterion: for example, immediate early genes, which are switched on shortly after the activation of a transcription factor, are more likely to be activated directly by that factor, because there has been little time for another gene to be activated and then for that to activate the target gene. This type of analysis is facilitated by the use of inducible gene expression, so the precise moment at which the transcription factor is activated and able to induce expression of downstream genes is known.

These methods provide further evidence that a target is direct but do not show that the transcription factor binds directly to a regulatory sequence in the gene; this can be tested by other approaches, such as the electrophoretic mobility shift assay (EMSA). This technique identifies binding of specific proteins to DNA sequences, and so can demonstrate direct binding of a transcription factor to the promoter region of its target gene. This in vitro method may not accurately reflect the situation in vivo, however, as binding is likely to be less tightly regulated in the assay.

This technique can be further improved by the use of protein-synthesis inhibitors, such as cycloheximide. Transcription factors that are already present within the cell are able to activate the expression of their target genes, but in the presence of cycloheximide the target genes cannot be translated, and so cannot switch on further downstream genes as indirect targets. Thus, only those genes upregulated in the presence of cycloheximide are direct targets. For instance, although microarray expression analysis identified 134 targets of Drosophila Clk, expression of a hormone-inducible form of Clk in cell culture in the presence of cycloheximide indicates that only nine of the genes are in fact direct targets.

To overcome this difficulty, two methods have been developed to demonstrate direct binding of a transcription factor to promoter regions of DNA in vivo: chromatin immunoprecipitation (ChIP) and Dam methylase identification (DamID. In addition to being used to ask whether a particular candidate gene is a direct target of a transcription factor these techniques can also be adapted to identify new target genes. For example, regulatory DNA sequences enriched by ChIP can be used as probes to identify the coding regions of direct target genes. Even these approaches have their limitations, however. In ChIP, protein-DNA interactions may not survive the procedure, and there is the risk of artifactual binding being introduced during the fixation process; similarly, expression of a fusion protein with DamID may not accurately replicate the situation in vivo. Nevertheless, these approaches prove to be very powerful and, as described below, can be scaled up to analyze the binding of transcription factors across the entire genome (so-called genome-wide location analysis).

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