Critical test
If you are interested in understanding the regulation of a particular gene, you need determine whether the expression of this gene is controlled at the transcription level. Allthough a Northern blot analysis is usually the first indication of a possible transcriptional control, it is complicated by posttranscriptional events, such as RNA processing, transport, editing, and mRNA degradation. Northern blot measures the net result of the above events; sometimes, it is called the steady state RNA concentration even though it is not exactly correct. The nuclear run-on experiment provides the most direct evidence because it measures the transcription efficiency in vivo. If the expression of this gene is really regulated at the transcriptional level, it is worth to clone the upstream sequence of this gene from a genomic library and further analyze the transcription.
Cis-acting elements
By analyzing the deletion mutants of this upstream sequence in vitro or in vivo, you can quickly identify the important cis-acting elements, such as promoter, enhancer, or silencer. An in vivo transient expression assay is frequently used because it is easier to set up than an in vitro run-off assay. Although making a chimeric gene of the promoter and a reporter gene need one more cloning step, this method greatly simplifies the preliminary assays. In addition, some of these constructs can be used in future cotransfection assays. By constructing linker scanning or site specific mutants, you can get a pretty good idea about the precise locaiton of the cis-acting elements.
Trans-acting factors
Usually, people will make crude cell extract and perform a footprint assay to find out whether there are any trans-acting factors bind to the cis-acting elements. After you have identified the factor binding site, you can use the run-off transcription to assay the chromatographic fractions and determine which fractions are required for transcription activity. As a result, the fraction containing each factor that is necessary for the transcription can be purified one by one. The reaction mechanism can be studied in the in vitro run-off system by using purified transcription factors.
The properties of each transcription factor can be studied by cloning the factor. If the factor binds to DNA sequence strongly, the gene of this DNA binding factor can be cloned by screening an expression cDNA library using South-Western method. Once you have the gene, you can make mutants to characterize this factor. Because of the convenience, the cotransfection assay is the most frequently strategy to define the functional domain of this factor. The domain information and these mutants will in turn be useful in understanding the transcription mechanism.
Discussion
At this point, the transcription regulation seems to be very clear. However, what controls the expression or activation of these transcription factors? If the activation is mediated by signal transduction, you can shift your interest to that direction. If the expression of some important transcriptional activators are again controlled at the transcriptional level, are you going to repeat the above exercise all over again? If you plan to do so, where is the end of this game? Be sure to think clearly before you step into this mess.