​Drosophila and I: A Journal of My Time with Flies 

The following crossing scheme utilizes a P-element called StanEx1 generated by Sangbin Park at Stanford in summer 2012. This P-element is a single copy of a Pprom-LHG construct integrated in the X-chromosome at the sprint locus. Pprom-LHG carries an expression cassette encoding a fusion protein consisting of the bacterial LexA DNA binding domain linked to the hinge region and transactivation domain of the Gal4 transcription factor from yeast. The fusion protein is from here on abbreviated LHG.

The expression cassette of Pprom-LHG  is under the control of a relatively weak P-element promoter (hence the Pprom). Without any surrounding genetic element fostering its expression (enhancers), the expression cassette is not transcribed, and no LHG protein is expressed. The P-element is “dark”. (Just to avoid confusion, the specific insertion of Pprom-LHG called StanEx1 is not dark, as it is inserted into a transcribed locus and displays expression in the ring gland and ventral nerve cord). There might be other reasons some P-elements are not expressed, like its location in a compact form of chromatin, called heterochromatin that does normally not foster transcription. But for reason of simplicity, we are not going to explore this any deeper.

At any rate, enhancer elements in range to the P-element have a chance to interact with our weak P-element promoter and impose its expression pattern upon the expression cassette of the P-element. This effect is called enhancer trapping, as the enhancer “traps” the expression cassette of the P-element. This is what the enhancer would say. From the perspective of the P-element, it’s it that traps the expression pattern of the enhancer.

The crux is that we can now use the expression cassette of the P-element that is transcribed and expressed in the tempo-spacial pattern imposed by the trapped enhancer to visualize this expression pattern. This is done routinely by crossing in a second P-element carrying a green fluorescent protein (GFP) construct that is transcriptionally controlled by the LexAop DNA element. The LexA:LHG protein binds to the LexAop DNA element and drives the transcription the GFP mRNA that is subsequently made into GFP protein. So only cells where the trapped enhancer is active light up under fluorescent light, because these are the only cells expressing GFP.

As a first order approximation this is correct, although we can easily think of scenarios in which these things do not align so neatly. For example, the enhancer might only be active for a very short time, and the LexA:LHG protein might be hanging around much longer than  the enhancer is active. That would result in cells that light up green although the trapped enhancer has gone long silent.

This is probably the right time to mention that at the present time it is still not easy to recognize an enhancer just by “looking” at primary DNA sequence. Efforts to map all enhancer elements in various genomes have been done, and more are on their way. Still, although we have trapped an enhancer, we are unable to put our finger on it and say: This is the DNA element we have trapped. Because we might have trapped more than one. Because it might be located a long distance from the P-element insertion site. Because we cannot recognize enhancers with high accuracy to begin with. The complexities are humbling.

Starting with StanEx1, we are aiming to generate a collection of P-elements, all inserted in different sites in the Drosophila genome and that are trapping different enhancers. To do so, we are mobilizing StanEx1 with transposase. Transposase is the enzyme that literally “cuts” the P-element out of its genomic insertion site and “pastes” it into a new location. Sometimes. If it happens, the P-element in its new insertion site has the potential to have trapped a new enhancer.

Very often, nothing happens at all, in some cases it just reinserts itself into the same location, in some cases the transposase works imprecise and takes out chunks of genomic DNA adjacent to one or both sites of the P-element and creates a deletion. That all means that we will have to go through a fair amount of fly work to get the events we are looking for.

P-element transposition events are semi-random. That means that P-elements have insertion chromatin/sequence preferences that puts them with a certain frequently close to 5’ ends of transcription units. They also show a higher insertion frequency into some loci compared to others.