Heat shock genes in Drosophila melanogaster provide an excellent model system for studying transcriptional regulation because they can be rapidly and robustly activated. Our lab has shown the recruitment of transcription machinery upon gene activation to the polytene chromosomes of salivary gland nuclei can be viewed in living tissue in real-time via Multiphoton Microscopy (MPM).  The deep-penetration and low background of MPM provides an ideal technique for examining protein recruitment and exchange on chromatin in intact tissues.  Furthermore, MPM can be coupled with Drosophila germline transformation to visualize GFP-tagged transcription factors expressed from stably incorporated transgenes.  These factors can then be detected at specific gene loci with high spatial and temporal resolution. Recently our lab observed the ‘compartmentalization’ of RNA Polymerase II (Pol II) during extended gene activation by heat shock in living salivary glands at the endogenous Hsp70 genes. Fluorescence recovery after photobleaching (FRAP) of GFP-tagged Pol II soon after gene activation showed complete recovery within two minutes; consistent with the time it takes Pol II to elongate through the Hsp70 gene. After extended gene activation however, there is little to no recovery of fluorescence, indicating that Pol II must be retained at the locus and efficiently recycled to account for the observed transcription. I have been using photo-activatable-GFP (paGFP) as a reciprocal and complimentary means of probing the properties of the transcription ‘compartment’ formed by polymerase. I demonstrate that paGFP-tagged Pol II molecules are quickly lost early in the heat shock response when new molecules are still being actively recruited.  Upon extended gene activation by heat shock, when few new molecules are recruited to the heat shock genes, nearly all Pol II molecules are retained. Preliminary data suggests that poly-(ADP)ribose polymerase-1 (PARP-1), a chromatin binding protein, may be involved in compartment formation. We hypothesize that compartmentalization of Pol II increases the local concentration around the gene eliminating recruitment as a rate-limiting step in achieving maximal transcriptional output.

Katherine Kieckhafer received her Bachelor’s degree at the University of Michigan in 2005.  There she worked in the laboratory of Dr. Jorge Iñiguez-Lluhí studying the SUMO-modification of transcription factors. She is currently a third year graduate student in John Lis’ laboratory in the field of Biochemistry, Molecular, and Cellular Biology in the department of Molecular Biology and Genetics. In the Lis Lab, Katherine is using quantitative fluorescent methods and live cell imaging to probe the dynamics of RNA Polymerase II during several stages of the transcription cycle including, pausing, activation, and compartmentalization.