Investigating the Effect of PARP Mediated Compartments on Transcription Dynamics

Abstract

Transcription of DNA into RNA is a fundamental, universal, and highly regulated process in all living organisms. RNA Polymerase II (Pol II), is the enzyme that catalyzes this reaction and it is highly regulated by the pausing factors DRB Sensitivity Inducing Factor (DSIF) and Negative Elongation Factor (NELF), and elongation factors, including Positive Transcription Elongation Factor b (P‐TEFb) and Polymerase Associated Factor 1 (PAF1). However, most previous studies of these factors have only provided snapshots of factor binding or snapshots of mRNA production following their depletion. While valuable, these studies provide limited information about the time frames in which Pol II and key transcription factors interact with each other and with the DNA. Using live‐cell imaging of amplified and interphase‐extended Drosophila polytene chromosomes with endogenously tagged transcription factors and Pol II, we can analyze the real time behavior of these molecules at single genetic loci and at high resolution. In particular, heat shock genes, like heat shock protein 70 (Hsp70), have been shown to be a robust system for studying transcription because of their inducible nature; they are able to transition quickly from a paused to a highly active state. Previous work from our lab utilizing this system suggested the formation of a transcription compartment during prolonged bouts of transcription. We propose that transcription compartments form around Hsp70 loci during an extended heat shock due to the activity of Poly ADP Ribose Polymerase (PARP) polymerizing large PAR chains that trap Pol II and other transcription factors, restricting their movement and preventing free diffusion with other molecules in the nucleus. We are tagging proteins of interest with photo‐activatable fluorescent proteins to minimize background by selectively activating a population of molecules only in the small 3‐D volume of specific loci. Utilizing this system we have begun measuring the dynamics of Pol II at specific loci and are working toward measuring the dynamics of key transcription regulating factors and further characterizing PARP‐mediated transcription compartments.