Identification Of A Novel, Rapid Mechanism To Alleviate The Nucleosome Barrier To Transcription

To efficiently transcribe genes, RNA Polymerase II (Pol II) must overcome the barrier imposed by nucleosomes and higher order chromatin structure. Many genes, including Drosophila melanogaster Hsp70, undergo changes in chromatin structure upon activation. It has long been thought that changes to chromatin structure occur co-transcriptionally as a result of Pol II movement through the gene and recruitment of Pol II associated factors that disrupt chromatin structure. In this dissertation, I demonstrate that, upon activation, the changes in chromatin structure of Drosophila melanogaster's Hsp70 gene occur in an extremely rapid manner that is independent of act ive transcription of the gene. In addition, these changes extend beyond the gene encoding unit to natural chromatin insulating elements. From a series of targeted RNAi screens I indentified four proteins necessary for the rapid, transcriptionindependent loss of nucleosomes at the Hsp70 locus following heat shock and an ordered mechanism through which they function. The first factor identified, heat shock factor (HSF), is the master transcriptional activator of heat shock genes and is rapidly recruited to the gene within seconds of heat shock, and binds cooperatively with the second factor, GAGA Factor, already bound to the gene. HSF is necessary for the recruitment of the third identified factor, dTip60, a histone acetyltransferase that acetylates histo ne H2A lysine 5. This acetylation is necessary for the enzymatic activation of the fourth and final factor, Poly(ADP)-Ribose Polymerase (PARP), which catalyzes the formation Poly(ADP-ribose). PARP is associated with the 5' end of Hsp70 before heat shock, and its enzymatic activity is rapidly induced by heat shock. This activation causes PARP to redistribute throughout the Hsp70 loci and Poly(ADP-ribose) to concurrently accumulate in the wake of PARP's redistribution. Both the protein PARP and its catalytic activity are necessary for the rapid loss in nucleosome structure of Hsp70 upon heat shock and full transcriptional activation of Hsp70. In this dissertation I propose a novel mechanism to overcome the nucleosome barrier to achieve full transcriptional activation through the enzymatic activation of PARP which results in a rapid, transcription-independent, locus-wide disruption of chromatin structure