Functional analyses of heat shock factor, GAGA factor and nucleosome remodeling factor in regulation of Drosophila HSP70

My doctoral work employed a transgenic approach to understand the roles of GAGA factor (GAF), Heat Shock Factor (HSF) and Nucleosome Remodeling Factor (NURF) in transcriptional control of the hsp70 gene in Drosophila. By fusing the activation domain of HSF to a modified form of the tetracycline repressor DNA-binding domain call Teton, I was able to mimic heat shock action by inducing Teton-HSF with doxycycline to bind transgenic target genes.;I used permanganate genomic footprinting to monitor the behavior of Pol II on my transgenic target genes in larval tissues. I was able to determine that the presence of a GAGA element in the transgene generated a paused Pol II, which turned out to be a prerequisite for the activation by Teton -HSF. I also discovered that promoter proximal pausing was not required for activation by Teton-VP16. However, the presence of paused Pol II greatly accelerated the rate of the induction by Teton-VP16 when binding was induced in tissues by addition of doxycycline.;I also investigated the role of the chromatin remodeling factor NURF. Previous work with reconstituted chromatin indicated that GAF and NURF function together to remodel chromatin so the transcriptional machinery can access the hsp70 promoter DNA. I made the surprising finding that paused Pol II was present on the hsp70 gene in NURF mutant salivary glands, suggesting that NURF is not required for transcription initiation. Instead, the NURF mutation seemed to reduce the stability of the paused Pol II normally found in the promoter proximal region prior to heat shock induction. Pol II tended to escape from the promoter region and to accumulate in the region 100 to 200 nucleotides downstream from the transcription start. Micrococal Nuclease analysis of chromatin structure indicated that NURF mutants cause a global defect of chromatin structure. We speculate that NURF stabilizes the paused Pol II on hsp70 by positioning a nucleosome in front of the Pol II.