Role of chromatin in promoter proximal pausing in Drosophila

Open Access
Author:
Achary, Bhavana Gopalakrishnan
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
November 19, 2012
Committee Members:
  • David Scott Gilmour, Dissertation Advisor
  • David Scott Gilmour, Committee Chair
  • Song Tan, Committee Member
  • Joseph C. Reese, Committee Member
  • Yanming Wang, Committee Member
  • Pamela Hankey Giblin, Special Member
Keywords:
  • chromatin
  • pausing
  • nucleosome positioning
  • histone deacetylases
Abstract:
RNA polymerase II pauses in the promoter proximal region of thousands of genes. Amongst the various factors that contribute to pausing, chromatin architecture in the promoter proximal region is thought to be one of them. In vitro experiments have shown that nucleosomes can impede elongation of RNA Pol II. Genome wide maps of Pol II and nucleosomes in Drosophila indicate a dynamic interplay between the two. Currently, there are two prevalent models regarding the relationship between paused Pol II and nucleosome positioning. The first model indicates that a positioned nucleosome could contribute to pausing of RNA Pol II. The second model suggests that it is the paused Pol II that contributes to determining nucleosome organization in the promoter proximal region of a gene. In this study, I asked the question, what is the effect on nucleosome position when the position of paused Pol II is changed? To answer this, I determined the position of paused Pol II in embryos from wild type flies and flies with a mutant form of RNA Pol II. Permanganate footprinting experiments showed that the mutant RNA Pol II is paused closer to the transcription start site than in wild type. I mapped the nucleosomes in wild type and mutant to see if the shift in the paused Pol II affected the nucleosome position. The results indicate that the position of +1 nucleosome does not change when the position of paused RNA Pol II is shifted. Pausing is widely recognized as an important step in regulation of gene expression and the mechanism of pausing and the factors involved are under investigation. The Drosophila hsp70 gene has long been regarded as a model gene to study promoter proximal pausing. Transcription of the hsp70 gene involves distinct steps, which include establishment of a paused Pol II, activation and release of the paused Pol II into productive elongation. Various factors are implicated in regulating these distinct steps. I have investigated the effects of depleting some of these factors on transcription of hsp70. Using a beta-galactosidase reporter assay as an initial screen, I chose several factors that when depleted, showed reduced hsp70 transcription. Some of the factors are known to be important in transcription of hsp70, such as the activator HSF and the Pol II CTD kinase, PTEF-b. One surprising result was the effect of depletion of HDAC3. HDAC3 depletion resulted in a decreased level of hsp70 expression, both in the beta-gal assay and mRNA measurements. Upon further investigation, I observed that HDAC3 depletion affected RNA Pol II recruitment and impaired pausing on the hsp70 gene.