Doctor of Philosophy (PhD)
Boundary elements consisting of barriers and insulators are genomic sequence elements that along with their associated DNA-binding proteins block the spread of heterochromatin into euchromatic regions or prevent the targeted activation of promoters from distal/proximal enhancers, respectively. In Saccharomyces cerevisiae, the deletion of RPD3, a histone deacetylase, results in an extended SIR protein-mediated silencing effect bypassing a tRNAthr barrier element adjacent to the cryptic mating locus, HMRa. We mutagenized rpd3Δ strains and identified suppressor mutants through a genetic screen that no longer displayed this enhanced silencing effect. Our results identified BRE1 and BRE2, which are either directly or indirectly responsible for the tri-methylation of histone H3K4 and H3K79, as effectors of the rpd3Δ extended silencing effect at HMRa. We hypothesize that the increased silencing effect in rpd3Δ mutants is the result of a redistribution of SIR proteins which become concentrated at the HMRa region in response to a global change in the acetylation and/or methylation state of histones contingent on RPD3, BRE1, and BRE2. ETC, or Extra-TFIIIC, sites are genomic elements which bind the RNA Polymerase III transcription factor, TFIIIC. ETC sites contain B-box promoter sequences normally associated with RNA Polymerase III promoters, and their locations are over-represented between divergently transcribed RNA Polymerase II genes. Our results show that the transcription of TFC6, which codes for a DNA-binding component of TFIIIC, is auto-regulated by TFIIIC which binds to the ETC6 site in the TFC6 promoter region. Inhibition of TFIIIC binding to the ETC6 site results in increased TFC6 expression from its own promoter, and transcription of TFC6 is inversely correlated with TFIIIC binding to the ETC6 site. The TFC6 promoter is also down-regulated when its own gene product is over-expressed. We present here a novel function of gene regulation where a Pol III transcription factor directly (auto) regulates a Pol II gene. Our results also point to how this regulation might be mediated by an insulator-like function of TFIIIC which can implicate the functionality of Extra-TFIIIC sites in other eukaryotes.
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Kleinschmidt, Richard Alton, "Regulation of gene expression by chromatin boundary elements" (2011). LSU Doctoral Dissertations. 3965.