Extra TF III C (ETC) sites are chromosomal locations bound in vivo by the RNA polymerase III (Pol III) transcription factor III C (TF III C) complex, but are not necessarily associated with Pol III transcription. Although the location of ETC sequences are conserved in budding yeast, and similar sites are found in other organisms, their functions are largely unstudied. One such site, ETC6 in Saccharomyces cerevisiae, lies upstream of TFC6, a gene encoding a subunit of the TF III C complex itself. Promoter analysis shows that the ETC6 B-box sequence is involved in autoregulation of the TFC6 promoter. Mutation of ETC6 increases TFC6 mRNA levels, whereas mutation immediately upstream severely weakens promoter activity. A temperature-sensitive mutation in TFC3 that weakens DNA binding of TF III C also results in increased TFC6 mRNA levels; however, no increase is observed in mutants of TF III B or Pol III subunits, demonstrating a specific role for the TF III C complex in TFC6 promoter regulation. Chromatin immunoprecipitation shows an inverse relationship of TF III C occupancy at ETC6 versus TFC6 mRNA levels. Overexpression of TFC6 increases association of TF III C at ETC6 (and other loci) and results in reduced expression of a TFC6 promoter-URA3 reporter gene. Both of these effects are dependent on the ETC6 Bbox. These results demonstrate that the TFC6 promoter is directly regulated by the TF III C complex, a demonstration of an RNA polymerase II promoter being directly responsive to a core Pol III transcription factor complex. This regulation could have implications in controlling global tRNA expression levels.T he eukaryotic RNA polymerase III (Pol III) system is responsible for synthesizing transfer RNA molecules and other transcripts, which in yeast include the U6 spliceosomal RNA, 7SL RNA, 5S ribosomal RNA, snr52 small nucleolar RNA, and the RNA component of RNaseP (1-3). Transcription by Pol III requires the activity of the multisubunit transcription factor III C (TF III C) complex, which binds to conserved A-box and B-box Pol III promoter elements and functions to overcome chromatin repression of Pol III transcription and to recruit the TF III B complex (4-6). Although Pol III and its transcription factors are thought to be dedicated to transcription of these specific genes, a growing body of evidence has shown that both partial and complete chromosomally bound Pol III complexes can have effects on nearby RNA polymerase II (Pol II) promoters (7-11). Chromatinbound Pol III complexes also mediate other extratranscriptional functions, including targeting Ty element integration (12-14), blocking replication fork progression (15), condensin and cohesin recruitment (16,17), and direct inhibition of transcription from nearby Pol II promoters (9,(18)(19)(20).Studies in both budding and fission yeast initially identified the presence of genome sequences that bind the TF III C complex, but not Pol III transcription factors TF III A and TF III B or the Pol III enzymatic complex itself (10,(21)(22)(23). Recently, simila...

In Saccharomyces cerevisiae, heterochromatic silencing occurs at the cryptic‐mating loci HMRa and HMLα, ribosomal DNA, and telomeres. Propagation of silent heterochromatin is sequence‐independent mediated by the Silent Information Regulator proteins (e.g. Sir3p) and other factors involved in repression. Rpd3p is part of a larger histone deacetylase complex that represses transcription when targeted by promoter‐specific transcription factors. In yeast, RPD3 deletion surprisingly enhances silencing at the cryptic mating loci and telomeres (Vannier et al. 1996), even overriding the tDNA barrier element adjacent to the HMR locus (Donze and Kamakaka, 2001, Jambunathan et al 2005). To understand the mechanism of enhanced silencing in strains lacking RPD3, we mutagenized rpd3D strains containing an ADE2 marker gene adjacent to HMR and identified suppressor mutants no longer displaying enhanced silencing. We identified seven genes which may affect heterochromatin formation in rpd3Δ backgrounds: BRE1, GDH2, BRE2, GAT3, QNS1, NPT1 and RXT3. After deleting each of these genes in the rpd3Δ background, only BRE1 or BRE2 deleted strains crossed with rpd3Δ strains showed a loss of extended silencing at the HMR and a marked telomere. BRE1 and BRE2 are indirectly and directly involved in the tri‐methylation of histone H3 lysine 4 respectively. Other studies of histone H3 lysine 4 methylation have suggested that mutations in this pathway lead to a re‐distribution of Sir proteins at silenced loci (Madhani et al. 2007). We hypothesize that the increased silencing in rpd3D mutants might also be due to redistribution of chromatin proteins. Future experiments will utilize ChIP‐Seq and DamID methodologies to determine Sir3p occupancy at HMR and genome‐wide euchromatic regions in WT vs. rpd3Δ strains. Support for this project was provided by a grant from the National Science Foundation.

Several studies have identified the paradoxical phenotype of increased heterochromatic gene silencing at specific loci that results from deletion or mutation of the histone deacetylase (HDAC) gene RPD3. To further understand this phenomenon, we conducted a genetic screen for suppressors of this extended silencing phenotype at the HMR locus in Saccharomyces cerevisiae. Most of the mutations that suppressed extended HMR-silencing in rpd3 mutants without completely abolishing silencing were identified in the histone H3 lysine 4 methylation (H3K4me) pathway, specifically in SET1, BRE1 and BRE2. These second site mutations retained normal HMR silencing, therefore appear to be specific for the rpd3Δ extended silencing phenotype. As an initial assessment of the role of H3K4 methylation in extended silencing, we rule out some of the known mechanisms of Set1p/H3K4me mediated gene repression by HST1, HOS2 and HST3 encoded HDACs. Interestingly, we demonstrate that the RNA Polymerase III complex remains bound and active at the HMR-tDNA in rpd3 mutants despite silencing extending beyond the normal barrier. We discuss these results as they relate to the interplay among different chromatin modifying enzyme functions and the importance of further study of this enigmatic phenomenon.

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