TFIIIC binding sites function as both heterochromatin barriers and chromatin insulators in Saccharomyces cerevisiae

Chromosomal sites of RNA polymerase III (Pol III) transcription have been demonstrated to have "extratranscriptional" functions, as the assembled Pol III complex can act as chromatin boundaries or pause sites for replication forks, can alter nucleosome positioning or affect transcription of neighboring genes, and can play a role in sister chromatid cohesion. Several studies have demonstrated that assembled Pol III complexes block the propagation of heterochromatin-mediated gene repression. Here we show that in Saccharomyces cerevisiae tRNA genes (tDNAs) and even partially assembled Pol III complexes containing only the transcription factor TFIIIC can exhibit chromatin boundary functions both as heterochromatin barriers and as insulators to gene activation. Both the TRT2 tDNA and the ETC4 site which binds only the TFIIIC complex prevented an upstream activation sequence from activating the GAL promoters in our assay system, effectively acting as chromatin insulators. Additionally, when placed downstream from the heterochromatic HMR locus, ETC4 blocked the ectopic spread of Sir protein-mediated silencing, thus functioning as a barrier to repression. Finally, we show that TRT2 and the ETC6 site upstream of TFC6 in their natural contexts display potential insulator-like functions, and ETC6 may represent a novel case of a Pol III factor directly regulating a Pol II promoter. The results are discussed in the context of how the TFIIIC transcription factor complex may function to demarcate chromosomal domains in yeast and possibly in other eukaryotes.