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.Eukaryotic genomes are organized into structurally and functionally distinct domains as one layer of transcriptional regulation to allow the expression of particular sets of genes when required and to restrict their expression when necessary. Mechanisms of activation usually involve DNA-bound transcription factors that recruit RNA polymerase or general transcription factors or recruit proteins that promote the formation of chromatin structures compatible with RNA polymerase preinitiation complex formation and transcriptional elongation. Repressive chromatin domains can inhibit gene expression at either of these stages. Chromatin boundary elements function to separate chromosomal domains so that regulatory regions of one domain do not inappropriately influence adjacent domains, either by insulating promoters from activation or by acting as a barrier to propagating repressive heterochromatin (55, 58).Evidence has accumulated over the past several years that RNA polymerase III (Pol III) promoter sequences, mainly studied using tRNA genes (tDNAs), can possess an intrinsic chromatin boundary activity. This was first demonstrated at the heterochromatic HMR locus in Saccharomyces cerevisiae, as the downstream tDNA is a critical component of the barrier that prevents the inappropriate spreading of silencing from HMR (16), and the characterization of this activity was the first demonstration of a natural chromatin boundary in yeast. Another yeast tDNA, TRT2, was shown to prevent the...