Nucleoporin mediated nuclear positioning and silencing of HMR.

The organization of chromatin domains in the nucleus is an important factor in gene regulation. In eukaryotic nuclei, transcriptionally silenced chromatin clusters at the nuclear periphery while transcriptionally poised chromatin resides in the nuclear interior. Recent studies suggest that nuclear pore proteins (NUPs) recruit loci to nuclear pores to aid in insulation of genes from silencing and during gene activation. We investigated the role of NUPs at a native yeast insulator and show that while NUPs localize to the native tDNA insulator adjacent to the silenced HMR domain, loss of pore proteins does not compromise insulation. Surprisingly we find that NUPs contribute to silencing at HMR and are able to restore silencing to a silencing-defective HMR allele when tethered to the locus. We show that the perinuclear positioning of heterochromatin is important for the NUP-mediated silencing effect and find that loss of NUPs result in decreased localization of HMR to the nuclear periphery. We also show that loss of telomeric tethering pathways does not eliminate NUP localization to HMR, suggesting that NUPs may mediate an independent pathway for HMR association with the nuclear periphery. We propose that localization of NUPs to the tDNA insulator at HMR helps maintain the intranuclear position of the silent locus, which in turn contributes to the fidelity of silencing at HMR.

Long-range communication between the silencers of HMR.

Gene regulation involves long-range communication between silencers, enhancers, and promoters. In Saccharomyces cerevisiae, silencers flank transcriptionally repressed genes to mediate regional silencing. Silencers recruit the Sir proteins, which then spread along chromatin to encompass the entire silenced domain. In this report we have employed a boundary trap assay, an enhancer activity assay, chromatin immunoprecipitations, and chromosome conformation capture analyses to demonstrate that the two HMR silencer elements are in close proximity and functionally communicate with one another in vivo. We further show that silencing is necessary for these long-range interactions, and we present models for Sir-mediated silencing based upon these results.

A tDNA establishes cohesion of a neighboring silent chromatin domain.

DNA replication generates sister chromatid pairs that are bound to one another until anaphase onset. The process, termed sister chromatid cohesion, requires the multisubunit cohesin complex that resides at centromeres and sites where genes converge. At the HMR mating-type locus of budding yeast, cohesin associates with a heterochromatin-like structure known as silent chromatin. In this report, we show that silent chromatin is necessary but not sufficient for cohesion of the replicating locus. A tRNA gene (tDNA) that delimits the silent chromatin domain is also required, as are subunits of the TFIIIB and RSC complexes that bind the gene. Non-tDNA boundary elements do not substitute for tDNAs in cohesion, suggesting that barrier activity is not responsible for the phenomenon. The results reveal an unexpected role for tDNAs and RNA polymerase III-associated proteins in establishment of sister chromatid cohesion.