Nucleoporin Mediated Nuclear Positioning and Silencing of HMR

Abstract

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.

Structure and Function in the Budding Yeast Nucleus

“…Intriguingly, mutations in nuclear pore constituents affect both gene activation and repression (Dilworth et al 2005;Therizols et al 2006;Ruben et al 2011). This dual role does not appear to stem from the boundary activity of nuclear pore proteins, as reported by Ishii et al (2002).…”

“…This dual role does not appear to stem from the boundary activity of nuclear pore proteins, as reported by Ishii et al (2002). Indeed, it was shown that, despite the fact that nuclear pore proteins can bind the native tDNA insulator adjacent to HMR, the loss of their binding did not compromise insulator activity, even though they contributed to silencing at this locus (Ruben et al 2011). To reconcile the apparently conflicting role between NPCs and repressive telomeric foci, one might propose that the impact of NPCs on gene expression is not simply pore or position dependent, but depends on the binding of transactivators or repressors to cis-acting elements (Ruben et al 2011).…”

“…Subsequently, a series of genomewide studies suggested that nuclear pore components bind highly active genes (Casolari et al 2004(Casolari et al , 2005Schmid et al 2006). While some promoter-bound pore components are not stably bound to NPCs (Dilworth et al 2005;Therizols et al 2006;Ruben et al 2011), the tagging and localization of specific inducible genes confirmed that some genes indeed shift to NPCs upon activation (for reviews, see Akhtar and Gasser 2007;Taddei 2007;Dieppois and Stutz 2010;Egecioglu and Brickner 2011) (Figure 6).…”

“…Indeed, it was shown that, despite the fact that nuclear pore proteins can bind the native tDNA insulator adjacent to HMR, the loss of their binding did not compromise insulator activity, even though they contributed to silencing at this locus (Ruben et al 2011). To reconcile the apparently conflicting role between NPCs and repressive telomeric foci, one might propose that the impact of NPCs on gene expression is not simply pore or position dependent, but depends on the binding of transactivators or repressors to cis-acting elements (Ruben et al 2011). Consistent with this notion, increasing the association of the HXK1 subtelomeric gene with the nuclear periphery through a neutral anchor improved both its repression of glucose medium and its activation in the absence of glucose (Taddei et al 2006).…”

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data

“…Several other studies have shown an association of NPCs with silenced genomic regions and chromatin boundary. Data from yeast and Drosophila revealed heterochromatin association with stable Nups, such as Nup100 or Nup84 (Galy et al, 2000;Ruben et al, 2011;Van de Vosse et al, 2013). In human HeLa cells, ChIP analysis of Nup93 also demonstrated that Nup93 interactions include silent gene regions marked by repressive histone modifications (Brown et al, 2008).…”