Nucleoporin Mediated Nuclear Positioning and Silencing of HMR

Ludwig-Maximilians-Universität München, Germany
PLoS ONE (Impact Factor: 3.23). 07/2011; 6(7):e21923. DOI: 10.1371/journal.pone.0021923
Source: PubMed

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.

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Available from: Rohinton T Kamakaka
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    • "In Saccharomyces cerevisiae, subtelomeric regions and silent mating type loci represent repressed regions. The ability of Nups to influence the efficient silencing and localization of these regions remains under debate and requires further clarification (Galy et al. 2000; Feuerbach et al. 2002; Hediger and Gasser 2002; Hediger et al. 2002; Gartenberg et al. 2004; Therizols et al. 2006; Ruben et al. 2011; Van de Vosse et al. 2013). However, recent reports have proposed that a subset of scaffold components, including Nup133, Nup84, and Nup170, might participate in the efficient silencing of subtelomeric regions (Therizols et al. 2006; Van de Vosse et al. 2013). "
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    ABSTRACT: Nuclear pore complexes (NPCs) are composed of several copies of ∼30 different proteins called nucleoporins (Nups). NPCs penetrate the nuclear envelope (NE) and regulate the nucleocytoplasmic trafficking of macromolecules. Beyond this vital role, NPC components influence genome functions in a transport-independent manner. Nups play an evolutionarily conserved role in gene expression regulation that, in metazoans, extends into the nuclear interior. Additionally, in proliferative cells, Nups play a crucial role in genome integrity maintenance and mitotic progression. Here we discuss genome-related functions of Nups and their impact on essential DNA metabolism processes such as transcription, chromosome duplication, and segregation. © 2015 Ibarra and Hetzer; Published by Cold Spring Harbor Laboratory Press.
    Preview · Article · Feb 2015 · Genes & Development
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    • "Paradoxically, fluorescence microscopy studies showed that tT(AGU)C localized to the nucleolus infrequently even though the gene is transcribed (Donze and Kamakaka 2001; Valenzuela et al. 2008). Evidence that tT(AGU)C instead associates with NPCs emerged with the discovery that Nups bind the tDNA and direct the gene to the nuclear periphery (Ruben et al. 2011). In this study, we show that NPC tethering is a general feature of yeast tDNAs that occurs in M phase when transcription of the genes elevates. "
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    ABSTRACT: tRNAs are encoded by RNA polymerase III-transcribed genes that reside at seemingly random intervals along the chromosomes of budding yeast. Existing evidence suggests that the genes congregate together at the nucleolus and/or centromeres. In this study, we re-examined spatial and temporal aspects of tRNA gene (tDNA) expression. We show that tDNA transcription fluctuates during cell cycle progression. In M phase, when tRNA synthesis peaks, tDNAs localize at nuclear pore complexes (NPCs). Docking of a tDNA requires the DNA sequence of the contacted gene, nucleoporins Nup60 and Nup2, and cohesin. Characterization of mutants that block NPC localization revealed that docking is a consequence of elevated tDNA transcription. NPC-tDNA contact falters in the absence of the principal exportin of nascent tRNA, Los1, and genetic assays indicate that gating of tDNAs at NPCs favors cytoplasmic accumulation of functional tRNA. Collectively, the data suggest that tDNAs associate with NPCs to coordinate RNA polymerase III transcription with the nuclear export of pre-tRNA. The M-phase specificity of NPC contact reflects a regulatory mechanism that may have evolved, in part, to avoid collisions between DNA replication forks and transcribing RNA polymerase III machinery at NPCs.
    Preview · Article · May 2014 · Genes & development
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    • "The 3D clustering of tDNAs at the nucleolus is also dependent on condensins [70] but the loss of condensin mediated tDNA clustering within the nucleus does not appear to lead to a change in tDNA transcription suggesting that clustering is separable from tDNA transcription. Interestingly, the tRNA gene insulator adjacent to the silenced HMR locus does not localize with the nucleolus or centromere but associates with the nuclear pore [64] [72] though pore localization is also not necessary for insulation . Whether this tDNA recruits condensins is not known but this tDNA insulator does recruit cohesins [66], and mutations in the cohesins affect insulation [9], though the exact mechanism by which cohesins contribute to insulator activity is also unknown. "
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    ABSTRACT: tRNA genes (tDNAs) have been known to have barrier insulator function in budding yeast, Saccharomyces cerevisiae, for over a decade. tDNAs also play a role in genome organization by clustering at sites in the nucleus and both of these functions are dependent on the transcription factor TFIIIC. More recently TFIIIC bound sites devoid of pol III, termed Extra-TFIIIC sites (ETC) have been identified in budding yeast and these sites also function as insulators and affect genome organization. Subsequent studies in Schizosaccharomyces pombe showed that TFIIIC bound sites were insulators and also functioned as Chromosome Organization Clamps (COC); tethering the sites to the nuclear periphery. Very recently studies have moved to mammalian systems where pol III genes and their associated factors have been investigated in both mouse and human cells. Short interspersed nuclear elements (SINEs) that bind TFIIIC, function as insulator elements and tDNAs can also function as both enhancer — blocking and barrier insulators in these organisms. It was also recently shown that tDNAs cluster with other tDNAs and with ETCs but not with pol II transcribed genes. Intriguingly, TFIIIC is often found near pol II transcription start sites and it remains unclear what the consequences of TFIIIC based genomic organization are and what influence pol III factors have on pol II transcribed genes and vice versa. In this review we provide a comprehensive overview of the known data on pol III factors in insulation and genome organization and identify the many open questions that require further investigation. This article is part of a Special Issue entitled: Transcription by Odd Pols.
    Full-text · Article · Sep 2012 · Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
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