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, Sep 28, 2015
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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.
    Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 09/2012; 1829(s 3–4):418–424. DOI:10.1016/j.bbagrm.2012.09.006 · 6.33 Impact Factor
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    • "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). "
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    ABSTRACT: Budding yeast, like other eukaryotes, carries its genetic information on chromosomes that are sequestered from other cellular constituents by a double membrane, which forms the nucleus. An elaborate molecular machinery forms large pores that span the double membrane and regulate the traffic of macromolecules into and out of the nucleus. In multicellular eukaryotes, an intermediate filament meshwork formed of lamin proteins bridges from pore to pore and helps the nucleus reform after mitosis. Yeast, however, lacks lamins, and the nuclear envelope is not disrupted during yeast mitosis. The mitotic spindle nucleates from the nucleoplasmic face of the spindle pole body, which is embedded in the nuclear envelope. Surprisingly, the kinetochores remain attached to short microtubules throughout interphase, influencing the position of centromeres in the interphase nucleus, and telomeres are found clustered in foci at the nuclear periphery. In addition to this chromosomal organization, the yeast nucleus is functionally compartmentalized to allow efficient gene expression, repression, RNA processing, genomic replication, and repair. The formation of functional subcompartments is achieved in the nucleus without intranuclear membranes and depends instead on sequence elements, protein-protein interactions, specific anchorage sites at the nuclear envelope or at pores, and long-range contacts between specific chromosomal loci, such as telomeres. Here we review the spatial organization of the budding yeast nucleus, the proteins involved in forming nuclear subcompartments, and evidence suggesting that the spatial organization of the nucleus is important for nuclear function.
    Genetics 09/2012; 192(1):107-29. DOI:10.1534/genetics.112.140608 · 5.96 Impact Factor
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    ABSTRACT: Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes.
    The EMBO Journal 11/2011; 31(2):330-50. DOI:10.1038/emboj.2011.406 · 10.43 Impact Factor
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