Nucleoporin Mediated Nuclear Positioning and Silencing of HMR

Citation: Ruben GJ, Kirkland JG, MacDonough T, Chen M, Dubey RN, et al. ( Nucleoporin Mediated Nuclear Positioning and Silencing of HMR Giulia J. Ruben 0 Jacob G. Kirkland 0 Tracy MacDonough 0 Miao Chen 0 Rudra N. Dubey 0 Marc R. Gartenberg 0 Rohinton T. Kamakaka 0 Axel Imhof, Ludwig-Maximilians-Universitat M unchen, Germany 0 1 Department of Molecular Cell Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America, 2 Department of Pharmacology, University of Medicine Dentistry New Jersey - Robert Wood Johnson Medical School , Piscataway , New Jersey, United States of America, 3 Department of Molecular Biology and Biochemistry, Rutgers University , Piscataway, New Jersey , United States of America 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. - Funding: This work was supported by a grant from the NIH (GM078068) to RTK, (T32-GM008646) to GR and (GM51402) to MRG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The regulation of gene expression is governed by the interactions between positive and negative transcription factors and DNA sequence elements. The higher order organization of chromatin domains within the nucleus also contributes to the regulation of gene activity [1]. Transcriptionally active genes adopt a more open chromatin conformation while silenced genes are present within highly inaccessible chromatin domains. These open and closed chromatin domains are not randomly distributed throughout the nucleus but localize to specific regions. In Saccharomyces cerevisiae heterochromatin is established and maintained through the coordinated actions of DNA sequence elements, known as silencers, and repressor proteins [2]. Silencers autonomously initiate silencing by the recruitment of sequence specific factors, which in turn recruit repressor proteins, including the Sir proteins, which deacetylate and bind histones forming a repressed chromatin domain. Sub-telomeric domains account for the majority of the heterochromatin in yeast. These domains cluster together and are tethered to the nuclear envelope in multiple foci [3,4,5] by Ku70/80, Sir4, Esc1 [3,6,7] and Mps3 [8]. The clustering of the telomeres at the nuclear periphery thus creates a silent compartment in the eukaryotic nucleus [9] located at the nuclear membrane but excluded from the nuclear pores [4,5,7] and while nuclear membrane association increases the likelihood of a locus becoming silenced, it is not essential for silencing [10]. The cryptic mating type loci HMR and HML are also silent in yeast. Repression at these loci is mediated by their cognate silencers, but also depends in part on their proximity to telomeric heterochromatin [11,12]. HMR and HML colocalize with telomeric foci at the nuclear periphery [10]. Repression can be restored to a silencer-crippled HMR locus by artificially recruiting it to the nuclear membrane, bringing it in spatial proximity to telomeric heterochromatin and the perinuclear-silencing compartment [13]. Numerous reports have recently shown that nuclear pore proteins (NUPs) play a role in regulating gene activity by affecting the intranuclear position of a genomic locus [14]. Numerous NUPs like Nic96, Nup2 and Nup60 physically associate with active genes [15,16] and it has been shown that some genes become NPC-associated upon activation [15,17,18,19]. The localization of genes at or near nuclear pore complexes (NPCs) has been shown to facilitate efficient transcription [17]. While association with the NPC is not necessary for gene activation, such association maximizes transcriptional activity. Interestingly, mutations in members of the Nup84 complex have been shown to alter telomere organization [20,21,22]. Furthermore, Nup2 is enriched at intergenic regions of subtelomeric genes and deletion of Nup2, a mobile basket NUP, leads to mild derepression of sub-telomeric loci [23] suggesting a direct or indirect role for NUPs in the regulation of these silenced loci. Taken together, these data provide evidence for a role for NUPs in transcriptional regulation of genes. Silenced and active chromatin domains reside adjacent to one another and insulator elements are DNA elements that functionally separate them. In yeast barrier insulators restrict the action of silencers, blocking the spread of repression into neighboring active regions. Barriers are often highly active promoters of genes or have promoter characteristics, recruiting various chromatin remodeling and modifying factors to block the spread of silencing [24]. Loss of barrier function, either by mutation of the insulator element or proteins important for insulator activity, results in a spread of silencing into the neighboring region [25,26,27,28]. Barrier activity is commonly monitored by sensitive assays using reporter genes or by fine mapping of Sir proteins across the region in question. Ishii et al. showed that ectopic NUP recruitment at a modified silenced HML locus could insulate a reporter gene from silencing, and suggested that NUPs functioned as barrier proteins by anchoring the base of chromatin loops to the pore thus separating an active chromatin loop from a silenced chromatin loop [29]. Based on studies with these synthetic constructs it was suggested that recruitment of an insulator element to the nuclear pore complex was a critical step for insulation. Unfortunately the role of NUPs and NPC association at native insulators was not explored in this study. In th (...truncated)