Role of Inner Nuclear Membrane Protein Complex Lem2-Nur1 in Heterochromatic Gene Silencing*

Heterochromatin in the fission yeast Schizosaccharomyces pombe is clustered at the nuclear periphery and interacts with a number of nuclear membrane proteins. However, the significance and the factors that sequester heterochromatin at the nuclear periphery are not fully known. Here, we report that an inner nuclear membrane protein complex Lem2-Nur1 is essential for heterochromatin-mediated gene silencing. We found that Lem2 is physically associated with another inner nuclear membrane protein, Nur1, and deletion of either lem2 or nur1 causes silencing defect at centromeres, telomeres, and rDNA loci. We analyzed the genome-wide association of Lem2 using ChIP sequencing and we found that it binds to the central core region of centromeres, in striking contrast to Chp1, a component of pericentromeric heterochromatin, which binds H3K9me-rich chromatin in neighboring sequences. The recruitment of Lem2 and Nur1 to silent regions of the genome is dependent on H3K9 methyltransferase, Clr4. Finally, we show that the Lem2-Nur1 complex regulates the local balance between the underln]Snf2/HDAC-containing repressor complex (SHREC) histone deacetylase complex and the anti-silencing protein Epe1. These findings uncover a novel role for Lem2-Nur1 as a key functional link between localization at the nuclear periphery and heterochromatin-mediated gene silencing.

Regulation of eukaryotic genome function involves the differential organization of chromatin into euchromatic and heterochromatic domains distinguished on the basis of their appearance, structure, localization and function (1)(2)(3)(4)(5). Heterochromatin is a silent chromatin structure that represses gene expression and recombination to maintain genome integrity. In the fission yeast Schizosaccharomyces pombe, heterochromatic loci are restricted to the pericentromeric DNA regions, mating type loci and telomeres. The centromeres are surrounded by DNA repeats that compose an outer repeat (otr) region (containing dg and dh repeats) and innermost repeat (imr) region, which flank the central core (cnt) domain. These repeats are thought to be transposon remnants and are required for centromere assembly and proper chromosome segregation (6 -8). Assembly of heterochromatin in fission yeast requires methylation of histone H3 at lysine 9 (H3K9) by the conserved Clr4 methyltransferase. H3K9 methylation requires components of the RNA interference (RNAi) machinery. RNA transcripts act as a scaffold for the assembly of RNAi and chromatin modifying factors that initiate the formation of heterochromatin. Methylation of H3K9 recruits chromodomain containing HP1 proteins, Swi6 and Chp2, which are critical for heterochromatic gene silencing (9 -15). Cytological studies have shown that heterochromatin has a tendency to associate within the nuclear periphery, raising the possibility that proximity to the nuclear envelope facilitates heterochromatin-mediated gene silencing (3,16). In S. pombe, for example, telomeres form clusters at the nuclear periphery. Localization of transcriptionally silent domains toward the nuclear periphery has also been observed in budding yeast flies and plants (17). Observations in higher eukaryotes also suggest a role for nuclear periphery in gene silencing. In human cells, gene-poor chromosomes as well as the inactive X chromosome or Barr body tend to localize at the nuclear periphery. In metazoans, lamina-associated domains (lamina-associated domains), which lies beneath the inner nuclear membrane, are enriched for repressed chromatin state (18). This peripheral association of these domains is linked to repressive H3K9 methylation, a hallmark of repressive chromatin. In humans, lamina-associated domains constitute around 40% of mammalian genome (19,20). Similarly, in Caenorhabditis elegans, lamina-associated domains constitute repeat-rich regions, and knockdown of lamina-associated proteins EMR-1 and LEM-2 leads to derepression of perinuclear heterochromatin (21,22). These studies suggest that attachment of chromosome domains to the nuclear periphery can affect its expression. In support of this, artificial tethering of transcriptionally active loci in yeast and mammals become repressed when tethered to the nuclear periphery (2). Lamins have been shown to interact with several inner nuclear membrane proteins (INM). 2 Several lamin-associated proteins contain a LEM domain (LAP2-Emerin-MAN1), a 40-amino acid helix-extension-helix motif that is conserved from yeast to humans (23,24). S. pombe contain three INM (Lem2, Man1, and Ima1) proteins that show homology with lamin-associated proteins (25). Lem2 and Man1 contain a helix-extension-helix motif homologous to metazoan LEM domain. Recently, Lem2 and Man1 have been shown to be important for nuclear structure integrity and telomere anchoring with nuclear membrane in fission yeast (26). Furthermore, in the Saccharomyces cerevisiae Lem2-Nur1 homologue, Heh1-Nur1 forms a CLIP complex (chromosome linkage INM proteins) that physically links rDNA repeats to nuclear periphery. CLIP is required for the maintenance of rDNA repeat stability and not for their silencing. Deletion of either heh1 or nur1 causes release of rDNA repeats from the nuclear periphery and leads to chromosome instability by promoting aberrant recombination events in the rDNA repeats. In addition, artificial tethering of rDNA repeats to nuclear periphery partially suppresses repeat instability in heh1 or nur1 mutant cells (27). Although the localization of fission yeast heterochromatin at the nuclear periphery is thought to be important for gene regulation, the functional significance and the factors that sequester heterochromatin at the nuclear periphery are not fully known. Here, we show that inner nuclear membrane protein Lem2 and its interacting partner Nur1 are required for heterochromatin silencing in S. pombe. We found that deletion of lem2 or nur1 causes silencing defects at centromeres, telomeres and at rDNA loci. We further determined the genome-wide localization of Lem2 and demonstrated that it associates specifically with silent regions of the genome in a heterochromatin-dependent manner. Furthermore, we show that the Lem2-Nur1 complex regulates the balance between chromatin binding of the histone deacetylase complex Snf2/HDAC-containing repressor complex (SHREC) and anti-silencing protein Epe1. Overall, our results uncover a novel role for the Lem2-Nur1 complex in heterochromatin gene silencing in S. pombe. These findings add a new perspective to the evolutionarily conserved LEM domain proteins and suggest that the mammalian homologues might also play crucial roles in heterochromatin gene silencing.

Lem2-Nur1 Complex Is Essential for Heterochromatic Gene
Silencing-To identify novel factors required for heterochromatin gene silencing and its localization toward the nuclear periphery, we identified two inner nuclear membrane proteins in fission yeast, Lem2, a conserved LEM domain protein, and Nur1, a nuclear rim protein. Homologs of Lem2 and Nur1 in S. cerevisiae have been shown to be required for peripheral localization of heterochromatin but not for silencing (27). We used co-immunoprecipitation assays to demonstrate that in fission yeast Lem2 and Nur1 were physically associated. We performed immunoprecipitation experiments from cells that expressed functional C-terminally TAP-tagged Lem2 and C-terminally Myc-tagged Nur1 expressed under the control of their endogenous promoters. As shown in Fig. 1A, Nur1-Myc co-immunoprecipitated with Lem2-TAP. To gain insight about the role of the Lem2-Nur1 complex in heterochromatin gene silencing, we deleted either lem2 or nur1 or both in a strain that has ura4 ϩ reporter inserted at the centromere (Fig. 1B). Deletion of either lem2 or nur1 leads to a loss of gene silencing that is observed with an imr1R::ura4 ϩ reporter gene and a loss of growth on 5-FOA medium (Fig. 1C, 5-FOA is toxic to cells that express ura4 ϩ ). The silencing phenotypes were verified by quantitative RT-PCR (qRT-PCR) to examine the levels of inserted ura4 ϩ reporter gene (imr1::ura4 ϩ ) or endogenous heterochromatic transcripts originating from the dg elements of centromeres. clr4⌬, in which heterochromatin is completely disrupted, was used as a positive control. We found that inserted ura4 ϩ and centromeric dg transcripts were derepressed in lem2⌬ and nur1⌬ cells, although to a lesser extent than in clr4⌬ cells (Fig. 1, D and E). Consistent with the silencing and qRT-PCR results, occupancy of polymerase II at the centromeric dg region increased in lem2 and nur1 mutant cells (Fig. 1F). Centromeric heterochromatin is important for chromosome segregation, and any perturbation of centromeric heterochromatin causes defects in chromosome segregation (31,32). We observed that like clr4⌬ cells, lem2⌬ and nur1⌬ cells showed hypersensitivity to the microtubule-destabilizing drug thiabendazole (TBZ) (Fig. 1G). These results indicate that the Lem2-Nur1 complex is required for heterochromatic gene silencing at pericentromeric heterochromatin.
Lem2-Nur1 Binds to Central Core Region of Centromeres, Telomeres, and rDNA Repeats-To gain further insight into the role of Lem2-Nur1 in heterochromatin organization, we constructed strains expressing Lem2 or Nur1 with a C-terminal TAP tag. We performed ChIP-seq analysis of Lem2 and Chp1. Interestingly, Lem2 was strictly confined to the central core cnt1 domain and was excluded from the pericentromeric regions, whereas Chp1 was enriched at the outer repeats (Fig. 2, B and C). The ChIP-seq data were further validated by chromatin immunoprecipitation (ChIP-qPCR) experiments at dg and central core regions (Fig. 2, D and E). Furthermore, ChIP analysis of Nur1 showed a similar binding pattern as that of lem2 (Fig. 2, F and G). In addition to centromeres, genome-wide distribution of Lem2 showed that it also associates with other silent regions of the genome-like telomeres and rDNA repeats (Fig. 3, A-D). Similar to pericentromeric heterochromatin, deletion of either Lem2 or Nur1 leads to derepression of telomeres (Fig. 3E). Interestingly, we did not see any enrichment of Lem2 or Nur1 to euchromatin regions of the genome. These findings indicate that regulation of heterochromatin gene silencing by Lem2 and Nur1 is through direct association with centromeric as well as with other silent regions of the genome.
Chromatin Binding of Lem2-Nur1 Is Dependent on Clr4 -Heterochromatin has been shown to serve as a platform for the recruitment of a diverse pool of cellular factors (33,34). Because Lem2-Nur1 complex specifically localizes to heterochromatic loci, we explored whether Lem2-Nur1 recruitment to heterochromatic loci is dependent on heterochromatin. We constructed strains expressing Lem2 or Nur1 with a C-terminal TAP tag in clr4 deletion background. ChIP-seq analysis revealed deletion of H3K9-specific methyltransferase, Clr4 abolished lem2 localization at centromeres (Fig. 4, A and B), telomeres, and the DNA repeats (data not shown). The ChIPseq experiments were further verified by ChIP experiments, which showed complete loss of Lem2-Nur1 binding in clr4deleted cells (Fig. 4, C and D). Thus, effects of mutations in heterochromatin assembly factor Clr4 impairs Lem2-Nur1 recruitment. These observations suggest that heterochromatin serves as a platform for binding of the Lem2 and Nur1 complex, which tethers heterochromatin towards the nuclear periphery that ensures proper silencing.
Lem2-Nur1 regulates SHREC and Epe1 Localization to Heterochromatin-In S. pombe H3K9 methylation is a key event in the formation of heterochromatin. This mark serves as a binding site for Swi6, which plays a critical role in maintaining chromatin structure and ensuring faithful chromosome segregation during cell division (15)(16)(17)(18). To gain insight into the molecular mechanism of the Lem2-Nur1 complex in heterochromatin gene silencing, we looked at the effect of deleting lem2 on H3K9me2 and Swi6 enrichment at centromeric heterochromatin. Cells lacking Lem2 displayed a drop in levels of H3K9me2 and Swi6 (Fig. 5, A and B). A similar effect was seen on levels of H3K9me2 and Swi6 in nur1-deleted cells. Because deletion of lem2 and nur1 did not greatly affect H3K9me and Swi6 levels, we speculated whether deletion of lem2 or nur1 impairs the recruitment of other factors that are important for silencing. To test this, we took a candidate-based approach and determined the recruitment of RNAi-linked Snf2-HDAC repressor complex (SHREC) and anti-silencing protein Epe1 in lem2 deletion cells, because these factors have previously been shown to work antagonistically in regulation of heterochromatin gene silencing downstream of H3K9me (35,36). SHREC belongs to Snf2/HDAC repressor complex family  SEPTEMBER 16, 2016 • VOLUME 291 • NUMBER 38 and contains the class II HDAC Clr3 that targets lysine 14 of histone H3. Epe1 is a conserved Jumonji C domain nuclear protein that antagonizes heterochromatization. Inactivation of Epe1 enhances reporter gene silencing and promotes heterochromatin spreading across boundary elements, while its overexpression abrogates heterochromatin structure and impairs centromere functions (36). We constructed strains expressing either FLAG-tagged Clr1, a component of SHREC, or FLAG-tagged Epe1 in lem2 deletion cells and looked at binding of SHREC or Epe1 in lem2-deleted cells by chromatin immunoprecipitation. Interestingly, we found increased association of Epe1 and decreased SHREC/Clr1 binding in lem2-deleted cells (Fig. 5, C and D). Consistent with the ChIP data, the acetylation levels were greatly increased in cells lacking either lem2 or nur1 (Fig. 5E). These results suggest that Lem2-Nur1 promotes SHREC binding while preventing Epe1 localization at centromeres.

Discussion
The eukaryotic genome within the nucleus is spatially segregated into euchromatin and heterochromatin, with heterochromatin often associated with the nuclear envelope (2, 3). These specific spatiotemporal distributions correlate with the cell's functional state (37)(38)(39). In fission yeast, heterochromatin domains are localized toward the nuclear periphery and interact with a number of nuclear membrane proteins (2,3,6). This localization is thought to present a sub-compartment enriched for factors required for heterochromatin silencing and with properties distinct from the nuclear interior (12,40). This perinuclear sequestration of heterochromatin is conserved from yeast to humans (41)(42)(43)(44). However, the significance of this peripheral localization in terms of heterochromatin stability and the proteins responsible for this perinuclear sequestration are not fully understood. This study identified and character-

Lem2-Nur1 in Heterochromatic Gene Silencing
ized a novel inner nuclear membrane protein complex, Lem2-Nur1, as being essential for heterochromatin-mediated gene silencing in fission yeast. Deletion of either lem2 or nur1 causes silencing defects both at centromeres and at telomeres. Compared with clr4 deletion, where heterochromatin is completely disrupted, lem2-and nur1-deleted cells show significant derepression of transcription within centromeric and telomeric heterochromatin (Figs. 1 and 3). Interestingly, ChIP and ChIPseq data showed that Lem2 and Nur1 strictly bind to the central core cnt1 domain of centromeres, which is involved in kinetochore assembly and deletion of lem2 and nur1 causes segregation defects possibly through defects in kinetochore assembly (Fig. 1). Interestingly, unlike lem2 and nur1, which are important for tethering and silencing of all the heterochromatin domains in S. pombe, the budding yeast homologs Heh1 and Nur1, which forms the CLIP complex are implicated in tether-ing and stability of rDNA repeats and not in silencing. It would be interesting to investigate whether the Lem2-Nur2 complex in S. pombe does play a role in stability of rDNA repeats.
Even though tethering and silencing have been mechanistically separated from each other, both processes reinforce each other (16,45). Indeed, the mechanisms used for the recruitment are often epigenetic marks established during heterochromatin formation. In case of budding yeast, deacetylation of histone H4 is necessary for SIR protein recruitment. Similarly, in C. elegans, methylation of H3K9 is required for SET-25 localization with peripheral heterochromatin (2,46). In tune with the above findings, Lem2-Nur1 recruitment to repressive chromatin depends on heterochromatin (Fig. 4).
On the mechanistic level, the function of Lem2-Nur1 involves the recruitment of the repressor complex SHREC to heterochromatin, which contributes to repression. SHREC is the Snf2-HDAC-containing repressor complex, which deacetylates histone H3 and H4 and remodels chromatin structure at heterochromatic regions, resulting in transcriptional gene silencing and heterochromatin spreading (Fig. 6) (35). Deletion of lem2 or nur1 causes an increase in binding of Epe1, a Jumonji C domain protein that blocks spreading of H3K9me at heterochromatic barriers (36). Epe1 counteracts SHREC and targeting of Epe1 causes an increase in transcription at heterochromatin and a defect in heterochromatin spreading. Furthermore, loss of lem2 or nur1 increases the acetylation levels at the silent regions (Fig. 5C). An independent study also found Lem2 being essential for silencing and heterochromatin localization to nuclear periphery. It demonstrated that silencing by Lem2 is epistatic with SHREC and opposes the function of anti-silencing protein Epe1. Furthermore, it elegantly shows that Lem2 collaborates with multiple factors like Csi1 to ensure proper heterochromatin silencing and its localization at the nuclear periphery (34). Regulation of SHREC and Epe1 binding by Lem2-Nur1 adds an unexpected functional layer in the regulation of heterochromatin silencing. We speculate that Clr4 mediated methylation of H3K9 and subsequent binding of Swi6 to methylated histones localizes heterochromatin to the nuclear periphery. Localization to the nuclear periphery depends on interaction with the inner nuclear membrane protein complex Lem2-Nur1, which then regulates binding of SHREC (Fig. 6). Given the evolutionary conservation of Lem2-Nur1, the mechanism described here may represent a widespread strategy for fine-tuning transcriptional gene silencing by localization of heterochromatin at the nuclear periphery.
In summary, we uncovered a multistep mechanism of heterochromatin gene silencing mediated by Clr4-dependent chromatin binding of Lem2-Nur1. Lem2-Nur1 regulates silencing by ensuring appropriate balance between binding of repressing SHREC and activating Epe1 factors. These factors modulate the levels of repressive and activating histone posttranslational modifications which in turn affect gene silencing.

Materials and Methods
Yeast Strains-Strains and plasmids used in this study are in Table 1 and were made by a PCR-based gene-targeting procedure (28).
Quantitative RT-PCR-Yeast cultures were grown in YES medium at 32°C to an A 600 of 0.5. Total RNA was isolated by hot phenol procedure and cleaned by using RNeasy kit (Qiagen) to remove potential genomic DNA contamination. Gene-specific primers for dg and tlh1 were used to prepare cDNA followed by quantitative PCR using Light Cycler. Relative RNA levels were calculated from C T values according to the ⌬C T method (Applied Biosystems) and normalized to act1 ϩ RNA levels.
ChIP and ChIP-Seq Assays-ChIP assays were performed as described previously (29). Cells were cross-linked with 10 mM dimethyl adipimidate and subsequently with 1% formaldehyde for 20 min at room temperature and quenched with 125 mM glycine for 5 min. Cross-linked chromatin was sonicated to yield DNA fragments of an average size of 200 -500 bp. Immunoprecipitations were carried out with anti-TAP (Applied Biosystems), anti-FLAG M2 (Sigma), and diMeH3K9 antibody (Abcam). Primers used in the PCRs were analyzed for linearity range and efficiency with a LightCycler (Applied Biosystems) to accurately evaluate occupancy (% of immunoprecipitation/input). The results shown with standard errors are based on three independent experiments. For ChIP-seq experiments, libraries for Illumina sequencing were constructed following the manufacturer's protocols, starting with ϳ5 ng of immunoprecipitated DNA fragments. Each library was generated with custom-made adaptors carrying unique barcode sequences at the ligating end (30). Bar-coded libraries were mixed and sequenced with Illumina HiSeq 2000. Raw reads were separated according to their barcodes and mapped to the S. pombe genome using Bowtie.  Mapped reads were normalized to reads per million and visualized in IGV.
Silencing Assays-Silencing assays were performed from overnight cultures grown in 5 ml of YEA (yeast extract supplemented with adenine). 10-Fold serial dilutions were made so that the highest density spot contained 1.2 ϫ 10 5 cells. Cells were spotted on normal YEA, 5-FOA, and TBZ plates (same as normal plates with the addition of 1g/liter 5-fluoro-orotic acid and 10 g/ml thiabendazole). The plates were then incubated at 32°C for 3 days.
Immunoprecipitations-For immunoprecipitations, nontagged control and Lem2 Tap-Nur1 Myc cells were harvested at A 600 ϭ 1.5-2 and resuspended in lysis buffer (20 mM Hepes-NaOH, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, pH 8.0, 0.5 mM DTT, 10% glycerol, 0.25% Triton X-100, 1 mM PMSF, complete Protease Inhibitor Mixture). Cells were disrupted by bead beating and were cleared by centrifugation at 13,000 rpm for 5 min. Protein concentrations were normalized using the Bio-Rad protein assay, and the supernatant was incubated with 30 l of IgG-conjugated Dynabeads for 2 h at 4°C. Beads were washed four times with ice-cold lysis buffer, and bound proteins were analyzed by SDS-PAGE followed by Western blotting using anti-Myc antibody.