SUMOylation regulates Lem2 function in centromere clustering and silencing

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TL;DR

This study reveals that hyper-SUMOylation at the nuclear periphery in fission yeast disrupts centromere silencing but enhances centromere clustering, with the inner nuclear membrane protein Lem2 as a key SUMO substrate. Mutation of Lem2 SUMOylation sites rescues silencing defects in nup132Δ cells, while SUMOylation of Lem2 partially rescues clustering defects in csi1Δ cells. These findings suggest SUMOylation acts as a regulatory switch for Lem2's diverse functions, with potential implications for understanding Lem2-related human diseases.

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**Background:** Centromere clustering and silencing are critical for genome stability. In fission yeast *Schizosaccharomyces pombe*, centromeres cluster at the nuclear periphery near the spindle pole body (SPB), a process involving Csi1 and the inner nuclear membrane protein Lem2. Lem2 also contributes to pericentromeric silencing. SUMOylation, a post-translational modification, is known to affect many centromere-associated proteins. Deletion of the nucleoporin Nup132 mislocalizes the SUMO protease Ulp1 from the nuclear periphery, leading to both reduced global SUMOylation (via Pli1 destabilization) and local hyper-SUMOylation. This study investigates the role of SUMOylation in centromere function, focusing on Lem2.

**Methods:** The study used *S. pombe* genetics, including gene deletions, point mutations (Pli1^K3R^, Lem2^K7R^), and overexpression constructs. Centromere silencing was assessed using an *imr1:ura4+* reporter assay (growth on 5-FOA) and RT-qPCR. Centromere clustering was analyzed by live-cell imaging of GFP-Cnp1 (centromere marker) and Sid4-RFP (SPB marker). Global ubiquitylation sites were identified by diGly-immunoprecipitation and LC-MS/MS. Protein levels and SUMOylation were analyzed by western blotting. Statistical significance was determined using χ² tests and Student's t-tests.

**Key Results:** 1. **Centromere defects in nup132Δ cells are not due to Pli1 destabilization:** Stabilizing Pli1 via the Pli1^K3R^ mutation (which blocks its ubiquitylation at K15, K169, and K214) did not rescue the silencing defect or TBZ sensitivity in *nup132*Δ cells, contradicting previous models. 2. **Hyper-SUMOylation drives centromere defects:** Overexpression of Ulp1 or a lysine-less SUMO (Pmt3^KallR^) partially suppressed TBZ sensitivity in *nup132*Δ cells. Tethering Ulp1 to the nuclear periphery (Nup107-Ulp1) rescued both TBZ sensitivity and silencing defects. 3. **Lem2 SUMOylation is key for silencing defects:** Mutation of seven known SUMOylation sites in Lem2 (Lem2^K7R^) rescued the centromere silencing defect in *nup132*Δ cells, restoring growth on 5-FOA and reducing *imr:ura4+* transcript levels to near wild-type. This rescue required the N-terminal domain of Lem2. Lem2^K7R^ did not rescue TBZ sensitivity. 4. **SUMOylation enhances centromere clustering:** *nup132*Δ cells showed mild centromere declustering. Surprisingly, deletion of *nup132+* substantially rescued the severe clustering defect in *csi1*Δ cells (85.0% normal clustering in double mutant vs. 63.9% in *csi1*Δ single mutant). This rescue was suppressed by reducing polySUMOylation (overexpression of Pmt3^KallR^ or Ulp1, or deletion of *pli1+*), but not by blocking STUbL pathways (deletion of *slx8+* or expression of *ufd1*ΔCt). 5. **Lem2 SUMOylation contributes to clustering rescue:** Expression of Lem2^K7R^ partially suppressed the rescue of centromere clustering in *nup132*Δ *csi1*Δ cells (reducing clustering from 85.0% to 76.5%). This was associated with enhanced Lem2 localization at the SPB in *nup132*Δ *csi1*Δ cells (85.2% vs. 75.0% in *csi1*Δ), which was reduced with Lem2^K7R^. Deletion of *bqt4+* also enhanced Lem2 SPB localization but did not rescue clustering defects in *csi1*Δ cells, indicating SUMOylation has an additional role beyond localization.

**Clinical Implications:** This study provides mechanistic insight into how SUMOylation regulates the dual functions of Lem2 in centromere clustering and silencing, proposing a model where SUMOylation acts as a switch between Lem2's partner protein interactions. Given that mammalian LEM2 variants are linked to human diseases including juvenile cataracts, arrhythmic cardiomyopathy, and progeria-like syndromes, understanding the regulation of Lem2 pathways is clinically relevant. The findings suggest that post-translational modifications like SUMOylation could modulate Lem2 function in response to cellular or environmental cues, potentially offering new avenues for therapeutic intervention in Lem2-related pathologies. The study also highlights the conserved role of SUMOylation in centromere organization, which may be relevant to cancer biology where centromere clustering is altered.