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. 2016 Jun 2;44(10):4871-80.
doi: 10.1093/nar/gkw354. Epub 2016 Apr 29.

Dimerization of SLX4 contributes to functioning of the SLX4-nuclease complex

Jinhu Yin  1 , Bingbing Wan  2 , Jaya Sarkar  1 , Kent Horvath  1 , Jian Wu  3 , Yong Chen  3 , Guangjuan Cheng  4 , Ke Wan  4 , Peiju Chin  1 , Ming Lei  5 , Yie Liu  6
Affiliations

Dimerization of SLX4 contributes to functioning of the SLX4-nuclease complex

Jinhu Yin et al. Nucleic Acids Res. .

Abstract

The Fanconi anemia protein SLX4 assembles a genome and telomere maintenance toolkit, consisting of the nucleases SLX1, MUS81 and XPF. Although it is known that SLX4 acts as a scaffold for building this complex, the molecular basis underlying this function of SLX4 remains unclear. Here, we report that functioning of SLX4 is dependent on its dimerization via an oligomerization motif called the BTB domain. We solved the crystal structure of the SLX4BTB dimer, identifying key contacts (F681 and F708) that mediate dimerization. Disruption of BTB dimerization abrogates nuclear foci formation and telomeric localization of not only SLX4 but also of its associated nucleases. Furthermore, dimerization-deficient SLX4 mutants cause defective cellular response to DNA interstrand crosslinking agent and telomere maintenance, underscoring the contribution of BTB domain-mediated dimerization of SLX4 in genome and telomere maintenance.

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Figures

Figure 1.
Figure 1.
Hydrophobic contacts located in the BTB domain mediate dimerization of SLX4. (A) Schematic showing domain mapping of human SLX4 protein. ZF, ubiquitin-binding zinc finger domain; XBR, XPF-binding region; BTB, Bric-a-brac, Tramtrack and Broad complex domain; TBM, TRF2-binding motif; SIM: SUMO-Interacting Motif; SAP, SAP motif, MUS81-binding region; SBD, SLX1-binding domain. (B) Overall structure of dimeric SLX4BTB. The monomers are colored in cyan and yellow. (C) An enlarged view of the SLX4BTB dimeric interface. Residues F681 and F708 essential to preserve the dimer interface are highlighted as stick models. (D) Surface view of the hydrophobic dimerization interface. Hydrophobic, basic and acidic residues are shown in white, blue and red, respectively. (E) Close-up view of the critical hydrophobic interaction region of panel (D). Main binding surface is located in α1 and α2 helices.
Figure 2.
Figure 2.
SLX4 forms a dimer. (A) Superposition of gel filtration chromatography profiles of wild-type and dimer-disrupting mutants of SLX4BTB. (B) SLX4BTB dimer formation was assessed by measuring β-galactosidase activity produced by the reporter gene in yeast two-hybrid system. The Y-axis depicts readings at OD420 with ortho-nitrophenyl-β-galactoside (ONPG) as substrate. Data are averages of three independent measurements. AD, activation domain; BD, DNA-binding domain. (C) SLX4BTB mutants impair oligomerization of SLX4 in cells. SLX4-depleted U2OS cells were transiently co-transfected with HA-SLX4WT and GFP-SLX4 (WT or mutants) plasmids. Western blot was performed on HA-SLX4 immunoprecipitates (IP) (right). Inputs for GFP-SLX4 are shown (left). GFP-SLX4WT and GFP-SLX4L1022A, but not GFP-SLX4 BTB mutants (SLX4ΔBTB, SLX4F708R, SLX4F681R/F708R and SLX4F681R/F708R/L1022A) were co-immunoprecipitated with HA-SLX4WT.
Figure 3.
Figure 3.
SLX4 dimerization is necessary for foci formation of SLX4. (A) Representative IF images, showing nuclear foci formation and colocalization of moderately expressed GFP-SLX4 (wild type or mutants) with TRF2 in SLX4-depleted U2OS cells. Bar: 5 μm. (B,C) Quantification of number of SLX4 foci per cell, and percentage of SLX4 foci overlapping with telomeres. SLX4ΔBTB, SLX4F708R and SLX4F681R/F708R are SLX4BTB domain mutants that are defective in dimerization. SLX4L1022A is a SLX4TBM mutant that does not interact with TRF2. SLX4F681R/F708R/L1022A contains mutations in both BTB domain and TBM of SLX4. SLX4ΔZF, SLX4ΔXBR, SLX4ΔSAP and SLX4ΔSBD are domain deletion mutants of the ZF, XBR, SAP and SBD domains of SLX4, respectively. Approximately 30 cells/genotype were examined. Error bars: SD; P-values: Student's t-test. *P < 0.0001.
Figure 4.
Figure 4.
Disruption of SLX4 dimerization causes failure of nuclease toolkit assembly at telomeres. Representative IF-FISH images, showing that the nuclease XPF form discrete nuclear foci that colocalize with SLX4 foci and telomeres in U2OS cells expressing wild-type SLX4, but not in SLX4ΔBTB, SLX4F708R, SLX4F681R/F708R and SLX4F681R/F708R/L1022A mutants. IF-FISH was performed on SLX4-depleted U2OS cells transiently expressing GFP (vector), GFP-SLX4WT or GFP-SLX4 mutants together with Myc-XPF. Bar: 5 μm.
Figure 5.
Figure 5.
Disruption of SLX4 dimerization causes telomere defects. (A) Telomere length, (B) frequency of fragile telomeres, and (C) T-SCEs per chromosome are all adversely impacted when SLX4 dimerization is disrupted in cells. U2OS cells stably depleted of SLX4 (shSLX4) were transiently transfected with GFP (vector) or GFP-SLX4 wild type or mutant plasmids. Mean telomere length for each genotype is derived from 30 metaphases and is indicated in green in panel (A). The frequencies of individual telomeres were plotted against the telomere signal intensity using arbitrary units (A.U.). Representative fragile and T-SCE positive chromosome images are shown in panels B and C, respectively. Two groups were compared using a one-way ANOVA in panel (A) and a Student's t-test in panels (B) and (C). * P < 0.0001.
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