Fig 1: Binding to C11orf84 and H3K4me3K9me3 is required for Spindlin1 to stimulate rRNA transcription.a C11orf84 colocalizes with Spindlin1 and RPA194 in the nucleolus. C11orf84 and RPA194 were visualized by indirect immunofluorescence whereas GFP-Spindlin1 by direct fluorescence. Nuclei were stained with DAPI. Scale bar: 5 μm. b Overexpression of Spindlin1 stimulates rRNA transcription. The 45S pre-rRNA level was determined by RT-qPCR in HEK293T mock cells and Spindlin1-overexpressed (Spindlin1 OE) cells. c RT-qPCR reveals that depletion of Spindlin1 or C11orf84 results in a reduced level of 45S pre-rRNA transcript in U2OS cells. d Ectopic expression of wild-type Spindlin1 but not the mutated forms that impaired the binding to either K4me3 or K9me3 of histone H3 restored the rRNA transcription level in Spindlin1 stable knockdown U2OS (Spindlin1 KD) cells. RT-qPCR was performed to determine the level of 45S pre-rRNA. Western blot shows the level of Myc-tagged wild type and mutant Spindlin1. e Ectopic expression of wild-type C11orf84 but not the truncated form that impaired the binding to Spindlin1 restored rRNA synthesis in C11orf84 stable knockdown U2OS (C11orf84 KD) cells. b–e Data are shown as mean ± SD (n = 3 independent repeated experiments). The p values were calculated by two-tailed unpaired t-test. f Knockdown of Spindlin1 or C11orf84 inhibits cell proliferation. Cell number was counted by Cell Counting Kit-8 (CCK-8) on a daily basis for a period of 5 days. Data are presented as mean ± SD (n = 5 independent experiments). The p values were calculated with two-way ANOVA test followed by Dunnett’s multiple comparisons test. Spindlin1 KD versus Mock, p values are 0.0036, 0.0005, 0.0045, and 0.004, respectively from day 2 to day 5; C11orf84 KD versus Mock, p values are 0.0019, 0.0004, 0.0004, and <0.0001, respectively, from day 2 to day 5. Source data are provided as a Source Data file.
Fig 2: Spindlin1/C11orf84 complex prefers to recognize non-canonical bivalent mark H3K4me3K9me3.a Quantitative ITC measurements suggest that Spindlin1 binds H3K4me3K9me3 dual mark with a fourfold increase in binding affinity over H3K4me3R8me2a. The insert lists the calculated dissociation constant (Kd). b The top panel shows protein architecture of Spindlin1 and C11orf84. The lower panel shows that Spindlin1 (aa: 50–262) forms a stable complex with C11orf84 (aa: 219–305) as analyzed by anion-exchange chromatography and SDS-PAGE gel of the peak fraction. c Coimmunoprecipitation (Co-IP) shows the region (aa: 219–305) of C11orf84 is responsible for Spindlin1 binding. d Immunoblotting shows the Spindlin1 protein level is dramatically reduced in C11orf84 stable knockdown (C11orf84 KD) U2OS cells, and ectopic expression of wild-type C11orf84 but not the C11orf84Δ253-280 mutant restored the Spindlin1 protein level. e ITC measurement shows the Spindlin1/C11orf84 heterodimer binds twofold stronger to H3K4me3K9me3 peptide than Spindlin1 alone. f ITC fitting curves for binding of Spindlin1/C11orf84 complex to various H3 peptides with dissociation constant (Kd) values indicated. The thermodynamic parameters are listed in Supplementary Table 1. Source data are provided as a Source Data file.
Fig 3: Spindlin1/C11orf84 complex displaces HP1γ from the rDNA foci enriched with H3K4me3K9me3 bivalent mark and facilitates the recruitment of RNA Pol I.a The top panel is a schematic diagram of a rDNA repeat. Arrows indicate the positions of eight primer pairs designed for qPCR analysis of immunoprecipitated DNA. The lower four panels are ChIP-qPCR measurement of the relative occupancy of Spindlin1, HP1γ, H3K9me3, and H3K4me3 on different regions of rDNA in HEK293T cells expressing Myc-tagged Spindlin1 or an empty vector (Mock). The precipitated DNA was analyzed by qPCR using primers that amplify the regions of H1: 5′ ETS (external transcribed spacer); H4: 18S rRNA coding; H8: 28S rRNA coding; H13: 3′ ETS; H18, H27, H42: IGS (intergenic spacer); Prom: promoter. The y-axis depicts the ChIP/input ratio. Data are presented as mean ± SD (n = 3 independent experiments). b Immunoblotting shows the protein level of RPA194 remains relatively stable in Spindlin1-depleted U2OS cells ectopically expressing Spindlin1 wild type or mutants. c ChIP-qPCR analysis of the recruitment of RPA194 (RNA Pol I subunit) to rRNA gene promoter and coding regions (H4 and H8) in Spindlin1 KD cells, Spindlin1 KD cells expressing Myc-tagged Spindlin1 wild type or indicated mutants compared with mock cells. Results are presented as mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.
Fig 4: Structural details of Spindlin1/C11orf84/H3K4me3K9me3 ternary complex.a Cartoon representation of the structure of Spindlin1/C11orf84 bound to H3K4me3K9me3 peptide. Spindlin1 Tudor 1, Tudor 2, and Tudor 3 domains are colored in light pink, green, and cyan, respectively. The C11orf84 segment and H3K4me3K9me3 peptide are colored in orange and yellow, respectively. b Detailed view of the interaction between C11orf84 and Spindlin1 Tudor 3 domain. Key residues involved in the interaction are depicted as stick models and labeled. Hydrogen bonds and salt bridges are shown in magenta dashed lines. c Structural comparison with the previous determined Spindlin1/H3K4me3R8me2a binary complex (PDB: 4MZF, colored in light gray) revealed that binding of C11orf84 fragment induced conformational changes in Spindlin1 Tudor 3 domain. The red dash box highlights the Y256-K260 segment of Tudor 3 adopts a β-strand conformation upon C11orf84 binding. In addition, the Tudor 3 β1–loop–β2 region was pushed outward with ~6 Å in distance to accommodate the C11orf84 fragment. d The detailed interaction network of Spindlin1/C11orf84 with H3K4me3K9me3 peptide. The H3K4me3K9me3 segment is depicted as yellow sticks. Hydrogen bonds and salt bridges are shown in magenta dashes. e ITC fitting curves for binding of Spindlin1/C11orf84 complex including Spindlin1 wild type and mutants to the H3K4me3K9me3 peptide, along with the calculated Kd. The thermodynamic parameters are listed in Supplementary Table 3.
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