EMBO/sd-character-level-ner huggingface dataset:
The GABA‐induced block in pexophagy and mitophagy is overridden by rapamycinPeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig .Figure 2APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A. B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1‐GFP grown in oleate medium to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA or to SD‐N with GABA and rapamycin for 6 h. Bar, 5 μm. C Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .
Fig.Figure 4A) For each gene set (top18, APPtg-Blue and GWAS p<0.001 (Marioni et al, 2018)) their expression was assessed in the different cell types listed on the left As can be viewed, the top18 genes are highly expressed in microglia. B) Based on the marker genes for each cell type enrichment of these marker genes was assessed among the three gene sets (top18, APPtg-Blue and GWAS p<0.001 (Marioni et al, 2018)). Significant enrichment of microglial marker genes can be observed for all three gene sets, while the APPtg-Blue module is also enriched for astrocyte marker genes. Data information: Significant enrichment : ***: p< 0.001; *: p<0.05. P-values are Benjamini-Yuketieli-adjusted in (B) C) Enrichment of transcription factor targets among the 'top 18' genes (red bars) and the GWAS genes with p<0.001 (green bars), using i-CisTarget (Imrichová et al, 2015). Data information: Significant enrichment : ***: p< 0.001; *: p<0.05. P-values are empirical and Bonferroni-adjusted in (C).
Figure 7Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with the indicated expression vectors, treated with either DMSO or 150 nM PLK4 inhibitor Centrinone. WT and Spata2 KO iBMDMs stably expressing Flag-tagged WT NEK7 were left untreated or pretreated with the PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4h. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204. HEK293 cells stably expressing GFP-PLK4Δ24 were transfected with a control siRNA or siRNAs targeting Spata2 or CYLD. GFP-PLK4Δ24 was immunoprecipitated under denaturing conditions and immunoblotted for K63-, K48-linked and linear ubiquitination. Relative levels of PLK4 ubiquitination were normalized to GFP-PLK4 and shown below. Co-immunoprecipitation analysis of the PLK4-NEK7 interaction in HEK293 cells co-transfected with Flag-NEK7 and HA-PLK4 in the presence (+) or absence (-) of GFP, GFP-Spata2, GFP-CYLD, or GFP-CYLD C601S, a catalytically inactive mutant. A model of NLRP3 inflammasome regulation by PLK4 and Spata2/CYLD. Polyubiquitination of PLK4 suppresses PLK4 binding to and phosphorylation of NEK7. This polyubiquitination-mediated suppression can be reversed by Spata2 and CYLD-mediated deubiquitination of PLK4, thereby allowing for PLK4 phosphorylation of NEK7, which attenuates NEK7 binding to NLRP3 and NLRP3 inflammasome activation. Spata2 or CYLD deficiency would cause accumulation of PLK4 polyubiquitination, reduction of NEK7 phosphorylation, increase of NEK7 binding to NLRP3, and eventually hyperactivation of NLRP3 inflammasome.
Figure 6(A) CRISPR-targeted follicular B cell proliferation after 3 days of CD40L stimulation. mCherry+ populations were quantified within individual DDAO peaks. N = 3 mice. P, statistical significance calculated by two-way ANOVA reached p<0.0001 for all proliferation peaks. Data information: All data show means ± SEM. (B) CD80 mRNA levels in B cells following 24 hours of CD40L culture, relative to untreated B cells. Data from RNAseq of N = 3 mice. (C-D) Isolated naïve B cells from CRISPR-targeted chimeras after 3-day culture in stated cytokines. (C) mCherry percentage in targeted primary cell cultures. N = 15 mice. *p<0.05, **p<0.01, ****p<0.0001 using two-way ANOVA. (D) AnnexinV surface stain in targeted cells. N = 3-4 mice. Data information: All data show means ± SEM. (E) mCherry+ cell percentage in CRISPR-targeted Ramos cells over time. N = 6 independent experiments. (F) Cell counts at passage 3-6 of CRISPR-targeted puromycin-selected Ramos cells. N = 4 independent infections. P, statistical significance from unpaired t test. Data information: All data show means ± SEM. (G) Oxygen consumption rate (OCR) in CRISPR-targeted Ramos cells. N = 2 independent infections. P, two-way ANOVA of basal and maximum OCR. (H) OCR in B cells isolated from Sh3gl1-/- and WT littermates. N = 9 mice. P, two-way ANOVA of basal and maximum OCR. (I) OCR in T cells isolated from Sh3gl1-/- and WT littermates. N = 4 mice. Cell cultures with full RPMI media supplemented with 50 μg/ml FeSO4 or 5 μM hemin chloride. (J) SH3GL1-targeted Ramos cells. N = 4 independent infections. P, statistical significance from two-way ANOVA from both treatments versus RPMI. (K) Control sgRNA-targeted Ramos cells. Cell cultures with full RPMI media supplemented with 50 μg/ml FeSO4 or 5 μM hemin chloride. (L) Sh3gl1-/- isolated B cells, maintained in CD40L culture. (M) B cells isolated from WT littermates, maintained in CD40L culture. (N) OCR in Sh3gl1-/- or WT B cells after overnight culture in CD40L, supplemented with 5 μM hemin chloride. N = 6 mice. P, two-way ANOVA with matched samples for basal and maximum OCR (O) TfR internalization assay in Ramos cells using biotinylated anti-CD71. 4 independent CRISPR infections in 2 experiments. P, significance calculated using two-way ANOVA and indicated in corresponding color. (P,Q) Mouse transferrin internalization in B cells from SRBC-immunized WT and Sh3gl1-/- littermates. N = 7 mice per genotype in 2 experiments. P, statistical significance analyzed using two-way ANOVA. (P) Naïve B cells. (Q) GC B cells.
Figure 5(A-C) U2OS ATG2A/B double knock out (DKO) cells or DKO reconstituted with ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA) were starved for 2 h, fixed and stained for LC3B (green) or WIPI2 (magenta), (B) ATG9A (magenta) or (C) GABARAP (Magenta) and imaged using a Zeiss 880 Airyscan super-resolution confocal microscope. Images are representatives of n=3 independent experiments. Scale bars 10µm. Transmission electron micrographs of ATG2A/B DKO cells (upper left), DKO + ATG2A-WT (upper right), DKO+ ATG2A-mLIR (lower left) and DKO+ATG2A-mYFS( lower right) starved (EBSS) for 2 h. Clustered small vesicles (open arrowheads; DKO and DKO-mLIR) and autophagosomes (closed arrow heads, DKO+ATG2A-WT and -mYFS) indicated. Images are representative of n=3 independent experiments. Scale bars 500nm. ER= endoplasmic reticulum; N = Nucleus; Mt = Mitochondria; G = Golgi; AP = Autophagosome. Model of ATG2 function based on the current knowledge. ATG2A localizes to ER membranes and facilitates lipid transfer from the ER to the growing phagophore. ATG2 interaction with GABARAP/GABARAP-L1 is essential for anchoring ATG2 to growing phagophore and mutation of the GABARAP interaction region results in the formation of immature phagophores.
Characterization of RNASEH1 and ISCU mutant fibroblasts A Western-blot of whole cell extracts for TIMM50 and GAPDH in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in either glucose or galactose containing medium.Figure 7A Western-blot of whole cell extracts for TIMM50 and GAPDH in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in either glucose or galactose containing medium. B Mitochondrial membrane potential in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001. C Western blot for representatives of all five respiratory complexes in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. GAPDH is from the same blot as panel A. Quantification based on two biological replicates for RNASEH1 and ISCU mutant fibroblasts Significant changes in protein levels in RNASEH1 and ISCU mutant fibroblasts compared to controls have been boxed in red. D Oxygen consumption measurements in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001. E Steady state levels of ROS related proteins SOD2 and ACO2 and mitophagy related proteins VDAC1, p62 and LC3 in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. GAPDH is from the same blot as panel A. F Quantification of the number of late apoptotic cells in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Data are shown as mean ± SD, n = 4 or more biological replicates; ∗*∗p < 0.001.
Analysis of Cdc25C/CLU complex in PC3 cells by co-immunoprecipitation, immunofluorescence microscopy and in situ proximity ligation assay (PLA).Figure 4(A) Co-immunoprecipitation from parental PC3 cells transfected with CLU and Cdc25C plasmid expression vectors. Proteins were immunoprecipitated with specific anti-Cdc25C (left) and CLU (right) antibodies and analyzed by immunoblotting. (B) Dual immunofluorescence staining with Cdc25C (green) and CLU (red) antibodies in PC3 cells. Confocal microscopy was used to identify the interaction (white arrows). DNA was counterstained with (DAPI) (blue). Scale bar represent 10 µm. (C) Duolink proximity ligation assay between Cdc25C and CLU in PC3 cells. Confocal microscopy was used to detect the interaction (red dots). DNA was counterstained with DAPI (blue). PC3 cells transfected with siCLU were used as a negative control. Scale bar represent 10 µm.
Figure 4(A) Diagram of the disintegration reaction and substrate used in (B). (B) Disintegration reaction (1 hour) performed by RAG1 aa 216-1008 R848M/E649V, RAG2 aa 1-361 and full length human HMGB1 with Mg2+ or Mn2+ at the indicated temperatures. Denaturing gel displays the fluorophore-labeled DNA strand from the RAG STC before (16 nt band, lane 2) and after (37 nt) the disintegration reaction. Lane 1, fluorophore-labeled 37 nt DNA marker. Representative of 2 independent experiments. (C,D) Cryo-EM maps of RAG TCC (C) at 3.8 Å and Paired STC (D) at 2.9 Å shown in both top and front views with protein colored white, target DNA colored red, and signal DNA colored blue. Insets highlight the different connectivity between signal and target DNA in the TCC and STC.
| NTRAS controls TJP1 splicing and endothelial barrier function.Figure 3(A) Scheme depicting the in vitro splicing of a TJP1 exon 19 - exon 20 splice substrate featuring an intronic splicing silencer (ISS). hnRNPL binding sites are highlighted in red. (B) In vitro splicing efficiency of the TJP1 splice substrate, comparing mock, NTRAS-depleted, and NTRAS-CA16 motif add-back conditions (n = 7-12 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. (B two-tailed unpaired t-test, (C) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-silenced HUVECs (n = 6 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test (D) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test, (E) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-CA16 motif overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test, (F) RT-PCR-based analysis of TJP1 exon 20 inclusion upon NTRAS overexpression and overexpression of the NTRAS-CA16 motif (n = 8 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. one-way ANOVA. (G) Endothelial resistance of NTRAS-silenced HUVECs (n = 3-5 independent biological replicates), analyzed by electrical cell-substrate impedance sensing (ECIS). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test (H) Endothelial resistance of hnRNPL-silenced HUVECs (n = 3 independent biological replicates), analyzed by ECIS. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test, (I) Analysis of TJP1 exon 20 inclusion by RT-PCR upon transfection of HUVECs with a control SSO or an SSO masking the exon 20 - intron 20 boundary (E20 SSO) (n = 9 independent biological replicates). Representative agarose gel on the right. Schematic outline at the top right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test, (J) Endothelial resistance of control SSO- or E20 SSO-transfected HUVECs (n = 4 independent biological replicates), analyzed by ECIS. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,
Figure 7A. RT-PCR results showed that hnRNP U, FUBP1, and ILF2 promote exon 3 skipping of Cdk1. We injected Control-LNA or LincGET-LNA2 together with siRNA for either Egfp, Hnrnpu, Fubp1, or Ilf2 or Control-LNA together with mRNA for either one of them at the pronuclear stage (12 groups) and assessed Cdk1 and Cdk1-3ExS levels at the late 2-cell stage (phCG 48 hours). For each lane, about 50 embryos were used. Three experimental replicates were performed. The results show that knockdown of either of Hnrnpu, Fubp1, or Ilf2, but not Egfp in the presence of LincGET had no effect on exon 3 skipping of Cdk1 (left 4 lanes). Knockdown of either Hnrnpu, Fubp1, or Ilf2, but not Egfp in absence of LincGET decreased the Cdk1-3ExS level (middle 4 lanes), especially si-Fubp1. Overexpression of Hnrnpu, Fubp1, or Ilf2, but not Egfp in the presence of LincGET promoted exon 3 skipping of Cdk1 (right 4 lanes), especially Fubp1. The band intensities were quantitated and are shown under the gel image. Two tailed student's t-test was used for the statistical analysis and different letters (a - d) mean p < 0.05. si-, siRNA. OE-, overexpression. B. Knockdown of either Hnrnpu, Fubp1, or Ilf2 in the presence of LincGET had no effect on preimplantation development. We injected Control-LNA together with siRNA for Egfp, Hnrnpu, Fubp1, or Ilf2 at the pronuclear stage and development was assessed at the 2-cell (2C), 4-cell (4C), 8-cell (8C), morula (M), and blastocyst (BL) stage. Three experimental replicates were performed (Table 1). Two tailed student's t-test was used for the statistical analysis. * means 0.01 < p < 0.05, and ** means p < 0.01. si-, siRNA. C. Fubp1 knockdown can partially rescue the 2C arrest caused by LincGET depletion to the blastocyst stage and Ilf2 knockdown can improve the development rate of 4C, but Hnrnpu knockdown cannot improve the development. We injected LincGET-LNA2 together with siRNA against Egfp, Hnrnpu, Fubp1, or Ilf2 at the pronuclear stage and development was assessed at the 2C, 4C, 8C, M, and BL stage. Three experimental replicates were performed (Table 1). Two tailed student's t-test was used for the statistical analysis. * means 0.01 < p < 0.05, and ** means p < 0.01. LNA2, LincGET-LNA2. si-, siRNA. D. and E. Fubp1 overexpression in the presence of LincGET causes 2C arrest and Hnrnpu or Ilf2 overexpression can lower the preimplantation development rate. We injected Control-LNA together with mRNA for Egfp, Hnrnpu, Fubp1, or Ilf2 at the pronuclear stage and development was assessed at the 2C, 4C, 8C, M, and BL stage. Images were acquired at the BL stage (E). Three experimental replicates were performed (Table 1). Two tailed student's t-test was used for the statistical analysis. * means 0.01 < p < 0.05, and ** means p < 0.01. OE-, overexpression.
Loss of SOX10 and MITF releases GATA6 expressionA.Figure 6A. qRT-PCR analysis showing average TBP-normalized fold expression of SOX10, MITF and GATA6 in 501mel treated with either siCTL or siSOX10 for 48h. Data information: , data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05. B. Seurat UMAP of MM074 treated with siCTL or siSOX10 (24h, 48h and 72h post-treatment). The arrow indicated the trajectory from control to 72h post siSOX10 transfection. C. UMAP of AUCell GATA6 regulon in MM074 shows that GATA6 regulon is up-regulated along the trajectory from siCTL to 72h post siSOX10 treatment (GSE116237) The arrow indicated the trajectory from siCTL to 72h post siSOX10 transfection. We considered cell with GATA6 regulon activity of AUCell > 0.15 as active D-I. Graphs showing the average expression of the SOX10 (D), MITF (E), GATA6 (F), SERPINE1 (G), AMIGO2 (H) and ABCG2 (i) per individual melanoma cell measured by AUCell on MM074 at different time points post-transfection of siSOX10 (GSE116237) Data information: data are presented as mean values + standard error of the mean (SEM) for six replicates (n=6).
Figure 5A. Detection of de novo Ty1 insertions upstream of the SUF16 and SEO1 genes by PCR using a primer in HIS3 (red triangle) and a primer in the locus of interest (blue triangle). Ty1 retrotransposition was induced in cells transformed with plasmids expressing WT or mutant (IN1 K617A, S621A and L622A) Ty1his3AI from the GAL1 promoter. Total genomic DNA was extracted from His+ cells obtained from independent cultures. B. Genome-Wide Ty1 insertion frequencies at each genomic feature are clustered in a heatmap. Score is computed in column Z-score. ORFs, all Pol II-transcribed genes except gene at subtelomeres; Pol I, one RDN37 copy; up 1kb Pol III, 1kb upstream of all Pol III transcribed genes; Subtelomeres, genomic coordinates corresponding to chromatin covered by Sir2 and Sir3, when they are co-overexpressed (Hocher et al, 2018); Random, 100 000 random Ty1 computed insertions in the genome. C. Detection of de novo Ty1 insertions upstream of RDN5 and in RDN37 genes by PCR as described for panel 5A. Four total genomic DNA preparation from panel A were randomly tested by PCR. D. Ty1 insertion profile upstream of tDNAs. Total genomic DNA extracted in (B) was prepared for Ty1 de novo integration event sequencing. Ty1 insertions are computed in a 1kb window upstream of all the 275 nuclear tDNAs (position 0 in the graph). Each position is divided by the number of insertions at this position (weight). The Smoothing curves indicate the general trend. Nucleosome center positions for the three first nucleosomes upstream of all tDNAs are from (Brogaard et al, 2012). E. Ty1 insertion frequencies for each left and right subtelomere of chromosomes are clustered in a heatmap. Score is computed in row Z-score. Random, as described for panel B. High level of WT Ty1-HIS3 integration in II-L is due to the presence of the tRNA gene tF(GAA) B. F. Genome browser visualization of WT and IN1 K617A mutant Ty1-HIS3 insertions into chromosome V left and chromosome VI right subtelomeres compared to the subtelomere boundaries defined by (Hocher et al, 2018) and (Yue et al, 2017) respectively. Red stars indicate the first essential gene of each subtelomere. The CAN1 gene is indicated on chromosome V.
Figure 6A.10-fold serial dilutions of the indicated yeast strains were grown on YPD at 23°C for 3 days before imaging. B. Schematic of Sna3-mCherry localization. Sna3 accumulates in the vacuole (V) or class E compartment (E) in cells with, or lacking, ESCRT function, respectively. C. Deconvolved fluorescence micrographs of Sna3-mCherry in the indicated genetic backgrounds. Scale bar is 5 μm. D. Deconvolved fluorescence micrographs (maximum intensity projections of a z-series of images) of GFP-Nup49 in the indicated yeast strains (arrows indicate SINCs).E. Plot of proportion of cells in the indicated strains with SINCs. Error bars are the SD from the mean from 3 independent replicates quantifying >300 cells for each strain. p values from un-paired student's T-tests where ns is p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
Figure 3(A) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies after extraction using 0.2% Triton X-100. The resulting samples were observed using a confocal laser microscope (Zeiss LSM 700). Immunostaining with pre-immune serum in (A) represents the negative control for immunoreactivity. Scale bars, 10 μm. The value of the relative neck thickness is shown in the lower left corner of the merged images. (B) Intercentrosomal (centrosome-to-centrosome) linear signal profiles of β-catenin/Plk1 (left panel) and β-catenin p-S60/Plk1 (right panel) from the XY plane of the immunostained cells in (A). (C) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies as described in (A), and the midbody localization of β-catenin/β-catenin p-S60 in late telophase cells was observed using a confocal laser microscope (Zeiss LSM 700). Scale bars, 10 μm.
DGAT1 and mitophagy synergise to safeguard cell and tissue integrity Iron depletion rapidly reshapes the cellular metabolome.Figure 8Iron depletion rapidly reshapes the cellular metabolome. DFP treatment alters glucose utilization to promote lipid biosynthesis and TAG storage in lipid droplets via DGAT1 activity, upstream of NIX-dependent mitochondrial clearance. Without DGAT1, fatty acids cannot be esterified TAG, compounding lipid dysfunction that impairs lysosomal homeostasis, leading to inefficient mitophagy and promoting cell death. Strikingly, genetic depletion of DGAT1 in vivo also impairs basal mitophagy, demonstrating the physiological relevance of our in vitro findings.
Figure 4A) The position of a CDK1 phosphorylation site, T651CENP-C, is indicated in the diagram of chicken CENPC-CT. EMSA was performed to examine the binding affinities of phosphorylated or non-phosphorylated CENPC-CT (left panel) and CM peptide (right panel) to the CENP-A nucleosome. B) Structure of the loop region harboring phosphorylated T651CENP-C. CENPC-CT is shown in magenta, except for the pink canonical CENPC motif. The cryo-EM density map is overlaid on the cartoon model. The main chain interactions forming a short β-sheet are indicated by black dotted lines. C) Magnified view of the structure around phosphorylated T651CENP-C. Key residues in this region are shown as stick models. The side chains of R656CENP-C and R71H2A are situated in the vicinity of the phosphoryl group of T651CENP-C in the distance ranges of 3.5-4 Å and 3.5-4.5 Å, respectively. D) All constructs analyzed in (E) were stably expressed in CENP-C knockout cells. E) Localization analysis of GFP-fused CENPC-CT, CENPC-CTR656E, and CENPC-CTR655E_R656E (green) on mitotic chromosomes in chicken DT40 cells. DNA was stained using DAPI (blue). Scale bar indicates 10 μm. F) Model indicating the functional role of T651CENP-C phosphorylation in the CENP-A-CENP-C interaction. Primarily, the CENPC motif associates with CENP-A nucleosome. Phosphorylation of T651CENP-C by CDK1 facilitates the loop conformation in the CM upstream region through an intramolecular bridge with the side chain of R656CENP-C. Subsequently, the N-terminal α-helix is folded over to stabilize the CENP-C-CENP-A nucleosome interfaces mediated by the CENPC motif and CM downstream regions.