EMBO/soda-nel huggingface dataset:
Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway (2020)
Figure 3

Figure 3. Centrosome defects reduce brain size through activation of the mitotic surveillance pathway.

(A) Representative whole mount images of WT, Sas4cKO, Sas4cKO;Trp53bp1-/-, Sas4cKO;Usp28cKO animals at P14. Arrows indicate the cerebellar hypoplasia resulting from the lack of primary cilia. Scale bar = 0.2 cm.

(B) Telencephalon area of P14 brains of the indicated genotypes. WT littermates N = 8, Sas4cKO N = 7, Sas4cKO;Trp53bp1-/- N = 6, Sas4cKO;Usp28cKO N = 10, Sas4cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure 1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals.

(C) Cortical thickness of P14 brains of the indicated genotypes. WT littermates N = 4, Sas4cKO N = 4, Sas4cKO;Trp53bp1-/- N = 4, Sas4cKO;Usp28cKO N = 4, Sas4cKO;Trp53-/- N = 4; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure EV1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals.

(D) Representative whole mount images of WT, Cep63T/T, Cep63T/T;Trp53bp1-/-, Cep63T/T ;Usp28-/- animals at P60. Scale bar = 0.2 cm.

(E) Telencephalon area of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 7, Cep63T/T;Trp53bp1-/- N = 6, Cep63T/T;Usp28-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure 1B and shown alongside for comparison.

(F) Cortical thickness of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 5, Cep63T/T;Trp53bp1-/- N = 3, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure EV1B and shown alongside for comparison.

(G) Body weight of P7 animals of the indicated genotypes. WT littermates N=18, Cep63T/T N=16, Cep63T/T;Trp53bp1-/- N=2, Cep63T/T;Usp28-/- N=10; one-way ANOVA with post-hoc analysis.

(H) Quantification of total number of cells within a 500 μm-width column of cortices at P60. WT littermates N = 4, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .

(I-J) Quantification of the number of cells in the (I) superficial layer (CUX1+) and (J) deep layer (CTIP2+) within a 500 μm-width column of P60 cortices. WT littermates N = 3, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .

(K) P60 cortices of indicated genotypes stained with antibodies against the deep layer marker CTIP2 (green), superficial layer marker CUX1 (red) and DAPI (blue). Scale bar = 200 μm.

Data information: All data represent the means ± S.E.M. *P < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001 and not significant indicates P > 0.05.

See also Figure EV3.
Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway (2020)
Figure 4

Figure 4. Inactivation of the mitotic surveillance pathway prevents NPCs depletion in brains with centrosome defects.

(A) Cortices from E14.5 brains stained with antibodies against the radial glial cell marker PAX6 (yellow), intermediate progenitor marker TBR2 (red) and DAPI (blue). Scale bar = 50 μm.

(B and C) Quantification of the number of (B) radial glial cells (PAX6+) and (C) intermediate progenitors (TBR2+) within a 250 μm-width column of E14.5 cortices. WT N=3, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 3, Sas4cKO;Usp28cKO N = 3; one-way ANOVA with post-hoc analysis.

(D) Cortices from E14.5 embryos stained with antibodies against Cleaved-Caspase 3 (CC3, white), TP53 (red) and DAPI. Scale bar = 50 μm.

(E and F) Quantification of the number of CC3+ apoptotic cells (E) and cells with TP53 activation (F) within a 250 μm-width column of E14.5 cortices. WT N = 5, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 4, Sas4cKO;Usp28cKO N = 4; one-way ANOVA with post-hoc analysis.

Data information: All data represent the means ± S.E.M. *P < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001 and not significant indicates P > 0.05.

See also Figure EV4.
Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway (2020)
Figure 5

Figure 5. Inactivation of the mitotic surveillance pathway rescues NPCs proliferation without alleviating the centrosome defects or mitotic delay.

(A) Representative images of E14.5 cortices stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). White and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 10 μm.

(B) Percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T;Usp28-/- n = 205 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 308 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT. WT data are from Figure 1F and shown alongside for comparison.

(C) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T;Usp28-/- N = 3, Sas4cKO;Usp28cKO N = 3; two-tailed Mann-Whitney t-test. 3 WT data points are from Figure 1H and shown alongside for comparison.

(D) Representative images from live-cell movies of Nuclear-mCherry+ WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. White arrows showing the nuclei of daughter cells following cytokinesis.

(E) Plot showing quantification of mitotic duration of WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Sas4cKO n = 138 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 191 cells, N = 3 embryos; two-tailed Welch's t-test. WT and Sas4cKO data are from Figure 1K and shown alongside for comparison.

(F) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO;Usp28cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. Sas4cKO;Usp28cKO n = 271 cells, N = 3 embryos.

(G) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in F; #, chi-square test with *, post-hoc analysis, comparisons are made to the 0-30min group.

(H and I) Data from Figures 2C and 2F shown alongside for comparison with F and G.

Data information: All data represent the means ± S.E.M. *P < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001 and not significant indicates P > 0.05.

See also Figure EV5.
Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway (2020)
Figure 6

Figure 6. TP53 is activated through a distinct pathway in a microcephaly model caused by the loss of Smc5.

(a) WT and Smc5cKO cortices at E14.5 stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). Insets showing zoomed in view of 2 representative cells. Scale bar: 25 μm.

(B and C) Quantification of the number of (B) Centrin foci and (C) γ-Tubulin foci in mitotic cells in the VZ of E14.5 cortices. WT n = 175 cells, N = 4 embryos; Smc5cKO n = 134 cells, N = 3 embryos. #, chi-square test. WT data are from Figures EV1F and EV1G and shown alongside for comparison.

(D) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the VZ and SVZ. Scale bar = 100 μm.

(E) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Smc5cKO N = 5; two-tailed Mann-Whitney t-test. WT data are from Figure 1H and shown alongside for comparison.

(F and G) Quantification of the number of (F) apoptotic cells (CC3+) and (G) cells with TP53 activation within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison.

(H) Representative whole mount images of WT, Smc5cKO, Smc5cKO;Usp28cKO, Smc5cKO;Trp53-/- brains at P21. Scale bar = 0.2 cm.

(I) Telencephalon area of P21 brains of the indicated genotypes. WT littermates N = 6, Smc5cKO N = 5, Smc5cKO;Usp28cKO N = 4, Smc5cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis.

(J) Model for two TP53-dependent pathways underlying primary microcephaly. In the first model, NPCs with mutations in centrosome genes take longer to assemble their spindle in mitosis. This delay triggers the activation of the mitotic surveillance pathway, which activates TP53 through 53BP1 and USP28. By contrast, in microcephaly caused by mutations in genes required for DNA repair or genome stability, centrosome function and mitotic duration are unaffected. Instead, accumulated DNA lesions trigger DNA damage signaling in NPCs to activate TP53. In both models, increased TP53 activity leads to apoptosis, thus depleting the NPC pool and decreasing the final number of neurons.

Data information: All data represent the means ± S.E.M. *P < 0.05; *** < 0.001; **** < 0.0001 and not significant indicates P > 0.05.

See also Figure EV6.

Expanded View Figure Legends
cest-2.2 overexpression alters lipid metabolism and extends longevity of mitochondrial mutants (2022)
Figure 2

Figure 2. CEST-2.2 O/E stimulates mitochondrial respiration in complex I deficient nematodes.

(A) Representative oxygen consumption rate (OCR) curve of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes as measured with a Seahorse XF24 Analyzer. Injection time of FCCP and NaN3 is indicated with arrows (n=9 biological replicates, from 3 independent experiments).

(B-C) Indices of (B) normalized basal OCR and (C) maximal respiration of wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes across (n=9, one-way ANOVA with Tukey's multiple comparison test, n=9 biological replicates, from 3 independent experiments).

(D) Relative ATP content of gas-1(fc21) and gas-1(fc21); cest-2.2 O/E nematodes (unpaired t-test, n=3 biological replicates).

(E-F) Representative (E) Seahorse experiment and statistical analysis of (F) basal OCR and maximal respiration of 5-day-old gas-1; cest-2.2 O/E mutant C. elegans fed with control and cest-2.2 RNAi since hatching (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=7-9 biological replicates, from 3 independent experiments).

(G) OCR profiles of wt, cest-2.2 O/E and cest-2.2(lf) nematodes upon sequential treatment with FCCP and NaN3 (n=9-10 biological replicates, from 3 independent experiments).

(H) Representative images of control, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E nematodes expressing mitochondria-targeted GFP in the intestine (scale bar= 5 μm).

(I) Representative high-resolution confocal images of C. elegans expressing mitochondria-targeted GFP in the body wall muscle cells (scale bar= 10 μm). Quantification of mitochondrial morphology (elongation) is reported in the right panel (one-way ANOVA with Holm-Sidak's correction, n=37-47 animals from 3 independent experiments).

(J) Representative western blot analysis and densitometry of zcIs13[hsp-6p::GFP] expression in control, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E animals (one-way ANOVA with Holm-Sidak's correction, n=3 biological replicates).

(K-L) Representative (K) Seahorse profile and (L) statistical analysis of basal OCR and maximal respiration of mutant nematodes (unpaired t-test, n=12-13 biological replicates, from 3 independent experiments).

(M) Immunoblot and statistical analysis of zcIs13[hsp-6p::GFP] expression in control and nuo-6(qm200) animals fed with empty vector (C RNAi) and cest-2.2 RNAi bacteria (unpaired t-test, n=3 biological replicates).

Data information: In A, E, G, K, points represent mean± SEM; in B, C, D, F, J (right), L and M (right), bars represent mean± SEM; in I (right), middle line represents the median, and the upper and lower lines represent the upper and lower quartiles, respectively. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.
cest-2.2 overexpression alters lipid metabolism and extends longevity of mitochondrial mutants (2022)
Figure 4

Figure 4. CEST-2.2 overexpression modulates fat storage and lipid composition in gas-1 mutant nematodes.

(A) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with Oil Red O (ORO) (scale bar= full image-100 μm; hindgut magnification-20 μm).

(B) Quantification of hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes stained with ORO (one-way ANOVA with Tukey's multiple comparison test, n=18-26 animals).

(C) Quantification of mean intensity of ORO staining in the hindgut of wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes at different time points, starting from L4 stage (one-way ANOVA with Dunnett's post hoc correction, n=18-26 animals).

(D) Mean intensity of ORO staining in the whole body of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes plotted against body area.

(E) Representative images of wt, cest-2.2 O/E and cest-2.2 (lf) animals stained with ORO. Arrows indicate mislocalized lipid droplets.

(F) Quantification (mean intensity ± SEM) of ORO staining in hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old nematodes (one-way ANOVA with Dunnett's post hoc correction, n=11-17 animals).

(G) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with LipidTOX (scale bar= 100 μm).

(H) Statistical analysis of whole-body LipidTOX intensity of 5-day-old nematodes (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=25-37 animals).

(I-J) Representative images of nematodes stained with LipidTOX (I) (scale bar= 100 μm), with quantification (J) (mean intensity ± SEM) of LipidTOX staining.

(K) Representative thin layer chromatography (TLC) plate of samples from 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes.

(L) Densitometry of TLC-separated free fatty acids (FFA) across three biological replicates in 5-day-old mutant animals.

(M) Schematic representation of free fatty acid mass spec analysis.

(N) Sum of TLC-separated FFA of 5-day old animals (n=5 biological replicates).

(O) Quantification of individual FFA detected after TLC separation in 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (n=5 biological replicates, n.d.= not detected).

Data information: In B, F, H, J, L, N and O, bars represent mean ±SEM ; in C, points represent mean ±SEM. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.
YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of stem cells (2018)
Figure 1

Figure 1. Conditional deletion of YY1 in muscle progenitor cells causes defect in cell expansion. (A) Breeding scheme for generating conditional knock out (YY1cKO), Pax7Cre/+; YY1f/f and control (Ctrl), Pax7+/+; YY1f/+ littermates. (B-C) Immunofluorescent (IF) staining of Pax7 and YY1 was performed on cryosections of myotome or limb bud from Ctrl or YY1cKO mice to confirm the ablation of YY1 in Pax7 expressing progenitors. Scale bar = 100 µm. (D) Representative images of Ctrl, Heterozygous (Het) and YY1cKO newborn (P0) mice or embryos at E18.5 day. (E) Excised lungs from P0 Ctrl or YY1cKO pups were placed in a beaker for sinking test. The lung from YY1cKO pup sank whereas Ctrl floated. (F) P0 lungs were stained with Hematoxylin and Eosin (H&E) and representative images are shown. Scale bar = 100 µm. (G) Representative images of diaphragms (Dps) isolated from P0 Ctrl or YY1cKO littermates. (H) The above Dps were used for H&E or Immunohistochemistry (IHC) staining for MyHC. Representative images from 3 pairs of littermates are shown. Scale bar = 50 µm. (I-J) The above harvested Dps were subjected to IF staining for MyHC or Troponin T (TnT) together with Laminin. Scale bar = 50 µm. Quantifications of positively stained fibers per area are shown on the right (n=3 mice, each). (K) Representative images of H&E staining of hind limb muscles isolated from Ctrl or YY1cKO embryos at E18.5 day. Scale bar = 400 µm (left) or 50 µm (right). (L-M) The above harvested limb muscles were subjected to IF staining for MyHC or TnT together with Laminin. Scale bar = 400 µm (left) or 50 µm (right). Quantifications of the numbers of MyHC+ or TnT+ fibers per area are shown on the right (n=3 mice, each). The above harvested limb (N) or diaphragm (O) muscles were subjected to IF staining for Pax7 and Laminin. Scale bar = 100 µm (N) or 50 µm (O). Quantification of the numbers of Pax7+ muscle progenitor cells per area are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.
YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of stem cells (2018)
Figure 3

Figure. 3. YY1 deletion aggravates dystrophic phenotype in mdx mice. (A) Outline of the TM administration scheme to obtain Ctrl or YY1dKO mice. (B) SCs were FACS sorted 4 months after the last TM injection; RT-qPCR detection of YY1 mRNA shows the ablation in YY1dKO cells. (C) Representative images of Ctrl or YY1dKO mice 4 months after the TM administration. (D) Representative images of limb muscles (Tibialis anterior, TA; Gastrocnemius, Gas; Quadruple, Quad) from Ctrl or YY1dKO mice. (E) Quantifications of muscle weight from three pairs of littermate mice. (F) Representative images of Dp muscles isolated from Ctrl or YY1dKO mice 4 months after the TM injection. (G) H&E or (H) MyHC staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice. Quantifications of the numbers of MyHC+ fibers per area of TA or Dp muscles are shown on the right (n=3 mice, each). Scale bar = 100 µm. (I) Collagen1 or (J) Masson's Trichrome staining was performed on TA or Dp muscles from Ctrl or YY1dKO mice. Scale bar = 100 µm. (K) The distribution of fiber size measured by CSA in TA muscles from 3 pairs of 6-month-old Ctrl and YY1dKO mice was calculated (n=3 mice, each). (L) Representative FACS plots showing the percentage of SCs sorted from 6-month-old Ctrl and YY1dKO mice (n=3 mice, each). (M) IF staining for Pax7 and Laminin on TA muscles of Ctrl or YY1dKO mice. Quantifications of the numbers of Pax7+ SCs per 100 fibers are shown on the right (n=3 mice, each). (N-O) EDL or SOL muscles from Ctrl or YY1dKO mice were subjected to measurement of maximal isometric tetanic force. Left: Representative trace images of normalized tetanic force. (Right): quantification of the force by normalizing with cross-sectional area (CSA). (n = 3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.
YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of stem cells (2018)
Figure 5

Figure. 5. Transcriptomic and binding profiling reveals YY1 repression of mitochondrial genes in SCs. (A) RNA-seq was performed with RNAs extracted from ASCs (FACS purified and cultured for 36hrs) of YY1iKO or Ctrl mice; scatter plot shows differentially expressed genes with a fold change >=2 (red dots) in YY1iKO vs Ctrl. (B) Gene ontology (GO) analyses of the above up-regulated genes. The top 10 enriched GO terms are displayed on the Y axis and adjusted P values on the X axis. (C) Genomic snapshots showing the examples of mitochondrial genes (Uqcr11, Mrp11, Apo5o and Nme4) up-regulated in YY1iKO vs Ctrl cells. (D) GSEA analysis shows oxidative phosphorylation gene set is enriched in YY1iKO vs Ctrl cells (p-value < 0.0001). (E) RT-qPCR validation of the expression of the selected up-regulated mitochondrial genes in YY1iKO vs Ctrl. (F) Protein levels of the respiratory chain complexes I-V components in YY1iKO vs Ctrl ASCs were detected using a mitoprofile antibody cocktail. β-actin was used as the loading control. (G) RNA-seq was performed using RNAs from freshly isolated SCs (FISCs); scatter plot shows differentially expressed genes with a fold change >=2 (red dots) in YY1iKO vs Ctrl. (H) GO analysis for the up-regulated genes in YY1iKO vs Ctrl. (I) Genomic snapshots show the selected mitochondrial genes (Cox8b, Nme4, Slc25a29, Nnt) up-regulated in YY1iKO vs Ctrl FISCs. (J) ChIPseq was performed using chromatins from WT ASCs and the genomic distribution of 2031 YY1 binding peaks is shown. (K) De novo motif prediction by DREME revealed the enrichment of canonical YY1 motifs in the above binding regions. (L) Venn diagrams show the overlapping (90 genes) between the above identified binding target genes (1387) and the upregulated genes from ASC RNAseq (979). (M) GO analysis of the above 90 genes revealed an extreme enrichment of mitochondrial related terms. (N) Genomic snapshots of two of the above identified mitochondrial genes (Tomm40I and Ndufa13) that are bound by YY1 in their TSS (ChIPseq tracks) and up-regulated by YY1 deletion (RNAseq tracks). (O) ChIP-qPCR validation of YY1 binding on some mitochondrial genes. Negative control (NC) represents a genomic region on Chromosome 11 with no YY1 binding peak identified. Enrichment fold was calculated as the amount of amplified DNA from YY1 binding sites normalized to values obtained from IgG control. (P-R) ChIP-qPCR was performed to show the enrichment of Ezh2 (P), Suz12 (Q) and H3K27 me3 (R) binding on selected mitochondrial genes. (S) A construct to express wild type YY1(WT), or two mutants lacking DNA binding activity, YY1(1-394) and YY1(S365D) were transfected into YY1iKO cells; the expression of mitochondrial gene was measured. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.