EMBO/soda-nel huggingface dataset:
Miz1 is required to maintain autophagic flux (2013)
f3

(a) Representative examples for ChIP-sequencing traces obtained from NPCs using an α-Miz1 antibody. Input traces are shown as negative control. For each gene, the intron/exon structure is shown below the traces with exons indicated as vertical bars and the transcriptional start site (TSS) at the left. An enlarged picture of the peak region is shown on the right. (b) Miz1 binds predominantly to core promoters. The panel shows a histogram of the percentage of promoters with the indicated distances between the peak of the Miz1-binding site and the next TSS. Of all the peaks, 140/261 (54%) are located in core promoters (±1.5 kb). (c) Miz1 binding is conserved across different cell types and species. The plot shows a Venn diagram documenting the overlap in Miz1-bound promoters between murine NPCs and human MDA-MB231 cells (a mammary epithelial tumour cell line). The P-value was calculated using a hypergeometric test. Note that different α-Miz1 antibodies (H190 and G18) were used for the analysis of mouse and human Miz1-binding sites. (d) Miz1-binding sites contain a conserved sequence element. Sequences (±50 bp) around all 261 Miz1-binding sites observed in NPCs were analysed by MEME-ChIP (‘Motif Analysis of Large DNA Data sets’). (e) Electrophoretic mobility shift assay documenting binding of Miz1 to the conserved sequence element (‘Miz1-binding motif’). Nuclear extracts from control HeLa cells (‘Mock’) or cells ectopically expressing Miz1 were incubated with radioactive oligonucleotides spanning the Miz1-binding motif of the Ambra1 and Vps28 promoters. Where indicated, binding reactions included a 25-fold excess of non-labelled oligonucleotides (‘Competitor’), the indicated mutants thereof or of an oligonucleotide containing an E-box sequence from the Ubap2 promoter. Supershift assays were performed using α-Miz1 (H190, G18 and C19), α-Myc (N262) or control antibodies. *Non-specific band. The sequences of the oligonucleotides used are shown above the panel; mutated nucleotides are shown in bold.
Miz1 is required to maintain autophagic flux (2013)
f4

(a) Miz1 activates the Rorc and Ambra1 promoters, and the POZ domain is required for activation. HEK293 cells were transiently transfected with plasmids containing promoter sequences of Ambra1 or Rorc in front of luciferase and with expression vectors encoding Miz1 or MizΔPOZ. Empty vectors were used as control. Values were normalized with CMV-βGal. Error bars are s.d. of biological triplicates. (b) Direct target genes of Miz1 are downregulated in cerebella of Miz1ΔPOZNes mice. Microarray analysis was performed from cerebella of mice at the indicated ages. The panels show box plots (line: median, box: 50% of data points, whisker: 1.5 interquantile range, outliers are not shown) of relative gene expression (plotted as log2-fold change) of the 140 Miz1 direct target genes comparing control and Miz1ΔPOZNes mice. The P-value was calculated using a Mann–Whitney–Wilcoxon test. Microarrays were performed as technical duplicates from RNA pooled from two animals for each genotype. (c) Gene-set enrichment analysis documenting significant downregulation of Miz1 target genes in cerebella of Miz1ΔPOZNes mice. NES, normalized enrichment score. Target genes of Miz1 were the only significant gene set in this analysis. (d) qRT–PCR documenting the expression of selected Miz1 target genes in cerebella of control and of Miz1ΔPOZNes mice at the indicated ages. Data represent the mean of two independent biological experiments each with three technical replicates. Error bars are s.e.m.
Miz1 is required to maintain autophagic flux (2013)
f5

(a,b) Immunostaining using an α-ubiquitin antibody of (a) PCs and (b) the molecular layer of Miz1ΔPOZNes and age-matched control mice (dotted area: higher magnification). Arrowheads designate ubiquitin-positive structures. Scale bar, (a) 25 μm; (b) 50 μm. (c) Immunofluorescence staining using an α-p62 and an α-ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age-matched control mice. Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) and overviews of the cerebella clarifying the selected areas are shown. Scale bar, 25 μm. (d) Quantification of a, b and c. Error bars represent s.e.m. derived from the indicated numbers of animals. P-values were calculated using an unpaired two-tailed Student’s t-test. (e) Immunoblots of Triton X-100 soluble (‘TX100’) and insoluble (‘1% SDS’) fractions of cerebella obtained from control and Miz1ΔPOZNes mice of the indicated ages. Blots were probed with antibodies recognizing either mono- or polyubiquitinated proteins. Staining of the membrane with amido black was used as loading control. (f) Transmission electron microscopy documenting the accumulation of large multilamellar bodies in PC bodies (‘PC’) and Purkinje dendrites (‘D’) of Miz1ΔPOZNes mice (N, nucleus). Such structures were not observed in age-matched control mice (as quantified in Supplementary Fig. S7b). Mean age of mice for all electron microscopy pictures is 13 months for Miz1ΔPOZNes and 12.5 months for control mice. Morphologically similar structures accumulate in neurons of mice deficient in late steps of autophagy. Scale bars, 2, 0.2, 0.5 and 1 μm, respectively.
Miz1 is required to maintain autophagic flux (2013)
f6

(a) Genomic PCR documenting recombination of the floxed Miz1 alleles in Cre-ER-positive and -negative MEFs. 4-OHT was added to the media for 4 days. (b) qRT–PCR assays of Miz1 target genes in Miz1flox/floxCre-ER− and Miz1flox/floxCre-ER+ MEFs. The experiment was performed independently from four embryos with identical results. Error bars depict s.d. of technical triplicates from one experiment. (c) Immunoblots using an α-Lc3 antibody detecting Lc3-I and conjugated Lc3-II. 4-OHT-treated and control-treated MEFs of the indicated genotype were incubated with leupeptin and ammonium chloride (lys PI) for 2 h in DMEM (+amino acids) or in EBSS (−amino acids) medium. The expression of α-tubulin (upper panels) or α-vinculin (lower panels) was used as loading control. (d) qRT–PCR assays of Miz1 target genes in control and Miz1ΔPOZ MEFs after incubation in DMEM or in EBSS. Error bars depict s.d. of technical triplicates from one experiment. (e) qRT–PCR assays documenting expression of the indicated Miz1 target genes in control and Miz1ΔPOZ neurospheres; assays were performed at passage three. s.e.m. values are derived from six biological replicates, each with technical triplicates. (f) Recombination of Miz1flox/floxCre-ER+ neurospheres was tested by genomic PCR. 4-OHT was added to the media for 3 days. (g) Immunoblots using an α-Lc3 antibody detecting Lc3-I and Lc3-II. 4-OHT-treated and non-treated Miz1flox/floxCre-ER+ neurospheres were incubated with leupeptin and ammonium chloride (lys PI) for 30 min. The expression of α-tubulin was used as loading control. (h) Fluorescent microscopy of SHEP cells stably infected with lentiviruses expressing Lc3-GFP and Lamp1-RFP (to mark lysosomes). Vps28 was depleted with siRNAs and a non-targeting pool (siNTP) was used as a control. Note the accumulation of Lc3 in vesicles that do not overlap with lysosomes upon depletion of Vps28. Scale bar, 25 μm.
Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly (2016)
Figure 1

Figure 1 - Role of yNap1 in nucleosome assembly on a genomic scale.

A Analysis of MNase–digested chromatin DNA fragments from wild–type (WT, sizes indicated in black) and nap1Δ (N, sizes indicated in red) cells by agarose gel electrophoresis. Diagram illustrating reduction of array length in nap1Δ cells.

B Frequency distribution of nucleosomal DNA size (WT in grey fill with nap1Δ in blue traces) from H3 ChIP-seq.

C Calculation of nucleosome fuzziness as the standard deviation of nucleosome (or tag) positions. See also Appendix Fig S1.
Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly (2016)
Figure 2

Figure 2 - Crystal structure of the yNap1–H2A–H2B complex.

A Domain architecture of yNap1, H2A and H2B. The Histone Binding Region (HBR1 and HBR2) of yNap1 and the yNap1 Binding Region (NBR1 and NBR2) of H2A are indicated. Regions involved in higher–order oligomerization are shown (IF1 and IF2).

B Ribbon view of the asymmetric crystallographic unit containing a (yNap1c2–H2AΔ14–H2BΔ28)6 assembly. For clarity only layer 1 of the complex is shown. Layer 1 contains three protomers A–C that are related by two perpendicular non–crystallographic dyads ().

C Side–view of the complex. The layer 2 omitted from panel B is shown in grey. The yNap1 dimer is shown in green and yellow, H2A in red and H2B in blue. See also Fig EV1.

D View of the yNap1–H2A–H2B protomer complex with labeling of secondary structure elements.
Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly (2016)
Figure 3

Figure 3 - Interactions of yNap1 with the basic DNA binding surface of the H2A–H2B heterodimer. A Overview of the yNap1c2–H2AΔ14–H2BΔ28 complex.

B Residues R17, R20, R29, R32, R35 and K36 of H2A make contacts with the acidic HBR1 in yNap1 comprising residues D201, D205 and E310.

C Residues K75, R77 and R81 of H2A make polar contacts with the HBR2 comprising residues E332, D333 and E339 in α8 of yNap1.

D In vitro GST–pulldown assay showing the role of residues in the HBR interfaces in H2A–H2B binding. The mutant labeled HBR1 (lane 6) contains the triple mutation D201R, D205R, E310R and HBR2 (lane 12) the triple mutations E332, D333, E336. HBR1+2 (lane 13) contains all six mutations. The mutant labeled HBR1+2A (lane 14) has Alanine replacements in the six HBR residues. Top panel: Bound. Bottom panel: Input.

E Disruption of the Nap1-histone interaction by H2A mutation. Bottom panel: Inputs of the coexpressed proteins (soluble extract) and top panel the material bound to the glutathione affinity column were analyzed by SDS-PAGE and Coomassie staining.

F Controls showing input (bottom) and bound (top) fractions. See also Fig EV2.
Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly (2016)
Figure 5

Figure 5 - Native Mass spectrometry and negative–stain EM of the yNap1–H2A–H2B particle.

A EM analysis of the yNap1c2–H2AΔ14–H2BΔ28 complex. Representative raw image of negatively–stained particles.

B Representative particles (1st row), class averages coming from the projection matching procedure (2nd row) and comparison of the class averages with projections from a final model generated after four projection matching cycles (3rd row).

C From left to right: Crystal structure of the (yNap1c2–H2A–H2B)6 complex in two different orientations (brown); 60 Å–filtered structure of the complex (magenta); 3D reconstruction of the complex from negatively stained particles (green) and fit of the crystal structure into the EM map.

D–I Mass spectra of yNap1 variants in the absence and presence of H2A–H2B. yNap1 in complex in the absence (D) or presence of H2A–H2B (E). yNap1c in the absence (F) or bound to H2A–H2B (G). The oligomerization mutant yNap1_IF1 alone (H) or in complex with H2A_IF2Δ14–H2BΔ28 (I). yNap1 is shown in green circles and H2A–H2B as red and blue asterisks.
Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly (2016)
Figure 6

Figure 6 - Nucleosome assembly in vitro and in vivo.

A Native PAGE gel showing the result of an affinity shift assay scanned to detect AF488– (left) or AF647–labeled DNA (right). 31 nM – 4 μM of yNap1–H2A–H2B (lanes 6–13) were incubated with a mixture of 0.8 μM AF488–labeled H3–H4–DNA tetrasome complexes and 0.8 μM AF647–labeled DNA (lane 5). Lanes 1–3 show migration of tetrasomes (tetra), nucleosomes (nucl) or free H2A–H2B obtained by salt deposition onto AF647–labeled DNA.

B Intensity of the nucleosome band (nucl) is plotted. The value at 4 μM of yNap1–H2A–H2B (lane 13) was set to 100%. For non–specific DNA binding, the value in the absence of H2A–H2B (lane 5) was set to 100% intensity and disappearance of the free DNA AF647 band was plotted. Each curve is representative of three independent experiments performed on different days. Standard deviations are shown.

C Analysis of MNase–digested chromatin DNA fragments from wild–type (BY4741), nap1Δ, and nap1Δ cells complemented with different yNap1 expression plasmids. Total MNase units per 50 ml of culture are indicated on the bottom of the lanes. Kb+: DNA Marker.

D Positional representation of MNase titrated nucleosomal arrays from panel C, comparing with BY4741 (blue traces) with NAP1 deletion (green traces) and other mutants (HBR1–black, HBR2–red, HBR1+2–purple, and IF1–gold traces). Color gradients indicate MNase concentrations (from high to low) and the yellow box represents mononucleosomes. Dashed lines represent nucleosome dyad with respect to BY4741 (WT). Traces are offset vertically for ease of visibility. See also Fig EV4 I.
Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain. (2008)
Figure 1.

Ref(2)P localization and expression in the adult brain of wild-type flies. (A) Confocal micrographs of adult brain of a young (2 d old) wild-type fly. Positive staining for Ref(2)P and ubiquitin is not evident. (B–D) Confocal micrographs of adult brain of an old (8 wk old) wild-type fly. Ref(2)P accumulates in protein aggregates that often colocalize with ubiquitin (arrows). The white rectangle in C indicates the area shown in D. (E and F) Western blot analysis of total cell lysates (E) and insoluble protein fraction (F) of wild-type young and old adult heads probed with anti-ref(2)P and anti-ubiquitin antibodies, demonstrating a significant increase in Ref(2)P and insoluble ubiquitinated protein levels in old flies. wt, wild type. Bars: (A–C) 100 μm; (D) 10 μm.
Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain. (2008)
Figure 2.

Ref(2)P localizes in protein aggregates in old flies during normal aging. (A–D) Electron micrographs of adult brain of old wild-type flies. Ref(2)P localizes in electron-dense structures ranging from 50 nm to 1 μm in diameter that are surrounded (arrows) or not (arrowhead) by a limiting membrane. (E and F) Electron micrographs of wild-type brain of old flies demonstrating that Ref(2)P forms shell-like structures that surround aggregated filamentous material (arrows). Bars: (A–C) 50 nm; (D) 100 nm; (E and F) 200 nm.
Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain. (2008)
Figure 3.

Ref(2)P localizes to protein aggregates in flies that have impaired autophagy or proteasome function. The formation of these aggregates is inhibited in atg8a/ref(2)P double mutant flies. (A and B) atg8a mutants exhibiting a striking positive staining for Ref(2)P and ubiquitin staining in all the regions of the adult brain. As it is shown in high magnification of the optic lobe (medulla region; B), Ref(2)P- and ubiquitin-positive aggregates are observed (arrows). (C) Normal phenotype is observed in β2 and 6 dominant-negative temperature-sensitive mutants at 25°C. (D) Accumulation of Ref(2)P- and ubiquitin-positive aggregates are observed in β2 and 6 mutants at 29°C (arrows). (E and F) double mutants for atg8a and ref(2)P loss-of-function alleles lacking the PB1 (E) or UBA (F) domain do not form ubiquitinated protein aggregates. Ubiquitin exhibits a cytoplasmic staining pattern (arrows). Genotypes: (A and B) atg8aKG07569/Y, (C and D) elavGal4/+; UAS-β6/+; UAS-β2/+, (E) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, and (F) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3. Bars: (A) 100 μm; (B–F) 10 μm.
Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain. (2008)
Figure 4.

Ref(2)P is a component of protein aggregates in flies defective in autophagy and D. melanogaster models of human neurodegenerative diseases. (A and B) Protein aggregates in atg8aKG07569 mutants exhibit sequestosome morphology and are positively labeled for Ref(2)P (A) and ubiquitin (B). Protein aggregates are surrounded by small electron-dense vesicles (arrows). (C) Western blot analysis of cell lysates of wild type and atg8aKG07569 young adult heads demonstrating a significant increase of Ref(2)P protein levels in atg8aKG07569 flies. (D, left) Ref(2)P protein migration profile in ref(2)P loss-of-function alleles. Ref(2)P mutant protein for the PB1 domain has an apparent molecular mass of 85 kD (ref(2)Pod2/ref(2)Pod2) and the Ref(2)P mutant protein for the UBA domain has an apparent molecular mass of 69 kD (ref(2)Pod3/ref(2)Pod3). (D, right) Western blot analysis of insoluble protein fractions of adult heads of double mutants for atg8a and ref(2)P probed for ubiquitin. Genotypes: (1) atg8aKG07569/Y; ref(2)Pod2/Cyo, (2) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, (3) atg8aKG07569/Y; ref(2)Pod3/CyO, and (4) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3. The insoluble ubiquitinated protein profile of the double mutants is diminished compared with the one of the respective control heterozygous for ref(2)P. (E and F) Neuronal cells mutant for atg1 (green) accumulate Ref(2)P- (E) and ubiquitin-positive (F) structures (arrows), whereas the normal surrounding cells do not exhibit positive staining. (G) Confocal micrographs of the optic lobe (medulla region) of young flies expressing a mutated form of human tau protein exhibiting Ref(2)P- and tau-positive aggregates (arrows). Genotypes: (E and F) yw/UAS-CD8-GFP, hs-flp; tubGal4/+; atg1Δ3D, FRT80/tubGal80, FRT80, and (G) elav/+;UAS-E14/+. Bars: (A) 500 nm; (B) 200 nm; (E) 2 μm; (F) 5 μm; (G) 10 μm.
Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2 (2022)
Figure 1

Figure 1. Sigmar1 deletion has no influence on bone mass under steady conditions.

(A) Microcomputed tomography (micro-CT) images of the proximal femur from 12-week-old male WT and Sigmar1 gKO mice. Scale bars, 1 mm.

(B) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp), trabecular thickness (Tb. Th), cortical region BV/TV (Ct. BV/TV) and cortical thickness (Ct. Th, mm) (n = 5 biological replicates).

(C) Coronal images of the fifth lumbar spine. Scale Bars, 1 mm.

(D) Quantification of trabecular bone parameters of lumbar spine (n = 5 biological replicates).

(E) TRAP staining of tibias from male WT and Sigmar1 gKO mice. Scale bars, 50 μm.

(F and G) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates).

(H and I) Representative images and quantitative analysis of calcein double labeling. Scale bars, 20 μm. (n = 6 biological replicates)

(J and K) Serum PINP (procollagen I N-terminal propeptide) and CTX-I (C-terminal telopeptide of type I collagen) concentrations measured by ELISA in male Sigmar1 gKO mice and their WT littermates (n = 6 biological replicates).

(L) MSCs from male WT or Sigmar1 gKO mice underwent osteogenic differentiation for 7 or 21 days and staining for alkaline phosphatase or alizarin red, respectively.

Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test (B, D, F-G, I and J-K) was used for statistical analysis.
Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2 (2022)
Figure 2

Figure 2. Sigmar1 deletion results in severe osteoporosis in the OVX model and promotes osteoclastogenesis in vitro.

(A) Micro-CT images of the proximal femur from female WT or Sigmar1 gKO mice that received sham or ovariectomy surgery for 6 weeks. Scale bars, 1 mm.

(B) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (n = 5 biological replicates).

(C) TRAP staining of femur sections from the four groups. Scale Bars, 200 μm.

(D and E) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 5 biological replicates).

(F) TRAP staining to detect osteoclastogenesis of BMMs from WT or Sigmar1 gKO mice. Scale bars, 200 μm.

(G and H) Quantification of the size and nuclei numbers of TRAP-positive multinuclear cells (n = 6 biological replicates for G and n = 3 biological replicates for H).

(I and J) Representative images and quantification of the relative pit resorption area of hydroxyapatite-coated plates. WT or Sigamr1 gKO BMMs were seeded on hydroxyapatite-coated plates and treated with 50 ng/mL RANKL (n = 6 biological replicates).

Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (B, D and E) and unpaired two-tailed Student's t-test (G-H and J) were used for statistical analysis.
Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2 (2022)
Figure 3

Figure 3. Overexpression of Sigmar1 rescues OVX-induced bone loss.

(A) Schematic illustrating the protocol for OVX-induced bone loss and AAV-Sigmar1 treatment. Briefly, 12-week-old mice were subjected to either sham or ovariectomy surgery together with different AAV intramedullary injection, and radiological analysis was conducted 6 weeks later.

(B) Micro-CT images of the proximal femur from sham or OVX mice with different AAV injections. Scale bars, 1 mm.

(C) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (n = 5 biological replicates).

(D) TRAP staining of femur sections from the three groups. Scale Bars, 200 μm.

(E and F) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 5 biological replicates).

(G) TRAP staining to detect osteoclastogenesis of BMMs treated with different Sigmar1 agonists (10μM) or vector. Scale bars, 200 μm.

(H and I) Quantification of the size and nuclei numbers of TRAP-positive multinuclear cells (n = 6 biological replicates for H and n = 3 biological replicates for I).

(J) WT MSCs underwent osteogenic differentiation for 7 or 21 days in the presence or absence of dimemorfan and was stained for alkaline phosphatase or alizarin red, respectively.

Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C, E-F, I, K-O) was used for statistical analysis.