EMBO/SourceData huggingface dataset:
Figure 3.(A) Indicated strains were transformed with plasmids encoding either Rpl25-GFP (Rpl25) or rpl25 K74,75R-GFP (rpl25KR) before the deletion of a genomic copy of RPL25. (top) Comparable expression levels of Rpl25-GFP or rpl25 K74,75R-GFP in whole cell lysates were confirmed with anti-GFP blotting. Purified 6His-ubiquitin-conjugated forms of Rpl25-GFP or rpl25 K74,75R-GFP (middle) and 6His-ubiquitin expression (bottom) were analyzed as in Fig. 2. Ub, ubiquitin.(B) Wild-type and ubp3Δ cells expressing Rpl25-GFP or rpl25 K74,75R-GFP were starved for the indicated period of time. (C) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts, and the ratio between cleaved GFP and full-length protein was quantified for all time points in four independent experiments. a.u., arbitrary units. (D) Localization of GFP-tagged proteins. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole(E) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti-Rpl3 (gift from V. Albanese, Institut Jacques Monod, Paris, France) or anti-Mex67 (as a control) antibodies. Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0. Significance of the differences observed for the ribophagy efficiency was evaluated using Student's t test. *, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001. White lanes indicate that intervening lanes have been spliced out. The errors bars correspond to standard deviations.
Figure 4.(A) Wild-type cells expressing Bre5-GFP, Ltn1-Flag, or ltn1-ΔRing-Flag were starved for the indicated periods of time.(B) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132. Expression of tagged proteins was analyzed by anti-GFP or anti-Flag blotting of whole cell extracts. Five times less extracts were analyzed for ltn1-ΔRing-Flag cells. A nonspecific band served as a loading control. White lanes indicate that intervening lanes have been spliced out. WT, wild type.
f1(a) Miz1ΔPOZNes mice move slowly and lack motor coordination. The panel shows the average speed of movement and time on a rotarod (maximum: 300 s) maintained at constant speed of control and age-matched Miz1ΔPOZNes mice. The average age of mice tested was 16 months. P-values were calculated using an unpaired two-tailed Student's t-test (the number of animals tested is shown below each graph).(b) Defect of cerebellar growth in Miz1ΔPOZNes mice. The panels show sagittal sections of brains of control and Miz1ΔPOZNes mice at the indicated age. Dotted lines show the circumference of the cerebellum. For each mouse, the cerebellar surface area was determined; numbers show the ratio between control and Miz1ΔPOZNes mice. The number of mice analysed for each genotype is indicated on the right. Scale bar, 1 mm.(c,d) Histology of the cerebellum. Sections of the cerebellum of 11-month-old control (c) and Miz1ΔPOZNes (d, please note different scale) mice were stained with antibodies directed against calbindin, a marker for PCs, and glial fibrillary acid protein (GFAP), a marker for Bergman glial cells. Boxes indicate the area of a detail picture in the overviews. GFAP staining is shown for adjacent section (arrowheads: regions of missing PCs). Scale bar, 500 μm (upper panels) and 100 μm (lower panels).(e) Enhanced apoptosis in the granular layer of cerebella in Miz1ΔPOZNes mice. The upper panel shows representative TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining of cerebella of age-matched control and Miz1ΔPOZNes mice. The lower panel shows a quantification of staining observed in four control and five Miz1ΔPOZNes mice. Error bars show s.e.m. P-value was calculated using an unpaired two-tailed Student's t-test.
f2(a) ISH documenting the expression of Miz1 mRNA in PCs of the cerebellum in wild-type mice. Left panel shows ISH, right panel shows haematoxylin and eosin staining. Scale bar, 50 μm. (b) IHC documenting the presence of Miz1 in the nuclei of PCs. The panel on the left shows staining with α Miz1 (H190) antibody, staining on the right is a control staining lacking primary antibody. Scale bar, 50 μm.
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.(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.
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.(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.
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.(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 monthsfor 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.
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.
Figure 2A: Brightfield and fluorescence images of a NTH/GFP zygote at 4h post-fertilization, showing dispersed extranuclear fluorescence. Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm. B: Image of an NTH/GFP ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows). Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm. D: Brightfield and fluorescence images of a zygote at 4h post-fertilization simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red). Scale bar = 5μm. E: An ookinete simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red), showing co-localization in the crystalloid. Scale bar = 5μm. F: Western blot of purified NTH/GFP ookinete samples with (+) and without (-) prior in vivo crosslinking. Mr markers (kDa) are indicated on the right-hand side.
Figure 3A: Brightfield images of a NTH-KO and NTH/GFP ookinete, showing normal ookinete morphology, but lack of pigment clusters associated with crystalloids (arrows) in the NTH null mutant. Scale bar = 5μm.B: Aligned dot plot of oocyst numbers in Anopheles stephensi infected with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20); ns, not significantly different (Mann-Whitney).C: Brightfield and fluorescence images of oocysts of parasite lines NTH/GFP and NTH-KO at 15 days post-infection, showing lack of sporulation in the NTH null mutant. Hoechst DNA staining (artificially depicted red) labels parasite nuclei. The large nuclei of the epithelial midgut cells also show. Scale bar = 20μm.D: Aligned dot plot of oocyst diameters in Anopheles stephensi at 15 days post-infection with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20). ** mark statistically significant differences (P<0.0001, Mann-Whitney); ns, not significant.E: Images of ookinetes of parasite lines NTH∆PP showing dispersed NTH∆PP::GFP fluorescence; LAP3/mCherry showing focal LAP3::mCherry fluorescence in crystalloids; Cross (carrying modified alleles encoding NTH∆PP::GFP and LAP3::mCherry), showing dispersed LAP3::mCherry fluorescence. Arrow marks pigment cluster associated with crystalloids. Scale bar = 5μm.G: PCR diagnostic for the presence of modified nth alleles in blood stage infections obtained after sporozoite transmission of a NTH∆PP x LAP3/mCherry genetic cross (lane NTH∆PP), amplifying an approximately 4.4kb fragment. NTH/GFP parasites are used as a PCR control, amplifying approximately 4.6kb. Parental parasites provide a negative control.H: Image of a NTH/ND500LK ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows) indicative of normal crystalloid formation. Scale bar = 5μm.
Figure 4A: Brightfield and fluorescence image of an unsporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm. B: Image of a sporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm. C: Image of a NTH/GFP sporozoite, showing NTH::GFP localisation in a tubular structure. Scale bar = 10μm.D: Image of a sporulated oocyst simultaneously expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast. Scale bar = 20μm and 10μm (inset).E: Image of sporozoites expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast (arrows). Sporozoites expressing only ACP::mCherry or NTH::GFP are also present. Scale bar = 10μm.F: Image of group of NTH/GFP sporozoites stained with MitoTracker Red CMXRos, showing distinct subcellular positions of apicoplasts (green) and mitochondria (red). Scale bar = 10μm.
Figure 1 (A) Immunostaining of ES cells (Oct4), NPCs (day4, Nestin), and neurons (day 8, Tuj1). Scale bar: 200µm. (B) Expression levels of candidate lncRNAs during three stages of differentiation, measured by RNA-seq. Gene models of lncRNAs appear in green, adjacent protein-coding genes appear in grey. Blue depicts transcripts from the '+' strand, orange represents transcripts from the '-' strand. All three RNA-seq tracks were scaled separately to the maximum coverage in each locus. (C) qRT-PCR of Reno1, Cox10as1, and lnc-Nr2f1 in ES cells (top) and following eight days of neuronal differentiation (bottom), following knockdown using two different shRNAs, normalized to sh-NT. Mean ± SEM is shown for 3-6 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test).
Figure 2 (A) Immunostaining using anti-TUJ1 antibody of ES-cell-derived neurons following infection with either non-targeting shRNA (sh-NT), or two different shRNAs targeting Reno1. Scale bar: 200µm. (B) Quantification of cell numbers and neurite lengths of ten images of non-overlapping fields for each shRNA. Mean ± SEM is shown, ** P<0.01 (unpaired two sample t-test).  (C) Mean expression levels of genes which were down- or upregulated following knockdown (KD) of Reno1 or lnc-Nr2f1 during an eight time point neuronal differentiation and maturation of mouse ES cells: Neurons1 corresponds to day in vitro (DIV)1; Neurons2 - DIV7; Neurons3 - DIV16; Neurons4 - DIV21; and Neurons5 - DIV28 (Hubbard et al., 2013). (D) Transcriptional response of cell cycle and neuron-related genes. Heatmaps show log2FC of cells infected with shRNAs targeting Reno1 or lnc-Nr2f1 compared to cells infected with sh-NT, following eight days of neuronal differentiation, and of day eight of differentiation compared to ES cells during differentiation of WT cells.
Figure 3 (A) Reno1 locus in six vertebrate species. Shaded region indicates the Reno1 locus. Blue gene models are from RefSeq or Ensembl, red gene models are based on PLAR transcript reconstructions (Hezroni et al., 2015). RNA-seq datasets from indicated tissues were taken from publicly-available datasets: BodyMap (Li et al, 2017a) (mouse), Reference Epigenome (human), FAANG (sheep), SRP011985 (opossum), SRP016501 (chicken), DRP000627 (coelacanth). RNA-seq data was scaled separately in each species to the highest coverage in the window, except for opossum and chicken, where a custom upper threshold was set as indicated.  (B) The mouse Reno1 locus. Expression levels during neuronal differentiation of ES cells, in different cellular fractions of ES cells (data from (Engreitz et al, 2016)) and motor neurons (GSE90913), and in forebrain during different stages of embryonic development (data from the ENCODE project). Each set of tracks was normalized separately.  (C) RENO1 locus in human. Expression levels in dorsolateral prefrontal cortex across six age groups, and in different cellular fractions in fetal and adult cells. Each set of tracks was normalized separately.  (D) Reno1 smFISH signal (red) and DAPI staining (blue) in WT and Reno1m/m cells at day eight of neuronal differentiation, imaged using 100x objective.  (E) Expression levels of Reno1 and Bahcc1 in 30 adult and embryonic tissues. Data from the ENCODE project, quantification was done using RSEM (Li & Dewey, 2011). (F) Targeted Chromosome Conformation Capture (4C) using the Reno1 promoter as bait. Top, smoothed trend lines and raw counts of the contact profile in ES cells (red) or differentiated neurons (black); bottom, domainogram showing mean contact per fragment end for a series of window sizes.
Figure 4(B) Expression levels of Reno1 (both the spliced and unspliced isoforms) and Bahcc1 following four days of differentiation of the indicated cells. Mean ± SEM is shown for 3-4 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test). (C) Left: Oct4 staining of the indicated cells. Scale bar: 200 µm. Right: Cell count of the indicated ES cells grown in ES medium. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments.  (D) Left: Nestin staining of the indicated cells following four days of differentiation. Scale bar: 200 µm. Right: Cell count of the indicated cells following four days of differentiation. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. ** P<0.01 (unpaired two sample t-test).  (E) Flow cytometry analyses of dead and live cells in the indicated ES cells following four days of differentiation. Y axis: forward scatter; X axis: PI fluorescence intensity.
Figure 5 (A). Heatmap showing log2-transformed fold changes of Reno1 depleted cells compared to their respective controls. All genes which were significantly differentially expressed (P<0.05) in any of the treatments are shown. (B) Boxplots indicating the median, quartiles, and 5th and 95th percentiles of changes in expression levels of Reno1m/m cells compared to WT cells, for genes with significant (P<0.05) change in cells where Reno1 was perturbed with the indicated method. Number of genes in each group is indicated in the x axis. ** P<0.01 (two-sided Wilcoxon rank sum test).  (C) Mean expression levels of genes in genes which were significantly (P<0.05) dysregulated by at least two out of three Reno1 perturbations at day 2 in fig. 5A (n= 168 down-regulated and 94 up-regulated), during an eight time point neuronal differentiation and maturation of mouse ES cells: Neurons1 corresponds to day in vitro (DIV)1; Neurons2 - DIV7; Neurons3 - DIV16; Neurons4 - DIV21; and Neurons5 - DIV28 (Hubbard et al., 2013).  (D) Expression levels of Reno1 following Dox addition in WT and Reno1m/m cells. Mean ± SEM is shown for three independent experiments, * P<0.05 (unpaired two sample t-test).  (E) Cell count of WT and Reno1m/m cells following four days of differentiation, with or without Dox addition. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. * P<0.05 (unpaired two sample t-test).  (F-G) Boxplots indicating the median, quartiles, and 5th and 95th percentiles of changes in expression levels of WT (F) or Reno1m/m (G) cell treated with Dox for the first two days of differentiation compared to untreated cells, in genes which were significantly (P<0.05) downregulated (n=252) or upregulated (n=223) by at least two out of three Reno1 perturbations at day 2 in fig. 5A, compared to all genes (n=16,511). ** P<0.01 (two-sided Wilcoxon rank sum test).
Figure 6 (A) Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ratios of ATAC-seq signal for the indicated group of peaks, comparing the indicated genotypes or treatments. Core promoter peaks are <300 nt from a TSS; Extended promoter are more than 300 nt but <2 Kb for a TSS; Gene body peaks overlap transcription units, and Intergenic do not. Each group of peaks was compared to the intergenic peaks. In addition, for each group of peaks, the peaks within 1 Mb of Reno1 where compared to peaks further away. P-values computed using two-sided Wilcoxon rank sum test, and shown only for cases where P<0.05. Number of data points (from left to right): 39; 10,777; 11; 3,550; 17; 14,380; 18; 20,930. (B) Changes in ATAC-seq signal for peaks assigned as falling within core or extended promoters, of genes in the indicated group (up/down-regulated by at least 30% and with adjusted P<0.05) in cells with Reno1 (left) or Bahcc1 (right) KD. P-values computed using two sided Wilcoxon rank sum test.  (C) Top: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of changes in ATAC-seq signal on day 2 of neuronal differentiation of peaks from indicated Reno1 perturbation, classified according to the underlying chromatin state in mouse ES cells; Bottom: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ChIP-seq coverage of the indicated chromatin marks in ENCODE mouse ES data in the peak regions. Number of peaks in each group is indicated at the bottom.  (D) Correlation between changes in chromatin accessibility on day 2 of neuronal differentiation in the indicated perturbations and H3K4me3 or Fam60a ChIP coverage from mouse ES cells (H3K4me3 is from ENCODE project and Fam60a from (Streubel et al., 2017)). Coefficient and P-value computed using Spearman's correlation. n=49,722 peaks.