EMBO/soda-nel huggingface dataset:
H2A.B is a cancer/testis factor involved in activation of ribosome biogenesis in Hodgkin Lymphoma (2021)
Figure 4

Figure 4. H2A.B is required for high levels of pre-rRNA transcription.

A qPCR of cDNA from L1236 and L428 HL cell RNA obtained 5 days post H2A.B knockdown. Amplification was performed using primers that anneal to the ETS and ITS spliced-out regions to detect nascent rRNA. ETS and ITS, external and internal transcribed regions, respectively. The relative levels of both ETS and ITS were normalised to the average expression levels of the B2M and HPRT1, and presented as mean ± SD for eight biological replicates. The significance of the difference was determined by student's t test and denoted as * p < 0.05; *** p < 0.001.

B Cell proliferation assay in the presence (shNTC) or during depletion of H2A.B (shH2A.B). The results are presented as mean ± SD for five biological replicates. The significance of the difference was determined by Student's t test and denoted as ns (not significant); **, p < 0.01; ***, p<0.001.
H2A.B is a cancer/testis factor involved in activation of ribosome biogenesis in Hodgkin Lymphoma (2021)
Figure 6

Figure 6. H2A.B is located at the TSS of active genes. Mean H2A.B and IgG.

A, B CUT&RUN coverage normalised by spike-ins from 1kb upstream to 1kb downstream for all ribosomal protein genes in L1236 HL cells (A) and L428 HL cells (B). CUT&RUN data analyses were performed on two biological replicates.

C, D Mean difference between H2A.B and IgG CUT&RUN coverage normalized by spike-ins aligned between -1 and +1 kb from the TSS ranked according to their level of expression in L1236 HL cells (C) and L428 HL cells (D). Below is the position of H2A.B nucleosomes relative to the TSS; the colour reflects nucleosome abundance (red reflects the highest abundant H2A.B nucleosome located at the TSS).

E Genome browser screen shots of gene examples (RPs RPLP1 and RPL23A, and HDAC5 and TAF10) that contain H2A.B at their promoters identified by the CUT&RUN peak calling and whose expression decreased significantly following H2A.B knockdown in the RNA-seq analysis. Green and purple indicates L1236 and L428cells, respectively.
H2A.B is a cancer/testis factor involved in activation of ribosome biogenesis in Hodgkin Lymphoma (2021)
Figure 7

Figure 7. Pre-mRNA splicing is altered following the depletion of H2A.B.

A Changes in exon usage between shNTC and shH2A.B L1236 HL cells. Highlighted in red are RP genes. The significance (-log10 (P value)) is plotted against the fold change of exon usage (log2FC).

B Differential exon usage of the CD44 gene.

C. Differential exon usage of the RPL17 gene.
H2A.B is a cancer/testis factor involved in activation of ribosome biogenesis in Hodgkin Lymphoma (2021)
Figure 8

Figure 8. Loss of H2A.B decreases chromatin accessibility in introns.

A Changes in ATAC-seq accessibility at H2A.B CUT&RUN peaks following H2A.B knockdown. The significance (-log10 (P value)) is plotted against the fold change in accessibility (log2 (shH2A.B/shNTC)). Red indicates H2A.B peaks that increased in accessibility (5,552). Blue indicates H2A.B peaks that decreased in accessibility (3,310). ATAC-seq data analyses were performed on two biological replicates.

B Differential accessibility changes of H2A.B CUT&RUN peaks following H2A.B knockdown for the peaks that increased in accessibility (red) and peaks that decreased in accessibility (blue) in Panel A. The change in mean ATAC-seq coverage (RPM) is plotted against the centre of the H2A.B peaks.

C Genomic distribution of H2A.B CUT&RUN peaks, shown as percentages, with increased (5,552 in total) or decreased (3,310 in total) accessibility following H2A.B knockdown.

D, E Two examples show increased accessibility at the promoter peak (NXF1) (D) and decreased accessibility at the peak across the intron-exon boundary (KDM4A) (E), respectively.

EXPANDED VIEW FIGURE LEGENDS
Termination of autophagy and reformation of lysosomes regulated by mTOR (2010)
figf1

a, LAMP1+ lysosome size and quantity in NRK cells (dotted outline) starved for hours (h). Error bars show s.e.m. (n = 3). b, Starved NRK cells expressing LAMP1–YFP show tubules (arrows). Box is expanded on the right. c, Immuno-TEM (gold marks LAMP1) of starved NRK cells. Continuity (arrow) illustrated between autolysosome (red star) and tubule. d, LAMP1–YFP expressing NRK-LC3 cells as in a. Arrows show reformation tubules. e, Three-dimensional reconstruction of a cell from d. f, Time-lapse images (seconds) of starved LAMP1–YFP-expressing NRK cells. g, Time-lapse images (minutes) of starved LAMP1–PAGFP or LAMP1–RFP-expressing NRK cells after photo-activation. Scale bars, 5 µm.
Termination of autophagy and reformation of lysosomes regulated by mTOR (2010)
figf2

a–c, Starved LAMP1–YFP NRK cells stained with Lysotracker or DQ-BSA. c, Right, TEM of purified lysosomes. d, Magnification (4×) of images shown in c. Arrows indicate reformation tubules and arrowhead indicates autolysosome. e, f, Immunofluorescence (e) and immuno-EM (f) using LAMP1 antibody of cells shown in c. g, Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D. Middle panel shows 8-h fractions 7 and 9 analysed by TEM. Fraction 10 is the gradient top. h, Starved LAMP1–PAGFP NRK cells were photo-activated (4 h) and imaged at 12 h with Lysotracker (upper) or DQ-BSA (lower). Boxes show enlargements. R(r) = Pearson’s coefficient. Scale bars, 5 µm.
Termination of autophagy and reformation of lysosomes regulated by mTOR (2010)
figf3

a, TEM (bottom; scale bar, 5 µm) and fluorescence (top; scale bar, 10 µm) of NRK-LC3 cells starved for times shown with enlargements (boxes). b, d, Immunoblots of cells shown in a with indicated antibodies (d, after transfection with non-specific (NS) or atg5 RNAi). c, Autophagy (black lines show independent experiments) and phospho-S6K percentage (densitometry of data shown in b, red line). e–g, LAMP1–YFP NRK-LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h (total, 6 h) or 6 h (total, 8 h) analysed by blotting (e) as in b or microscopy (f; scale bar, 5 µm) with quantification (g). Error bars, s.e.m.; n = 3.
Termination of autophagy and reformation of lysosomes regulated by mTOR (2010)
figf4

a, LAMP1/LC3 NRK cells were starved for 4 h and treated with GTP&ggr;S for 6 h of further starvation. b, Rab7Q67L–GFP-transfected LC3–CFP/Lamp1–RFP NRK cells starved for 10 h. c, Rab7–CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation. d, LAMP1–YFP NRK-LC3 cells starved for times shown with 1 µg ml–1 leupeptin and blotted with indicated antibodies. e, Cells in d imaged.. f, Fibroblast cells from patients with Scheie syndrome (GM01256) or healthy controls starved and blotted with indicated antibodies. g, Cells in f imaged. Lysosome size classified by Image-ProPlus (IPP) software. h, Provisional model of the autophagic lysosome reformation (ALR) cycle. Scale bars, 5 µm.
DHPR α1S subunit controls skeletal muscle mass and morphogenesis (2009)
Figure 1

Generation of a local knockdown of α1S subunit. (A) Schema of exonic chaining of pre‐mRNA, exons 15, 16 and 17 are represented by blue boxes. Sequences of exon acceptor site SA (in blue) and exon splicing enhancer ESE (in black) of exon 16 were targeted by antisense sequences introduced in U7smOPT cassettes. (B) qRT–PCR analysis using primers overlapping exons 15 and 16 permitted to quantify the non‐skipped α1S form: 18±6% (**P⩽0.001, n=19) and 10±7% (**P⩽0.001 n=19) (black bars) of the total α1S mRNA (grey bars) at 2 and 6 months post injection in TA of AAV1‐(U7‐ESE) and AAV1‐(U7‐SA):ΔDHPR or AAV1‐(U7‐Ctrl): c as a control, respectively. (C) Six months post injection, lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for α1S or α‐actin for four mice. Graph depicts mean±s.e.m. of relative expression of α1S subunit determined by densitrometry and nomalized to the α‐actin expression for each muscle. Results were expressed in protein levels of α1S subunit in TAΔDHPR normalized to TACtrl for each mice, **P⩽0.001, n=4. (D) Longitudinal cryo‐sections from TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red) and anti‐laminin (green) antibodies, nuclei were visualized by Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.
DHPR α1S subunit controls skeletal muscle mass and morphogenesis (2009)
Figure 2

Consequence of α1S subunit knockdown. (A) Six months post injection, TAs from eight mice were dissected and weighed. (B) The internal diameters (shortest diameter) from all fibres throughout the total muscle section were recorded and analysed. Muscles from five different animals were examined. The bar graph presents mean±s.e.m. of the number of myofibres by fibre diameter class for TAΔDHPR (black) and TACtrl (grey). (C) Transversal sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with haematoxylin and eosin, bars represent 100 μm. (D) To quantify fibrosis, transversal sections of total muscle were stained with Red Sirius and quantified using Histolab Software (marked in blue), data were normalized with total surface of each muscle (orange line surrounding the sections), bars represent 500 μm. (E) Quantification is presented in bar graph and showed 4.1±0.1 fold increase of fibrosis in TAΔDHPR (ΔDHPR) compared to TACtrl (c) (**P0.001, n=4).
DHPR α1S subunit controls skeletal muscle mass and morphogenesis (2009)
Figure 3

nNOS membrane dissociation, FoxO3A accumulation in myonuclei and upregulation of autophagy‐related genes as consequences of α1S subunit loss. (A) mRNA from TAΔDHPR and TACtrl tissues were extracted and nNOS expression was quantified by qRT–PCR. Results are expressed as mean±s.e.m., **P0.001, n=4. (B) Transversal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐nNOS (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm. (C) The tissues from extracts were analysed by western blot with FoxO3a‐P antibody and normalized with α‐actin antibody. Graph depicts mean±s.e.m. of relative expression of FoxO3a‐P determined by densitometry and nomalized to the α‐actin expression for each muscle, *P⩽0.005, n=3. (D) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐FoxO3a (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm. (E) mRNA from TAΔDHPR and TACtrl tissues were extracted and regulation of autophagy genes expression was followed by qRT–PCR. Bnip, CathepsinL, LC3 and PI3KIII expression (noted in red) were significantly increased in TAΔDHPR compared with the controlateral TACtrl, **P0.001, n=4.
DHPR α1S subunit controls skeletal muscle mass and morphogenesis (2009)
Figure 4

Formation of autophagosomes in TAΔDHPR. (A) Ultrathin sections were imaged by electron microscopy, M, mitochondria; C, collagen fibres; T, T‐tubule. Arrows show double‐membrane vesicules called auphagosomes. (B) Transversal cryo‐sections were stained with anti‐LC3b (red), anti‐dystrophin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Upper panel: TACtrl (Ctrl) and lower panel: TAΔDHPR (ΔDHPR). Bars represent 20 μm. (C) Myofibres isolated from control (Ctrl) or 6 months post‐injected FDB (ΔDHPR) muscles were processed for immuno‐fluorescent labelling for P62 (green) and LC3b (red) and imaged by confocal microscopy. Scale bars, 10 μm.
DHPR α1S subunit controls skeletal muscle mass and morphogenesis (2009)
Figure 5

Impact of the α1S subunit knockdown in maintenance of muscle ultrastructure. (A) Ultrathin sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were imaged by electron microscopy. Cis, terminal cisternae; T, tubule; SR, sarcoplasmic reticulum. Bars: 500 nm. (B) Myofibres isolated from Ctrl or ΔDHPR FDB muscles 6 months post injection were processed for immuno‐fluorescent labelling for RyR1 (green), SERCA (red), Dapi (blue) and imaged by confocal microscopy. Scale bars, 10 μm.
DHPR α1S subunit controls skeletal muscle mass and morphogenesis (2009)
Figure 6

α1S subunit knockdown and RyR1 expression. (A) Lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for RyR1 or α‐actin. (n=4). Graph depicts mean±s.e.m. of relative expression of RyR1 determined by densitometry and nomalized to the α‐actin expression for each muscle. Results were expressed in protein levels of RyR1 in TAΔDHPR normalized to TACtrl for each mice, *P⩽0.005, n=4. (B) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red), anti‐RyR1 (green) antibodies and imaged by confocal microscopy. Bars=20 μm.
DHPR α1S subunit controls skeletal muscle mass and morphogenesis (2009)
Figure 7

Localization of α 1S subunit. Whole skeletal muscle fibres enzymatically isolated from Ctrl (A–D) or ΔDHPR (E–H) FDB muscles 6 months post injection were processed for immuno‐fluorescent labelling for α1 S subunit (red) and laminin (green). Scale bars, 10 μm; Arrows indicate α1 S subunit expression on sarcolemma; antibody labelling was visualized by serial confocal microscopy and represented as movies of the optical sections (Supplementary Movies S5A and S5B). (D, H) Present projections of confocal Z‐series (step between each frame is 1 μm) along XZ and YZ planes as indicated.
Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor (2021)
Figure 1

Figure 1. SERA6 is rapidly proteolytically processed into multiple protein species at egress.

(A) Top: SERA6 architecture. Positions of the papain-like domain, catalytic triad residues, SUB1 cleavage sites and epitopes for the antibodies used are indicated. Positions of the inserted mTAP or myc3 epitope tags (between codons Asn886-Val887) introduced in SERA6-mTAP:loxP parasites are shown. Bottom: time-course analysis of egress by western blot, showing processing of SERA6-mTAP into multiple protein species (representative of 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2-mediated arrest. Nomenclature for each protein species was based on apparent molecular mass or predicted composition. Note that the predicted mass of the p40 species is ~27 kDa, but its migration on SDS PAGE may be aberrant due to its acidic nature; the predicted pI of sequence between SUB1 site 1 and the central papain-like domain (Asp371-Lys605) is ~4.4 (see https://web.expasy.org/compute_pi/).

(B) Coomassie-stained SDS PAGE gel showing proteins immunoprecipitated from schizonts expressing SERA6-mTAP, sampled at the indicated times following C2 washout or arrested with E64 (50 μM). Control, proteins immunoprecipitated from SERA6:loxP (Thomas et al, 2018) schizonts, which express non-epitope-tagged SERA6, sampled at 5 min following C2 washout. Arrowed, species analysed by mass spectrometric peptide mapping. Fragments A-H were identified as SERA6-mTAP-derived, while fragments I and J were derived from a different protein, PF3D7_1014100.

(C) Schematic of the likely identities of the different SERA6-mTAP species identified from peptide mapping, relative to full length SERA6-mTAP and the papain-like domain (hatched). The predicted mass of each species based on its amino acid composition is indicated. Detection of a semi-tryptic SERA6-derived peptide (filled block arrowhead) unambiguously confirmed cleavage at SUB1 site 1 while the coverage allowed mapping of up to five residues upstream of SUB1 site 2 (fragment E). No evidence was found for processing at a third putative SUB1 cleavage site (Ruecker et al., 2012). The N-terminus of fragment H (assumed to correspond to PAP-mTAP) could not be precisely mapped; attempts to assign its N-terminus by Edman degradation were unsuccessful. Also see Figs EV1 and EV2.
Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor (2021)
Figure 2

Figure 2. Maturation of SERA6 p65 to the terminal PAP form is autocatalytic.

(A) Complementation constructs introduced into SERA6-mTAP:loxP parasites for episomal expression of wild-type (WT) or catalytically-dead (Cys644Ala) SERA6. The papain-like domain (hatched) and myc3 epitope tag are indicated. SERA6 expression from these constructs was under the control of its native promoter.

(B) Western blot analysis of egress time-course, comparing maturation of the different SERA6 variants from DMSO- and RAP-treated SERA6-WT-myc3 [SERA6-mTAP:loxP] and SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites (representative of 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2, or arrested with E64-d (50 μM), a membrane-permeable derivative of E64 which is a more potent inhibitor of SERA6 maturation (Appendix Fig S1). Samples were probed with anti-myc to detect the myc3-tag, anti-HA to detect the mTAP tag, or anti-SERA6 (S6C1) to detect a different region of SERA6 respectively (see Fig 1A for antibody epitope positions). One of the blots was stripped and re-probed with anti-AMA1 as a loading control. Nomenclature for each protein species was based on apparent molecular mass or predicted composition.

(C) Giemsa-stained thin films and western blot of DMSO- and RAP-treated SERA6-WT-myc3 [SERA6-mTAP:loxP] and SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites showing that cleavage of host RBC β-spectrin (red arrowhead) does not occur unless a functional copy of SERA6 is present, and is tightly associated with egress (reproducible in 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2. Scale bar, 5 μm. Also see Fig EV2.
Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor (2021)
Figure 4

Figure 4. MSA180 is an essential cofactor required for maturation of SERA6, host RBC β-spectrin cleavage and egress.

(A) Replication of DMSO- and RAP-treated MSA180-HA3:loxP parasites over three erythrocytic cycles. Parasitaemia values are averages from replicates in different blood sources. The similar growth rate of DMSO-treated parasites and the parental B11 clone shows that the genetic modifications to generate MSA180-HA3:loxP parasites did not affect parasite viability. Error bars, ± S.D (B11: n=3; DMSO- and RAP-treated MSA180-HA3:loxP: n=6 each).

(B) Stills from simultaneous time-lapse DIC and fluorescence microscopic examination of DMSO- and RAP-treated MSA180-HA3:loxP cycle 0 schizonts following removal of C2-arrest (representative of 2 independent experiments). Note the defect in RBCM rupture and merozoite release in RAP-treated parasites despite normal PVM rupture (indicated by a sudden increased visibility and motility of merozoites) and RBCM poration (green ring indicating access and binding of phalloidin to the host RBC cytoskeleton). Scale bars, 5 μm.

(C) Western blot of DMSO- and RAP-treated MSA180-HA3:loxP parasites showing that host RBC β-spectrin truncation (red arrowhead) does not occur in the absence of MSA180 (reproducible in 2 independent experiments). Schizonts were sampled at the indicated times following removal of C2-arrest.

(D) Time-course comparing fate of SERA6 from DMSO- and RAP-treated MSA180-HA3:loxP parasites (representative of 2 independent experiments). Schizonts were sampled at the indicated times following removal of C2-arrest, or arrested with E64-d (50 μM). RAP-treated SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites were included in the blot to allow comparison with processing of catalytically-dead SERA6 (SERA6-Cys644Ala-myc3). Note the accumulation of SERA6 p65 and poor conversion to SERA6 p40 in the RAP-treated (ΔMSA180) parasites.

(E) Schematic model of SERA6 maturation.
Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor (2021)
Figure 5

Figure 5. SERA6 and its partner MSA180 form transient multi-molecular aggregates during egress.

(A) Representative IFA images demonstrating the dynamic changes in localisation of SERA6-mTAP during egress (reproducible in 4 independent experiments). SERA6-mTAP:loxP schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green; ankyrin is a major component of the RBC cytoskeleton). Scale bar, 5 μm.

(B) Z-stack series of a representative schizont demonstrating that SERA6 aggregates observed during egress are distributed throughout the schizont. SERA6-mTAP:loxP schizonts sampled 5 minutes following removal of C2-arrest were fixed and co-stained with anti-HA (red) and anti-ankyrin (green), then imaged on an Olympus FLUOVIEW FV3000. Scale bar, 5 μm.

(C) Representative IFA images demonstrating localisation of MSA180-HA3 during egress. MSA180-HA3 [B11] schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green). Scale bar, 5 μm. See also Fig EV5.
Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor (2021)
Figure 6

Figure 6. SERA6 maturation is a druggable process.

(A) Structures of two compounds identified as inhibitors of SERA6 autoprocessing and egress.

(B) E64-d, MMV676881 and Compound 31 inhibit RBCM rupture and merozoite release despite normal PVM rupture, indicated by loss of a continuous ring of EXP2-mNeon signal (green) and RBCM poration (red ring indicating access and binding of phalloidin to the host RBC cytoskeleton) (reproducible in 2 independent experiments). P. falciparum EXP2-mNeon schizonts were allowed to reach the point of egress in the presence of C2 (1 µM), E64-d (10 µM), MMV676881 (2 µM), Compound 31 (2 µM) or DMSO (0.1% v/v; control) then examined by DIC and fluorescence microscopy. Scale bar, 5 μm.

(C) Western blot comparing dose-response of E64-d, MMV676881 and Compound 31-mediated inhibition of maturation of SERA6-mTAP from SERA6-mTAP:loxP parasites (reproducible in 3 independent experiments).

(D) Western blot of P. falciparum and P. knowlesi parasites showing that host RBC β-spectrin truncation during egress (red arrowhead) is inhibited by all three P. falciparum SERA6-maturation inhibitors (concentrations as in panel B), matching the egress inhibition in panel E (reproducible in 2 independent experiments).

(E) Giemsa-stained thin films showing inhibition of rupture of P. knowlesi schizonts in human RBCs by E64-d, MMV676881 or Compound 31 (concentrations as in panel B; representative of 2 independent experiments). The increased space between individual merozoites still contained within the host RBC in parasites treated with E64-d, MMV676881 and Compound 31 (compared to C2-arrested parasites) suggests that PVM rupture has occurred. Note that P. knowlesi schizonts generally form fewer merozoites (~10-12) than P. falciparum schizonts. Scale bar, 5 μm.