EMBO/soda-nel huggingface dataset:
Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis (2018)
Figure 1

Figure 1. Mutations in hag1 cause severe defects in de novo shoot regeneration.

A Callus, de novo shoot, and root formation in wild-type (WT; Col), hag1-6, hag1-7, and 35S::FLAG:HAG1 hag1-7 root explants. Root explants were transferred onto CIM for 1 week (w) and then transferred onto fresh CIM, SIM, or RIM for callus, de novo shoot, or root induction, respectively. Scale bar: 1 cm. Graph on the right shows the percentage of explants with shoots formed as scored at 18 days (d) on SIM. 36 explants of each genotype were used for scoring.

B Shoot regeneration assay using Col and hag1-6 root explants on media with different cytokinin (2IP) to auxin (IAA) ratios. Col and hag1-6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg/ml IAA-containing media supplemented with 0, 500, or 5,000 mg/ml 2IP. Pictures were taken at 27 d after transfer onto each media using representative explants. Scale bar: 2 mm.

C Root explant-derived calli of Col and hag1-6 at 4 w on CIM (left). Scale bar: 1 mm. 4-w-old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight (right).

D Histochemical GUS staining of CYCB1;1:GUS in Col (left) and CYCB1;1:GUS in hag1-6. Shown are root explants at 4 d on CIM. Scale bar: 1 mm. See Appendix Fig S1E for GUS signal quantification.

E Statistic chart of differentially expressed genes (DEGs) in Col and hag1-6 roots (R), 4 d CIM-incubated root explants (C), 4 d CIM-incubated + 2 d SIM-incubated root explants (S), and 4 d CIM-incubated + 2 w SIM-incubated root explants (S2W). Green: up-regulated genes, log2 ratio ≥ 1; red: down-regulated genes, log2 ratio ≤ -1; FDR < 0.01.

F Immunoblot analysis of HAG1:HA protein. Samples are as described in (E). 1 w CIM-incubated Col root explants (Col) were used as negative control. Ponceau staining was performed as loading control.

Data Information: In (A and C), data are presented as means ± SE. Student's t-test (two tailed) *P ≤ 0.001, **P ≤ 0.01.

Source data are available online for this figure.
Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis (2018)
Figure 2

Figure 2. WOX5 and SCR expression is reduced in hag1.

A pWOX5::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with propidium iodide (PI) staining (red).

B pSCR::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).

C pSCR::GFP-ER expression in Col (WT) and hag1-6 root tips at various time points on CIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).

D, E WOX5 transcript levels in Col and hag1-6 hypocotyl (D) and cotyledon (E) explants at indicated days on CIM.

F, G SCR transcript levels in Col and hag1-6 hypocotyl (F) and cotyledon (G) explants at indicated days on CIM.

Data Information: Scale bars: 50 μm (A-C). In (A-C), at least 25 independent explants from each genotype were used for observation to confirm representative expression patterns of each marker in each genotype. In (D-G), data are presented as means ± SE of three biological replicates. Col levels at CIM 0 d were set to 1 after normalization with UBQ10. Student's t-test (two tailed) *P ≤ 0.01, **P ≤ 0.005.
Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis (2018)
Figure 3

Figure 3. WOX5, SCR, PLT1, and PLT2 loci are direct histone acetylation targets of HAG1.

A-D H3Ac enrichment at the WOX5 (A), SCR (B), PLT1 (C), and PLT2 (D) loci in Col (upper black diagram) and hag1-6 (lower red diagram) calli (1 w CIM) as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 5e-2 for WOX5 (A) and 1e-5 for SCR (B), PLT1 (C), and PLT2 (D). ChIP-seq data were visualized using Integrated Genome Browser (IGB; http://bioviz.org/igb/index.html). The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position, respectively. Schematic representation of each locus is depicted at the bottom. Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines. 5' and 3' UTRs are represented as gray boxes. Transcription start sites are indicated with arrows.

E-H ChIP-qPCR analysis of H3Ac levels at the WOX5 (E), SCR (F), PLT1 (G), and PLT2 (H) loci in Col and hag1-6. Root (R), 1 w CIM (C), and 1 w CIM + 2 w SIM (S) samples were used for assays. Regions tested are indicated in the schematics in (A-D).

I-L Direct targeting of HAG1:HA protein to WOX5 (I), SCR (J), PLT1 (K), and PLT2 (L) chromatin as determined by ChIP-qPCR. Samples were prepared as described in (E-H).

Data Information: Data are the means ± SE of three biological replicates as relative values to UBQ11 after normalization to input DNA (E-H) or the means ± SE of three biological replicates obtained after normalization to input DNA (I-L). Student's t-test (two tailed) *P ≤ 0.05, **P ≤ 0.01 (E-L).
Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis (2018)
Figure 4

Figure 4. HAG1-regulated WOX5, WOX7, and WOX14 are essential for de novo shoot regeneration.

A, B Transcript levels of WOX7 (A) and WOX14 (B) in Col and hag1-6 roots (R), 4 d CIM explants (C), 4 d CIM + 2 d SIM explants (S), and 4 d CIM + 2 w SIM explants (S2W). Levels of Col R were set to 1 after normalization with UBQ10.

C, D De novo shoot regeneration of Col, Nossen (No), wox5-3, wox7-1, wox14-1, and wox5-3 wox7-1 wox14-1 root explants. Col is the WT control for wox5-3 and wox7-1, whereas No is the WT control for wox14-1. The picture was taken at 3 w on SIM using representative explants of each genotype. Scale bar: 1 cm. Percentage of explants with shoots was scored at indicated weeks on SIM (D).

E H3Ac enrichment at the WOX14 locus in Col (upper black diagram) or hag1-6 (lower red diagram) calli as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 1e-5. See Figs 3A-D for schematic.

F ChIP-qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1-6. Regions tested are indicated in the schematic in (E). Sample preparation and data presentation were performed as described in Figs 3E-H.

G Direct targeting of HAG1:HA protein to WOX14 chromatin as determined by ChIP-qPCR. Regions tested are indicated in the schematic in (E). See Figs 3I-L for sample preparation and data presentation.

Data Information: In (A, B, D, F, and G), data are presented as means ± SE. In (A, B, F, and G), three biological replicates were used for statistical analysis. Student's t-test (two tailed) *P ≤ 0.05, **P ≤ 0.001. In (D), n = 168 for Col, 168 for No, 89 for wox5-3, 106 for wox7-1, 168 for wox14-1, and 168 for wox5-3 wox7-1 wox14-1.
Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis (2018)
Figure 5

Figure 5. Mutations in SCR cause severe defects in de novo shoot regeneration.

A-C De novo shoot regeneration using Col and scr mutants; scr-3 (A and B) and scr-6 (A and C). Explants with shoots were scored at indicated days on SIM using Col and scr-3 root explants that had been pre-incubated on CIM for 2 w (B) or Col and scr-6 hypocotyl explants that had been pre-incubated on CIM for 1 w (C). Scale bar: 1 mm.

D-F De novo shoot regeneration of Col, wox5-3, scr-3, and wox5-3 scr-3. The percentage of root explants with shoots (E) and the number of shoots per root explant (F) were scored at 15 d on SIM. The explants had been pre-incubated on CIM for 2 w before transfer onto SIM. Scale bar: 1 cm.

G, H Transcript levels of PLT1 (G) and PLT2 (H) in Col, wox5-3, scr-3, and wox5-3 scr-3 roots (R), 1 w CIM explants (C), and 1 w CIM + 2 w SIM explants (S2W) as determined by RT-qPCR. Levels of Col R were set to 1 after normalization with UBQ10.

Data Information: In (B, C, and E-H), data are presented as means ± SE. In (G and H), three biological replicates were used for statistical analysis. Student's t-test (two tailed) *P ≤ 0.001, **P ≤ 0.05. n = 36 (B), 14 (C), and 37 (E and F).
Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis (2018)
Figure 6

Figure 6. Activation of WOX5 and SCR rescues the shoot-regeneration defect of hag1.

A, B Activation of WOX5 by DEX treatment of 35S::GVG-WOX5 in Col and hag1-6. Root explants of 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX. DEX was treated for 3-d-pulse period on CIM or/and SIM (see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure). Morphology of shoots regenerated from 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 root explants (A). Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX (B). C, D Activation of SCR by DEX treatment of UBI::mCHERRY-GR-SCR in Col and hag1-6. Sample preparation and data presentation are as described in (A and B).

E, F Activation of WOX5 and SCR by DEX treatment of 35S::GVG-WOX5 UBI::mCHERRY-GR-SCR in Col and hag1-6. Sample preparation and data presentation are as described in (A and B).

Data Information: Scale bars: 1mm (A, C, and E). In (B, D, and F), data are presented as means ± SE. Student's t-test (two tailed) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005. n > 42 (B), n > 33 (D), and n > 26 (F) for each genotype and treatment.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 1

CTNS-deficient cells and tissues have increased number of autophagosomes

Expression levels of the autophagosome marker LC3B-II were evaluated in wild-type (WT) and Ctns−/− fibroblasts by Western blot (WB). Data are representative of five different experiments with similar results.

WT and Ctns−/− fibroblasts were stained using LC3B antibodies and analyzed by fluorescence microscopy. The number of LC3B-positive puncta was quantified by the ImagePro software and normalized per area arbitrary units. Results are mean ± SEM (n = 27 WT and 24 Ctns−/− cells). ***P < 0.001 (unpaired t-test).

Immunofluorescence analysis of liver (C) and kidney (D) tissues from WT and Ctns−/− GFP-LC3 transgenic mice using anti-GFP antibodies. Quantification of LC3 puncta was obtained by counting the number of GFP-positive structures relative to the number of nuclei in the same field, using the ImagePro software. GFP-LC3 puncta were counted by analyzing 6 to 10 fields per tissue section (200–400 nuclei per field), in a total of 3 WT and 3 Ctns−/− mice expressing GFP-LC3. Results are mean ± SEM. In (C), **P = 0.0029; in (D), **P = 0.0025 (unpaired t-test). Scale bar: 5 μm.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 2

Analysis of the macroautophagic flux in Ctns−/− cells

Representative images of wild-type (WT) and Ctns−/− mouse fibroblasts transfected with the ptfLC3 vector under resting conditions (fed), or after serum starvation (Serum Starv.) in the presence or absence of the alkalinizing drug chloroquine (CQ). GFP and RFP staining was analyzed by confocal microscopy. Examples of red-only puncta (mature autophagosomes) are indicated with arrows. Scale bar: 5 μm.

The percentage of mature autophagosomes (red-only vesicles) was calculated based on the ratio between the number of red-only puncta and the total number of autophagosomes (number of green and red + red-only puncta). The graph is representative of three different experiments with similar results. Results are mean ± SEM (n = 7 cells per condition). In addition to the experimental conditions shown in (A), the Aa/serum starvation condition is included.

WT and Ctns−/− fibroblasts under resting conditions were treated with 100 nM bafilomycin A (BafA) for 2 h and LC3B-II levels were analyzed by Western blot (WB).

Phosphorylation levels of the mTOR complex kinase 1 substrate S6K and LC3B-II levels in WT and Ctns−/− fibroblasts were measured by WB under resting conditions (−), withdrawal of both amino acids and serum (Aa/Ser. Starv.) and subsequent recovery by replacement of starvation medium with normal cell growth medium (Rec), in the presence or absence of 100 nM BafA for the indicated time.

WB analysis showing phosphorylation levels of the mTOR substrate S6K and expression of LC3B-II in resting or serum-starved WT,Ctns−/− and Ctns−/− fibroblasts expressing GFP-CTNS.

Source data are available online for this figure.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 3

Degradation of SQSTM1/p62 and long-lived proteins is not impaired in CTNS-deficient cells

Analysis of the expression of endogenous SQSTM1 in wild-type or Ctns−/− mouse fibroblasts under fed (serum) or starved (15 h serum starvation) conditions. In these assays, 20 μg of total protein lysates were resolved by SDS-PAGE and analyzed by Western blot (WB; A). Equal loading was established by silver staining (B). Quantification (mean ± SEM) of five different areas of each silver-stained lane are shown in (C).

Degradation of long-lived proteins using metabolic labeling was performed as described in Supplementary Materials and Methods. The data represent the mean ± SEM of nine independent samples analyzed in three independent experiments. *P = 0.009 for WT and *P = 0.0013 for Ctns−/−, respectively. NS, not significant (unpaired t-test).

Source data are available online for this figure.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 4

The CMA receptor LAMP2A is downregulated in CTNS-deficient cells

The expression levels of the endogenous LAMP2A and LAMP1 proteins were analyzed in wild-type (WT) and Ctns−/− mouse fibroblasts (A), kidney (B) and liver lysosomes (C) by Western blot. Quantitative densitometry analysis of immunoblots obtained from different experiments was performed by calculating the ratio between LAMP2A and actin signals in each lane. Results are mean ± SEM of four independent experiments with mouse fibroblasts, 5 WT and 6 Ctns−/− mouse kidneys, 3 WT and 3 Ctns−/− mouse livers (lysosomal extracts).

Comparative analysis of the amount of LAMP2A in wild-type or Ctns−/− liver lysosomes was performed using two different anti-LAMP2A antibodies. 15 μg of lysosomal proteins were loaded in each lane. Ab1, anti-LAMP2A antibody raised against the twelve amino acids of the cytosolic region of rat LAMP2A largely validated to recognize mouse LAMP2A but not other LAMP2 isoforms (Cuervo & Dice, 1996). Ab2, anti-LAMP2A Abcam Ab18528. Decreased lysosomal LAMP2A was evident using either antibody. LAMP1 was used as a loading control. Representative of two independent experiments with similar results.

Source data are available online for this figure.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 5

The CMA receptor LAMP2A is mislocalized in CTNS-deficient cells

Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (A) Confocal microscopy analysis. White arrows represent LAMP1-positive circular structures (lysosomes) which were visible in both WT and Ctns−/− cells. Colocalization of LAMP1 and LAMP2A was observed in WT fibroblasts (white arrows, merged panel) but rarely in Ctns−/− cells (red arrow). Most LAMP2A showed a punctate pattern and lack of colocalization with LAMP1 in Ctns−/− cells (arrowheads, lower panels). Scale bars: 5 μm. Inset scale bars: 2 μm. (B) High-resolution stochastic optical reconstruction microscopy (STORM) analysis of the localization of LAMP1 and LAMP2A in wild-type and Ctns−/− cells. In Ctns−/− cells, LAMP2A was detected near (arrowheads, estimated distance > 50 nm) but not always adjacent (10–50 nm) to LAMP1 or at LAMP1-negative structures (arrows). Scale bar: 0.5 μm. (C) Immunofluorescence showing colocalization of endogenous LAMP1 and LAMP2B in both wild-type and CTNS-deficient fibroblasts (arrows). Scale bar: 20 μm. (D) Immunofluorescence analysis of LAMP1 and total LAMP2 (antibody ABL93 DSHB). Arrows show colocalization. Arrowheads show LAMP2-positive LAMP1-negative vesicles.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 6

Abnormal localization of LAMP2A at trafficking vesicular compartments in CTNS-deficient cells

Immunofluorescence analyses of endogenous LAMP2A, ER (Grp78/94) (A), which is expanded in cystinotic cells (white arrow), VAMP7-positive vesicles (B) and Rab11a-positive structures (C) was performed using wild-type or Ctns−/− fibroblasts as described in Materials and Methods. At least 2 independent experiments were performed for each vesicular trafficking or ER marker. (B, C) Results are expressed as the percentage of LAMP2A that colocalizes with VAMP7 or Rab11. For Rab11/LAMP2A colocalization, 29 wild-type and 15 Ctns−/− cells were quantified. For VAMP7/LAMP2A colocalization (white arrows), 49 WT and 57 Ctns−/− cells were analyzed. Data are expressed as mean ± SEM. *P < 0.001 (unpaired t-test). Scale bars: 20 μm.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 7

Lysosomal localization of LAMP2A in Ctns−/− cells is rescued by inhibition of lysosomal proteases

WT,Ctns−/− or Ctns−/− mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine (CQ/Leu) or bafilomycin A (BafA) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods. Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated (middle lower and bottom panels) but not in untreated (middle upper panels) Ctns−/− cells. Scale bars: 5 μm. Inset scale bars: 2 μm.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 8

Chaperone-mediated autophagy is impaired in CTNS-deficient mice

Schematic representation of lysosomal isolation and in vitroCMA assay [adapted from Kaushik and Cuervo (2008)].

Lysosomes were isolated from livers of starved wild-type (WT) and Ctns−/− mice as described in Supplementary Materials and Methods and incubated at 37°C for 30 min with the CMA substrate GAPDH, in the presence or absence of ATP (necessary for CMA) and protease inhibitors (Prot. Inhib.). A fraction of the CMA reactions was then mixed with sample buffer and boiled at 95°C for 5 min, followed by SDS–PAGE and GAPDH and LAMP1 immunoblotting.

Quantitative densitometry analysis of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT and 10 Ctns−/− mice. Results are mean ± SEM. ***P = 0.0005; **P = 0.0041; N.S., not significant (unpaired t-test).

The ability of lysosomal proteases to mediate GAPDH degradation was assessed by incubating Triton X-100-treated WT and Ctns−/− lysosomes with GAPDH for 30 min in acidic reaction buffer and compared with input (reaction with no lysosomes). Quantitative densitometry analysis of independent reactions performed with lysosomes from a total of 9 WT and 9 Ctns−/− mice shows no significant difference in protease activity. Results are mean ± SEM.

Source data are available online for this figure.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 9

CTNS-deficient cells are more susceptible to oxidative stress-induced cell death

WT and Ctns−/− mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat (PQ) for 4 h and stained with (A) FITC-annexin V or (B) propidium iodide (PI) and analyzed by FACS. Results from 3 different experiments are shown as mean ± SEM. In (A), *P = 0.05; ***P = 0.0002. In (B), *P = 0.046; **P = 0.0065. Unpaired t-test.

WT and Ctns−/− cells were treated as described above and samples analyzed by Western blot using antibodies recognizing cleaved (active) caspase 3. Data are representative of 2 independent experiments with similar results.
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 10

LAMP2A mislocalization is independent of cystine-induced lysosomal overload

Cystine content in WT,Ctns−/−, cysteamine-treated Ctns−/− fibroblasts or Ctns−/− fibroblasts expressing GFP-CTNS was assessed by mass spectrometry.

WT,Ctns−/−, Ctns−/− fibroblasts treated with 1 mM cysteamine (48 h), or Ctns−/− fibroblasts expressing GFP-CTNS were fixed and stained with anti-LAMP1 and LAMP2A antibodies as indicated. For better visualization of LAMP1/LAMP2A colocalization, GFP-CTNS was pseudocolored as magenta and LAMP1 was pseudocolored as green (lower panels). Some lysosomes are indicated with arrows. Arrowheads indicate LAMP2A distribution to structures different from lysosomes (only observed in Ctns−/− cells and in cysteamine-treated Ctns−/− cells). Rescue of the LAMP2A localization to lysosomes was observed in Ctns−/− cells expressing GFP-CTNS. Scale bar: 2 μm.

Quantification of the colocalization analysis described in (B). Calculation of the Pearson's colocalization coefficient was done by analyzing 113 WT, 251 Ctns−/−, 88 cysteamine-treated and 164 GFP-CTNS-expressing Ctns−/− cells, by using the ZEN 2010 software. Results are mean ± SEM. ***P < 0.001; NS, not significant (one-way ANOVA, Bonferroni's multiple comparisons test).
Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. (2015)
Figure 11

Cysteamine treatment does not rescue CMA impairment in Ctns−/− mice

Cystine content in WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers was assessed by mass spectrometry. Results are mean ± SEM (n = 4). ***P < 0.001; **P = 0.0026 (one-way ANOVA, Bonferroni's multiple comparisons test).

Western blot analysis of LAMP2A expression in lysosomes isolated from WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers.

Lysosomes, isolated as in (B), were incubated with the CMA substrate GAPDH, and CMA activity was evaluated as in Fig8 and as described in Materials and Methods.

Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors. Results are mean ± SEM (n = 4). *P = 0.0126 for WT versus Ctns−/− and *P = 0.0436 for WT versus Ctns−/− + cysteamine; NS, not significant; unpaired t-test.

Source data are available online for this figure.