EMBO/soda-nel huggingface dataset:
Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG (2006)
figf5

(a) Autophagic activity of UVRAG mutants. HCT116 cells expressing wild-type or mutant UVRAG were transfected with GFP–LC3 expression vector and incubated under normal or starvation conditions for 3 h. Autophagy was quantified as described in Fig. 3. (b) Effect of wild-type and mutant UVRAG on Beclin1-mediated autophagy. MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells were cotransfected with wild-type UVRAG or its mutant expression vector together with GFP–LC3 expression vector. At 16–18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as described in Fig. 3. The scale bars represent 20 μm. (c, d) Interdependence between UVRAG and Beclin1 for autophagosome induction. HCT116.vector and HCT116.UVRAG cells (c) or MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells (d) were transfected with control siRNA, Beclin1 siRNA or UVRAG siRNA together with GFP–LC3 expression vector as indicated. Autophagy was quantified as described in Fig. 3. WCLs of these cells were used for immunoblotting with anti-UVRAG, anti-Beclin1 or anti-tubulin. The scale bars represent 20 μm. In all figures, data represents mean ± s.d. of three experiments. The raw data for c and d are presented in the Supplementary Information, Fig S5.
Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG (2006)
figf6

(a) Increased Beclin1–PI(3)KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells were used for immunoprecipitation with anti-PI(3)KC3 followed by immunoblotting with anti-Beclin1. WCLs were used for immunoblotting with anti-Beclin1 and anti-PI(3)KC3. HCT116.UVRAG cells expressing Flag-tagged UVRAG were incubated under nutrient-rich conditions (lanes 1 and 2), starvation for 4 h (lane 3), or 2 μM rapamycin treatment for 2 h (lane 4). WCLs were then used for immunoprecipitation with anti-Beclin1 (lanes 2, 3 and 4) or preimmune antibody (lane 1) followed by immunoblotting with anti-Flag. The raw data for this experiment are presented in the Supplementary Information, Fig. S5. (b) Increased PI(3)KC3 activity by UVRAG. At 48 h posttransfection with Flag-tagged PI(3)KC3 with Beclin1 (lane 1), with Beclin1 + UVRAG (lane 2) or with Beclin1 + UVRAGΔCCD vector (lane 3), WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by an assay for PI(3)KC3 lipid kinase activity, as described in the Methods. At 16 h posttransfection with p40(phox) PX–EGFP fusion, HCT116.vector and HCT116.UVRAG cells were detected using an inverted fluorescence microscope. p40(phox) PX–EGFP-positive vesicles were quantified as the mean ± s.d. of combined results from three independent experiments. The scale bars represent 10 μm. (c) Suppression of autophagy by γHV68 vBcl-2. At 18 h posttransfection with GFP–LC3 vector, NIH3T3.vector, NIH3T3.vBcl-2 and NIH3T3.vBcl-2AAA, cells were incubated under normal or starvation conditions for 4 h. Subsequently, GFP–LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s.d. of combined results from three independent experiments.
Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG (2006)
figf7

(a) Proliferation rates. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 105 per well) were seeded into 6-well microtitre plates. Cell numbers were counted on the days indicated. The results represent means of three independent assays. (b) Anchorage independence. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 104) were incubated in soft agar as described in the Methods. After ten days, colonies were photographed and counted. The scale bars represent 50 μm. (c) Tumorigenicity. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells (1 × 107) were subcutaneously injected into 5 nude mice, separately. At 16 days postinoculation, mice and tumours were photographed and tumour weights were measured. Arrows indicate location of tumours. Colony number (b) and tumour mass (c) were quantified as means ± s.d. of combined results from five independent experiments.
The PEAT protein complexes are required for histone deacetylation and heterochromatin silencing (2018)
Figure 1

Figure 1. EPCR1 and EPCR2, two enhancer of polycomb-related proteins, are required for transcriptional silencing and development.

(A) The transcript level of solo LTR in the wild type and the epcr1 mutant. Error bars are standard deviations of three technical replicates. The experiment was independently performed three times and similar results were obtained.

(B) The developmental phenotype of the epcr1 and epcr2 single mutants and the epcr1/2 double mutant relative to the wild type. Two-week-old seedlings grown on vertical MS medium plates are shown.

(C) The transcript levels of solo LTR, SDC, AtSN1, AtGP1 and AtCOPIA28 in the wild type, epcr1 and epcr2 single mutants, epcr1/2 double mutant, and two individual EPCR1 transgenic lines in the epcr1/2 double mutant background. Error bars are standard deviations of three biological replicates.
The PEAT protein complexes are required for histone deacetylation and heterochromatin silencing (2018)
Figure 2

Figure 2. EPCR1/2 interact with ARID2/3/4, PWWP1/2/3, and TRB1/2, and form complexes.

(A) The interaction between EPCR1 and ARID2 or ARID3. Arabidopsis plants carrying EPCR1-Flag and ARID2-Myc or ARID3-Myc transgenes were used for co-IP.

(B) The interaction between TRB1 and ARID2. Arabidopsis plants carrying TRB1-Flag and/or ARID2-Myc transgenes were used for co-IP.

(C) The interaction between TRB1 and EPCR1. Arabidopsis plants carrying TRB1-Flag and/or EPCR1-Myc transgenes were used for co-IP.

(D) The diagram of protein-protein interactions that were detected in yeast two-hybrid assays. A line represents an interaction between two proteins. If a protein was indicated to interact with another protein when it was fused with both GAL4-BD and GAL4-AD, both straight and curved lines are shown between the two proteins. A lariat loop indicates that a protein can form a homodimer.

(E) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull-down assay.

(F) The interaction between EPCR1, PWWP2, ARID2, and TRB1 was detected by an HIS pull-down assay. The full-length PWWP2, ARID2, and TRB1, and the EPCR1-N terminal (1-500 aa) were used for the interaction assay.

(G) The diagram indicates the interactions among EPCR1, PWWP2, ARID2, and TRB1 as determined by pull-down assay, yeast two-hybrid, and co-IP. The solid line and the broken line indicate, respectively, direct and indirect protein-protein interactions.
The PEAT protein complexes are required for histone deacetylation and heterochromatin silencing (2018)
Figure 3

Figure 3. The PEAT complexes are required for transcriptional silencing and development.

(A) The developmental phenotype of the arid2/3/4 mutant as compared to the wild type. Two-week-old seedlings grown on MS medium are shown.

(B) The developmental phenotype of the pwwp1/2/3+/- mutant as compared to the wild type. One-month-old plants grown in soil are shown.

(C, D) The effect of the arid2, arid3, and arid4 mutations, and the effect of the pwwp1, pwwp2, and pwwp3 mutations on the silencing of solo LTR, SDC, and FWA as determined by qPCR. Showing are results of three biological replicates with standard deviations.

(E) The effect of the trb1 mutation and the TRB2 knockdown (TRB2-KD) on the silencing of solo LTR, as determined by qPCR (right panel). The transcript level of TRB2 was evaluated to confirm the TRB2 knockdown (left panel). Error bars represent standard deviations of three biological replicates.
The PEAT protein complexes are required for histone deacetylation and heterochromatin silencing (2018)
Figure 4

Figure 4. ARID2/3/4 and EPCR1/2 are required for transcriptional silencing.

(A) Venn diagrams showing up- and down-regulated genes and TEs in the epcr1/2 and arid2/3/4 mutants relative to the wild type, as determined by RNA-seq. The data are from three independent biological replicates. All the overlaps are highly significant (P<2.610-30) as determined by hypergeometric test.

(B) Heat maps showing differentially transcribed genes and TEs in the arid2/3/4 and epcr1/2 mutants relative to the wild type.

(C) Distribution of up-regulated TEs in nrpe1, epcr1/2, and arid2/3/4 on the Arabidopsis chromosomes.

(D) Genome browser snapshots showing the transcript patterns of ROS1 and IBM1 in arid2/3/4 and epcr1/2 relative to the wild type.

(E) Effects of arid2/3/4 epcr1/2 on the transcript levels of ROS1 and IBM1-L as determined by qPCR. The expression of ROS1 and the longer version of IBM1 (IBM1-L) was evaluated by qPCR. ACT7 was amplified as an internal control. Bars represent SD from two independent experiments, each with three technical replications.
The PEAT protein complexes are required for histone deacetylation and heterochromatin silencing (2018)
Figure 5

Figure 5. The interaction of EPCR1, ARID2, ARID3, ARID4, and TRB1 with the histone acetyltransferases HAM1 and HAM2 and the histone deacetylases HDA9 and HDA6.

(A) The interaction of EPCR1 with HAM1 and HAM2. EPCR1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants. The progeny were used to evaluate the interaction between EPCR1 with HAM1 and HAM2 by co-IP.

(B) The interaction of ARID2, ARID3, and ARID4 with HAM1 and HAM2. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HAM1-Flag and HAM2-Flag transgenic plants and their progeny were used for co-IP.

(C) The interaction of TRB1 with HAM1 and HAM2. TRB1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants and their progeny were used for co-IP.

(D) The interaction of HDA9 with ARID2, ARID3, and ARID4. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HDA9-Flag transgenic plants and their progeny were used for co-IP.

(E) The interaction of HDA6 with EPCR1 and ARID2. EPCR1-Flag and ARID2-Flag transgenic plants were crossed to HDA6-Myc transgenic plants and their progeny were used for co-IP.
The PEAT protein complexes are required for histone deacetylation and heterochromatin silencing (2018)
Figure 6

Figure 6. ARID2/3/4 and EPCR1/2 regulate histone acetylation, heterochromatin condensation, and siRNA accumulation.

(A) Overlap among H4K5 hyperacetylated regions in the arid2/3/4 and epcr1/2 mutants relative to the wild type. The overlap is significant (P=4.410-78) as determined by hypergeometric test.

(B) Metaplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Histone H4K5 acetylation levels are represented by normalized reads that obtained in the H4K5Ac ChIP-seq analysis. TSS represents transcription start site and TTS represents transcription termination site.

(C) Boxplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Asterisks indicate that H4K5Ac levels is significantly higher in the arid2/3/4 (P=2.4210-13) and epcr1/2 (P=1.5210-8) mutants than in the wild type as determined by Welch's Two-Sample t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.

(D) The nuclei with condensed, intermediate, or decondensed heterochromatin status, as marked by DAPI staining and H3K27me signals.

(E) Percentages of nuclei with condensed, intermediate, or decondensed heterochromatin signals in the wild type and the arid2/3/4 mutant. n=113.

(F) Abundance of Pol IV-dependent siRNAs in the epcr1/2, arid2/3/4, nrpd1, nrpe1, and hda6 mutants relative to the wild type [(Mut-WT)/(Mut+WT)] from chromosome 3 of Arabidopsis.

(G) Venn diagrams showing the numbers of differentially expressed Pol IV-dependent 24-nt siRNA clusters (P4-siRNAs) in arid2/3/4, epcr1/2, and hda6 relative to the wild type. All the overlaps are highly significant (P→0) as determined by hypergeometric test.

(H) Boxplot showing levels of Pol IV-dependent siRNAs that are up-regulated in the hda6 mutant. Asterisks indicate that siRNA levels in the mutants are significantly (P→0) up-regulated compared to the wild type as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.

(I) Distribution of the Pol IV-dependent siRNA regions in which siRNAs are up-regulated in the mutants relative to the wild type. The pericentromere refers to 6 million base pairs of a pericentromeric region on each chromosome.
The PEAT protein complexes are required for histone deacetylation and heterochromatin silencing (2018)
Figure 7

Figure 7. Effect of the epcr1/2 and arid2/3/4 mutations on DNA methylation.

(A) Metaplot of CG, CHG, and CHH methylation of genes and TEs in the genomes of wild type, epcr1/2, and arid2/3/4 plants. H indicates C, A, or T.

(B) Metaplot of CG, CHG, and CHH methylation on chromosome 3 of Arabidopsis in wild type, epcr1/2, and arid2/3/4 plants.

(C) Distribution of arid2/3/4 and epcr1/2 hyper-DMRs of CG, CHG, and CHH sites in pericentromeric regions and two chromosome arms. A pericentromeric region refers to 6 million base pairs of a centromere-flanking region on each chromosome.

(D) Boxplot of DNA methylation in the wild type, nrpe1, epcr1/2, and arid2/3/4 mutants. The hypo-DMRs identified in the nrpe1 mutant was defined as RdDM target loci. Asterisks indicate that DNA methylation is significantly (P<0.001) decreased in the mutants as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.

(E) Heat maps of CG, CHG, and CHH methylation at RdDM target loci. The hypo-DMRs identified in the nrpe1 mutant were defined as RdDM target loci and analyzed in the wild type, nrpe1, eprc1/2, and arid2/3/4 mutants. Black and light yellow indicate low methylation and high methylation, respectively.
The PEAT protein complexes are required for histone deacetylation and heterochromatin silencing (2018)
Figure 8

Figure 8. The PEAT complexes mediate heterochromatin silencing independently of affecting DNA methylation.

(A) Scatter plots showing the effect of the epcr1/2 and arid2/3/4 mutations on CG, CHG, and CHH methylation at the co-upregulated TEs and genes in the epcr1/2 and arid2/3/4 mutants. TE methylation and gene DNA methylation refer, respectively, to methylation of the TE body and methylation of the 1-kb-gene-promoter region.

(B) Box plots of CG, CHG, and CHH methylation in the wild type, arid2/3/4, and epcr1/2 mutants at the co-upregulated TEs and genes in the arid2/3/4 and epcr1/2 mutants. Asterisks indicate statistical significance as determined by paired student t test. CHG methylation of co-upregulated TEs is significantly increased in arid2/3/4 (P=1.2610-4) and epcr1/2 (P=6.310-9); CHH methylation of co-upregulated genes is significantly decreased in arid2/3/4 (P=3.510-6) and epcr1/2 (P=6.410-7). Horizontal lines represent the median, the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.

(C) Overexpression of DRD1 fails to restore the silencing of EPCR1/2 target loci in the epcr1/2 mutant background.

(D) Model for the role of the PEAT complexes in chromatin regulation. In heterochromatin regions, the PEAT complexes either directly mediate heterochromatin condensation or interact with the histone deacetylases HDA6 and HDA9 and thereby mediate heterochromatin condensation by histone deacetylation. Thus, the PEAT complexes repress transcription mediated by both Pol II and Pol IV. The repression of Pol II transcription is required for transcriptional silencing in heterochromatin regions. Moreover, the repression of Pol II and Pol IV transcription prevents overproduction of siRNAs that are responsible for DNA methylation. In euchromatin regions, we predict that the PEAT complexes may regulate histone acetylation by interaction with the histone acetyltransferases HAM1 and HAM2. However, further studies are required to elucidate how the molecular mechanisms of the regulation.

Expanded View Figures
A single cell atlas of the human liver tumor microenvironment (2020)
Figure 3

Figure 3 - Mesenchymal heterogeneity in the liver malignant sites

(A) Key marker genes for the four mesenchymal clusters (RBP1, COL1A1, RGS5, and MYH11). Light gray dots denote cells originating from non-tumor samples. Dark gray dots denote cells originating from the tumor samples.

(B) Left - Representative smFISH image of patient p1 stained for RGS5 and COL1A1 showing distinct spatial localization of CAFs and Pericytes. Scale bar 10µm. Dashed lines mark the shortest distance of cells (2a) and (2b) from the cell (1). Middle - zoom-in of (1) from left panel, showing a blood vessel like structure formed by endothelial cells marked by PDGFB (magenta) wrapped by pericytes marked by RGS5 (green). Dashed lines are two consecutive cell layers of endothelial cells and pericytes. Scale bar 2.5µm. Right - zoom-in of (2a and b) from the left panel, showing two distant CAFs expressing high COL1A1 signal but not RGS5. DAPI used for nuclei staining. Scale bar 2.5µm.

(C, D) Violin plot of the distance from blood vessels of low/high RGS5 expressing cells (n=358 and n= 360 respectively) and low/high COL1A1 expressing cells (n=359 and n= 359 respectively). 'p' is the p-value determined by Wilcoxon rank-sum test. Empty circles are the medians over all repeats.

(E) Schematic representation of the top-ranked interaction (bona-fide) detected by NicheNet (Methods). Results are sorted by the prior interaction potential between pericytes and tumor LVECt cells.

(F) Pathway enrichment analysis for all bona-fide genes (Datasets EV4) using Enrichr tool. Images in this figure are representative images out of eight independent experiments over four patients.
Linc-MYH configures INO80 to regulate muscle stem cell numbers and skeletal muscle hypertrophy. (2020)
Figure 1

Figure 1: linc-MYH is located in nuclei of proliferating and differentiated myocytes and interacts with the chromatin remodeler complex INO80

A RNA-seq. of freshly isolated, proliferating and differentiating satellite cells reveals an increased expression of linc-MYH in proliferating and differentiating MuSCs, but no expression is found in in freshly isolated MuSCs.

B, C Expression of linc-MYH is confined to skeletal muscle in mouse (B) and human (C) tissues.

D Expression of linc-MYH is not detected in limb buds at E10.5 (n = 3), E13.5 (n = 3) and E15.5 (n = 3). Expression of linc-MYH in hindlimb muscle of E18.5, newborn and 7 day old animals (n = 3/3/3). Expression of linc-MYH in m. soleus (soleus), m. tibialis anterior (TA) and and m. extensor digitorum longus (EDL) muscle at 3 weeks (n = 3) and 8 weeks of age (n = 5). Biological replicates were used for all PCR experiments, data are mean ± S.E.M.

E Murine linc-Myh is localized in nuclear extracts of C2C12 cells. Xist (nuclear) and Gapdh (mainly cytoplasmic) were used as controls (n = 2 nuclear / 3 cytoplasmic biological replicates.) Data are mean ± S.E.M. F-H RNA-FISH identifies linc-Myh in nuclei (DAPI, blue) of C2C12 myoblasts. (G) Adipor (cytoplasmic) and (H) Xist (nuclear) were used as controls.

I, J GO enrichment (I) analysis of proteins pulled down by in vitro transcribed RNA from nuclear extracts of C2C12 myoblasts. Linc-MYH RNA and three control RNAs were used in duplicate. (J) Heat map of proteins pulled down in the same experiments as in I, demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc-MYH. Blue color indicates pull-down of the respective protein, white color indicates that the protein was not detected in the respective sample.

K, L RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from murine C2C12 cells (K) and human HSMM cells (L). INO80 and INO80 II in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.
Linc-MYH configures INO80 to regulate muscle stem cell numbers and skeletal muscle hypertrophy. (2020)
Figure 2

Figure 2: linc-MYH keeps the number of MuSCs in check and limits muscle fiber growth

A, B Body weight (A) of male linc-MYH KO and ctrl mice at 10 weeks (n = 24 KO/17 WT; Student's t test, two-tailed, **p < 0.01) and tibia length (B) of male linc-MYH KO and ctrl mice of 10 weeks (n = 9 KO/13 WT; Student's t test, two-tailed, not significant). Data are mean ± S.E.M.

C, D Weight of m. tibialis anterior (TA) and m. digitorum longus (EDL) of male linc-MYH KO and ctrl mice at 10 weeks (n = 9 KO/10 WT for TA, n = 10 KO/11 WT for EDL; Student's t test, two-tailed, **p < 0.01, ***p<0.001). Data are mean ± S.E.M.

E-J Size distribution of fibers in cross sections of TA (E, F) and EDL (G, H) muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.

K Numbers of myonuclei in cross sections of TA and EDL muscle (TA: n = 6 KO/6 WT, EDL n = 3 KO/3 WT animals, Mann-Whitney test one-tailed, **p<0.01, *p = 0.05) Data are mean ± S.E.M.

L-O Number of myonuclei / fiber and of Pax7pos (red) MuSCs (red arrows) on myofibers isolated from flexor digitorum brevis muscle. Nuclei were stained using DAPI (blue). Scale bar in L indicates 50 µm for L and M. (N) The number of myonuclei / isolated fiber is increased in linc-MYH KO compared to WT myofibers (n = 4 KO/4 WT, > 26 fibers/animal; Mann-Whitney test two-tailed, * p < 0.05). (O) Number of MuSCs on isolated fibers from linc-MYH KO and WT mice (n = 4 KO/4 WT animals, Mann-Whitney test two-tailed; ****p < 0.0001). Data are mean ± S.E.M.

P-S Number of Pax7 (red) positive MuSCs in cross sections of tibialis anterior muscle (P-R); the relative amount of quiescent MuSCs identified by double staining (yellow arrows) for Pax7 and CalcR (green) does not significantly differ between WT and linc-MYH KO animals (S). Nuclei were stained with DAPI (blue). Scale bar in P indicates 25 µm for P and Q. (n = 3 KO/3 WT animals, Mann-Whitney test one-tailed; *p = 0.05, ns: not significant). All data are mean ± S.E.M.

T The number of MuSC-related events in FACS experiments is significantly increased in linc-MYH-KO animals (n = 7 KO/7 WT, Student's t test, two-tailed, *p = 0.0126). Data are mean ± S.E.M.

U-V Structure of MuSCs and the heterochromatin content of MuSC nuclei is not changed in linc-MYH KO (V) compared to WT mice (U).