EMBO/soda-nel huggingface dataset:
Snail enhances arginine synthesis by inhibiting ubiquitination-mediated degradation of ASS1 (2021)
Figure 1

Figure 1. LOC113230 is downregulated in CRC metastatic tissue and repressed by TGF-β/Snail axis.

(A) Identification of the potential LncRNAs repressed by Snail from CRC metastatic tissue by using bioinformatics approach to analyze the promoter regions of lowly expressed LncRNAs, and 26 LncRNAs were discovered due to their promoter regions containing multiple copies of E-box elements, among which LOC113230 contained 7 E-boxes in its proximal promoter region.*: previously reported LncRNAs in cancers. P-value by Student's t‐test.

(B) Diagram showed the positions and nucleotide constituents of 7 E-Boxes in the proximal promoter region of LOC113230.

(C) The expression of LOC113230 in SW-1116-Vector, SW-1116-Snail1, HCT116-Vector or HCT116-Snail cells examined by qRT-PCR assays.

(D, E) ChIP assays showed that Snail specifically bound E-boxes E2 and E5. ChIP assays were performed with anti-Snail antibody or IgG as a control. Enriched DNA fragments flanking E-boxes were examined by semi-quantitative PCR or qPCR assays by using specific primer sets.

(F) Wild type and E-box deletion mutants (E2 and E5) of LOC113230 promoter luciferase reporters were transiently transfected into SW-1116 cells and the luciferase activity was normalized to the value of β-gal activity.

(G) qRT-PCR assays showed the expression of LOC113230 in SW-1116 -Vector or SW-1116 -shSnail stimulated with 5 ng/ml TGF-β for 24 and 48 hrs.

(H) qRT-PCR assays showed the expression of Snail in SW-1116 -Vector or SW-1116 -shSnail stimulated with 5 ng/ml TGF-β for 24 and 48 hrs.

(I) Kaplan-Meier analysis of the correlation between expression level of LOC113230 and overall survival of colon adenocarcinoma (COAD) patients from TCGA database. P<0.001. P-value for Kaplan-Meier curves was determined using log-rank test.

(J) qRT-PCR analysis of LOC113230 expression level in 65 paired CRC samples.

(K) Representative RNA-Scope immunostaining images of LOC113230 levels in CRC tissue and corresponding normal tissue from TMA chips. Scale bar represents 100 μM.

(L) Summary of LOC113230 expression on TMA chips. **P<0.01. P-value of the RNA scope scoring results on TMA chips was analyzed by the McNemar test and Pearson's chi-square test. (M) Kaplan-Meier analysis for overall survival of CRC patients with high and low expression of LOC113230 from TMA chips. *P < 0.001. P-value for Kaplan-Meier curves was determined using log-rank test.

Data information: Data from (C, E, F, G, H, J) are shown as mean ± SEM. Data from are from three independent experiments, *P < 0.05 ,**P < 0.01 and ***P < 0.001 by Student's t‐test, NS: no significant difference. The LOC113230 expression levels were normalized to the GAPDH level.
Snail enhances arginine synthesis by inhibiting ubiquitination-mediated degradation of ASS1 (2021)
Figure 2

Figure 2. LOC113230 inhibits CRCs cell migration.

(A) Stable expression of LOC113230 in SW-1116 and HCT116 cells. LOC113230 was introduced into the cells via viral infections followed by puromycin selection and the resulting cells as mass pools were examined for LOC113230 level and subsequently used for cell migration assay.

(B ,C) Overexpression of LOC113230 inhibited cell migration in SW-1116 and HCT116 cells. Cell migration was detected by transwell assays. Scale bar represents 100 μM.

(D- F) Depletion of LOC113230 in Caco-2 cells using specific shRNAs and stable cells were established with puromycin selection. The resulting cells as mass pools were used for cell migration assays. Scale bar represents 100 μM.

(G) Overexpression of LOC113230 in SW-1116 cells inhibited lung metastasis. Left: Exponentially growing SW-1116 - LOC113230-Luc and SW-1116-Vector-Luc cells were injected into the tail vein of 8-week-old nude mice and intensities of lung metastatic tumor cells at the 8th week were analyzed by in vivo photon flux imaging equipment. The red circles in images are to help to see the tumors. Right: Box plot (central band: median; box limits: first and third quartile; whiskers: minimum and maximum) of lung photon flux at the 8th weeks.

(H, I) 5 ng/ml TGF-β and DMSO were applied to stimulate SW-1116 -Vector or SW-1116 -LOC113230 cells for 24 and 48 hrs, respectively. Cell migration was detected by transwell assays. Scale bar represents 100 μM.

Data information: Data from (A, C, D, F, I, G) were shown as mean ± SEM. Data from three independent experiments. Data G from six independent experiments, *P < 0.05, NS: no significant difference. (Student's t-test). The LOC113230 expression levels were normalized to the GAPDH level.
Snail enhances arginine synthesis by inhibiting ubiquitination-mediated degradation of ASS1 (2021)
Figure 3

Figure 3. LOC113230 300-800 contains full suppressive activity as that of the full length LOC113230.

(A) Diagram showed the full-length LOC113230 and the four ΔATG mutants (Δ1, Δ2, Δ3 and Δ1+2+3). Bottom: agarose gel image showed the in vitro-transcribes of ΔATG mutants (Δ1, Δ2, Δ3 and Δ1+2+3) were in correct sizes.

(B,C) ΔATG mutants (Δ1, Δ2, Δ3 and Δ1+2+3) showed no apparent effect on the cell migration. Scale bar represents 100 μM.

(D) Diagram showed the full-length LOC113230 and its truncations. Bottom: agarose gel showed that the in vitro transcribed full-length LOC113230 and the truncations were in correct sizes.

(E,F) Stable expression of LOC113230 300-800 resulted in decreased cell migration. Cell migration was detected by transwell assays. Scale bar represents 100 μM.

(G) Diagrams showed LOC113230 300-800 and its antisense strand. Bottom: agarose gel image showed the in vitro-transcribed LOC113230 and the antisense strand were in correct sizes.

(H,I)Stable expression of LOC113230 300-800 inhibited cell migration, while LOC113230 antisense showed no apparent effect on cell migration. Scale bar represents 100 μM.

Data information: Data from (C, F, I) were shown as mean ± SEM. Data from three independent experiments, *P < 0.05, NS: no significant difference. (Student's t-test).

Data from (A,D,G ) of markers indicate the length 1300 bp, 800 bp and 500 bp (bp, base pair).
Snail enhances arginine synthesis by inhibiting ubiquitination-mediated degradation of ASS1 (2021)
Figure 4

Figure 4. LOC113230 interacts with ASS1 to promote its degradation via ubiquitination.

(A)Top: Images taken by confocal microscopy showed the localization of LOC113230 detected by RNA scope assays in SW-1116. Scale bar represents 50 μM. Bottom: qRT-PCR assays showed LOC113230 expression level in the nuclear and cytoplasmic fractions extracted from SW-1116 cells.

(B) Silver staining images showed ASS1 as a novel LOC113230 interacting protein. The LOC113230 interacting proteins were identified by incubation biotin labeled LOC113230 with cytoplasmic extracts prepared from SW-1116 cells and the co-eluted proteins were identified by Mass spectrometry. NC, Negative control.

(C) RIP assays were employed to validate the association of ASS1 with LOC113230 in SW-1116 cells. Antibodies against HuR, GAPDH or control IgG were served as controls.

(D) Western blot assays showed that LOC113230 specifically precipitated ASS1 in SW-1116 cells. PC, Positive control.

(E) Western blot assays showed level of ASS1 in SW-1116-Vector and SW-1116-LOC113230.

(F) Top: western blot assays showed level of ASS1 in SW-1116 cells stably expressing LOC113230 or vector treated with cycloheximide (CHX, 20 μg/mL) or vehicle for the indicated periods of time. Bottom: the gray scale-analysis of ASS1 level was conducted and GAPDH protein level was used as a loading control. Results are shown as means±SD.

(G) SW-1116 cells stably expressing LOC113230 or vector were treated with MG132 (5 μM) or vehicle for 4, 8 or 12 hrs, respectively. Cell lysates were analyzed by western blotting assays.

(H) SW-1116 cells bearing stable expression or knockdown of LOC113230 were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub), ASS1 and α-Flag antibodies, respectively.

(I) Tandem mass spectrometry analysis identified the ubiquitinated K234 residue of ASS1 in the presence of LOC113230 expression.

(J) The protein level of ASS1 was examined by immunoblotting in SW-1116-Vector and SW-1116-LOC113230 cells overexpressing ASS1WT or ASS1K234R .

(K) SW-1116 cells stably expressing ASS1WT, or ASS1 K234R and LOC113230 or not were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub) and α-Flag antibodies, respectively.

Data information: Data from (A, C) were shown as mean ± SEM. Data from (A,C,D,F) are from three independent experiments. *P < 0.05, NS: no significant difference. (Student's t-test). HuR served as a positive control. Band intensities (E,G,H,J,K) were quantified relative to GAPDH (n=3).
Snail enhances arginine synthesis by inhibiting ubiquitination-mediated degradation of ASS1 (2021)
Figure 5

Figure 5. LOC113230 inhibits arginine sythesis.

(A) Urea production from each group was determined by a commercially available urea assay kit.

(B) GC-MS analyses of the major metabolites of Urea cycle in SW-1116-Vector, SW-1116-LOC113230 or SW-1116-shASS1 cells, respectively.

(C) Enzymatic activity of ASS1 was examined by di-phosphate release from the argininosuccinate synthesis reaction. whole cell extracts prepared from SW-1116 cells expressing LOC113230, shLOC113230, or vector were subjected for in vitro ASS1 enzymatic assays, and purified enzymatic dead mutant ASS1 K165Q and ASS1 K176Q proteins were used as controls.

(D) Arginine production from each group was determined by a commercially available Arginine assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs.

(E) Urea production for each group was determined by a commercially available urea assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs.

Data information: Data from (A, B, C, D, E) were shown as mean ± SEM. Data from three independent experiments. *P < 0.05, NS: no significant difference. (Student's t-test).
Snail enhances arginine synthesis by inhibiting ubiquitination-mediated degradation of ASS1 (2021)
Figure 6

Figure 6. Elevated ASS1 promotes CRCs cell migration

(A) qRT-PCR assays showed expression of ASS1 in Caco-2, SW620, SW480, HCT116 and SW-1116 cells.

(B) Western blotting assays showed the expression of ASS1 in Caco-2, SW620, SW480, HCT116 and SW-1116 cells.

(C, D) qRT-PCR and western blotting assays showed stable expression or depletion of ASS1 in SW-1116 and Caco-2. NC, Negative control.

(E,F) Depletion of ASS1 decreased cell migration in SW-1116 cells. Scale bar represents 100 μM.

(G) qRT-PCR assays showed stable expression of LOC113230 in Caco-2.

(H) Western blotting assays showed stable expression of ASS1WT and ASS1 K234R via an viral infections into Caco-2 or Caco-2 LOC113230 FL cells, respectively .

(I, J) Transwell assays showed the effect of ASS1WT and ASS1K234R on Caco-2-Vector or Caco-2-LOC113323 cell migration. Scale bar represents 100 μM..

(K) The expression of ASS1 in 65 paired CRC samples were analyzed by qRT-PCR.

(L) Representative immunostaining images of ASS1 levels in CRC samples on TMA chip. Scale bar represents 50 μM.

(M) Summary of ASS1 expression on TMA chips. P<0.01. P-value of the IHC scoring results on TMA chips was analyzed by the McNemar test and Pearson's chi-square test.

(N) Kaplan-Meier analysis for overall survival of patients with high or low expression of ASS1. P < 0.001. P-value for Kaplan-Meier curves was determined using log-rank test.

(O) Pearson's correlation analysis showed that a significant negative correlation between the level of ASS1 and LOC113230 in CRC samples. R2=-0.53. P < 0.05.

The LOC113230 and ASS1 expression levels were normalized to the GAPDH level, respectively.

Data information: Data from (A, C, F, G, J, K) were shown as mean ± SEM. Data from (A, C, F, G, H, J, K) are from three independent experiments. *P < 0.05, (Student's t-test). The LOC113230 and ASS1 expression levels were normalized to the GAPDH level. Band intensities (B,D) were quantified relative to GAPDH (n=3).
Snail enhances arginine synthesis by inhibiting ubiquitination-mediated degradation of ASS1 (2021)
Figure 7

Figure 7. LOC113230 recruits LRPPRC-TRAF2 E3 complex to ubiquitinate ASS1

(A) Identification of LRPPRC as a novel ASS1 interacting protein. Cell extracts prepared from SW-1116 cells expressing Flag-ASS1, Flag-ASS1/LOC113230 300-800 or empty vector were incubated with M2 beads and the co-eluted proteins were resolved on SDS-PAGE gel and detected by silver staining assays. The specific band was collected and the potential proteins were identified by Mass spectrometry.

(B) RIP assays showed association of LOC113230 with LRPPRC in SW-1116 cells. Antibodies against GAPDH and control IgG served as controls.

(C) LOC113230 strengthens the interaction between LRPPRC and ASS1. HA-LRPPRC, Flag-ASS1, together with LOC113230 were co-expressed in SW-1116 cells, and co-IP assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with Flag, HA antibodies, respectively.

(D) The endogenous interaction between LOC113230, ASS1 and LRPPRC was detected in SW-1116 cells by co-IP assays.

(E) The plasmids encoding HA-TRAF2, HA-TRAF3, Flag-TRAF6, Flag-ASS1 or LOC113230 were co-transfected into SW-1116 cells and western blot assays were performed using antibodies against ASS1, HA, Flag or GAPDH.

(F) in vitro binding assays showed the binding activity of TRAF2 and LRPPRC. GST-TRAF2 and His-LRPPRC proteins were prepared from E. coli BL21 cells.

(G) The interaction of endogenous TRAF2, ASS1 and LRPPRC was detected in SW-1116 cells by co-IP assays. GST-TRAF2, HA-LRPPRC, His-ASS1 and Biotin-LOC113230 were expressed in E. coli BL21 cells, and were used for in vitro binding assays.

(H) SW-1116-LOC113230 cells transiently transfected with siRNAs to LRPPRC or TRAF2 or scrambled control (SiRNA-NC) were treated with MG132 (5 µM) for 12 hrs. Cell lysates were immunoprecipitated with α-Flag antibody. The precipitate proteins and input proteins were analyzed by immunoblotting. NC: Negative control.

(I) Average urea production from each group was determined by a commercially available urea assay kit.

(J) The model describing the role of LOC113230 as a tumor suppressor by degrading ASS1 and inhibiting arginine synthesis in CRC cells.

Data information: Data from (B and I) were shown as mean ± SEM. Data from (B, F,G,H,I) three independent experiments. *P < 0.05, NS: no significant difference. (Student's t-test).

Band intensities (C) were quantified relative to Input (Flag) (n=3).Band intensities (D) were quantified relative to Input (LRPPRC) (n=3). Band intensities (E) were quantified relative to Input (GAPDH) (n=3)

Expanded View Figure Legends
H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing (2021)
Figure 1

Figure 1. Establishing H3K27me3 mintbody to visualise H3K27me3 in living cells.

(A) Schematic representation of the mintbody. H3K27me3-specific single-chain variable fragment (scFv) is genetically fused with a fluorescent protein (FP).

(B) Immunofluorescence (IF) validation of mintbody specificity. Mouse MC12 cells, which stably express H3K27me3-mintbody (sfGFP), are labelled with antibodies specific for H3K27me3 (Cy5) and H3K9me3 (Cy3). DNA is stained with Hoechst33342. Single confocal sections are shown. Arrows mark Xi. Scale bar = 10 μm.

(C) Time-lapse imaging of a dividing MC12 cell stably expressing H3K27me3-mintbody (sfGFP). Projection images of 7 confocal sections with 2μm intervals are shown with elapsed time (hh:mm). Scale bar = 10 μm.
H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing (2021)
Figure 2

Figure 2. Simultaneous visualisation of histone marks and X-chromosome loci in living cells.

(A) Schematic diagram of CRISPR/dCas9-3×sfGFP targeting loci on mouse X chromosome. sgRNA mX26 and mX8 target microsatellite repeats on Dxz4 and Xist loci, respectively.

(B) Experimental design: female ESCs are engineered to stably express two sgRNAs, dCas9-3xsfGFP, and a nuclear localization signal (NLS)-fused H3K27me3-mintbody (mCherry). Upon ESC differentiation by LIF withdrawal H3K27me3 accumulation occurs at the inactivating X (Xi) but not at the active X (Xa). Green foci allow marking of two X-linked loci (Dxz4 and Xist).

(C) Live imaging of female mESCs as in (B) in undifferentiated conditions (+LIF) or upon 3 days of LIF withdrawal. Double arrowheads mark Xa and single arrowhead marks Xi. Shown are maximum intensity projections of 11 z-plane confocal sections. Scale bar = 10 μm.

(D) ESCs as in (B) were engineered to express H3K27me3-mintbody (SNAP/JF646) and H4K20me1-mintbody (mCherry). Cells were cultured in the absence of LIF for 2 days. Time-lapse images for 12 z-plane confocal stack were acquired every 1 h. Maximum intensity projection images are shown with elapse time (hh:mm). Arrowheads mark Xi. Scale bar = 10 μm
H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing (2021)
Figure 4

Figure 4. Native ChIP-seq reveals H4K20me1 and H3K27me3 co-accumulation during XCI.

(A) Schematic representation of the experimental design. The hybrid TX1072 mouse female ESC line was used, in which Xist can be induced from the endogenous B6 allele. Time course nChiP-seq for H4K20me1 was performed and compared to a published H3K27me3 dataset (GSE116480)(Zylicz et al., 2019).

(B) Quantification of average H4K20me1 (blue) and H3K27me3 (red) enrichment at the Xi (B6 allele) compared with t = 0 h in 10-kb windows spanning the whole chromosome. Shading is the interquartile range.

(C) As in (B) but shown is normalised B6 enrichment to the average accumulation at 24 hrs. (D) Pairwise comparison of H4K20me1 and H3K27me3 accumulation dynamics (ED50) at the X chromosome. All 10-kb windows with ED50 < 24 h are plotted. * aired Wilcoxon rank sum test P-value <0.05.

(E) H4K20me1 (blue) and H3K27me3 (red) accumulation across the Xi after 12 h of DOX treatment. The black line is a locally estimated scatterplot smoothing (LOESS) regression on all 10-kb windows (dots). Below each plot shown is the Xist locus (green bar) and Xist entry sites (black bars).

(F) Correlation between H3K27me3 and H4K20me1 accumulation after 24h of DOX treatment at initially active gene bodies (left), inactive gene bodies (middle) and 10-kb intergenic windows spanning the X chromosome (right). All scales are logarithmic. All correlations (ρ) are with p<0.01 from Pearson's correlation test.
H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing (2021)
Figure 5

Figure 5. H4K20me1 accumulates intergenically.

(A) Genome browser tracks showing H3K27me3 (top) and H4K20me1 (bottom) accumulation at a gene silenced rapidly (Rnf12) or more slowly (Pgk1). Allele-specific tracks were overlaid (B6 in red and Cast in blue). Note strong H4K20me1 bi-allelic pre-marking at gene bodies.

(B) Average H3K27me3 (top) and H4K20me1 (bottom) enrichment at the B6 allele over initially active or inactive genes ± 30kb at the X chromosome. Shown is data for all time points.

(C) Average H4K20me1 enrichment at the B6 allele over early and late silenced genes ± 30kb at the X chromosome. Shown is data for all time points.

Data information: TSS : transcription start site ; TES : transcript end site.
H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing (2021)
Figure 6

Figure 6. H4K20me1 accumulation relies on Xist B+C repeat region

(A) Schematic representation of the experimental design. By DOX addition, male TXY mouse ESC lines allow for expression of full length XistFL or XistΔBC. Cells induced for 48h in the absence of LIF were used for combined IF and RNA FISH.

(B) Representative image of IF/RNA FISH for Xist RNA and H4K20me1 in cells expressing XistFL or XistΔBC. Arrowheads point to the Xist RNA domains. Scale bar = 5 μm.

(C) Graph represents the mean % +/- StDev of Xist RNA domains enriched for H4K20me1 (left) or H3K27me3 (right) in cells expressing XistWT or XistΔBC. Shown are averages from at least 2 independent experiments; minimum of 50 Xist RNA domains were counted per experiment; only P-values corresponding to significant differences from unpaired Student's t-test comparing mutants to Xist FL are indicated as * (P-value < 0.05). Data for H3K27me3 was extracted from published data (Bousard et al., 2019).

Expanded View Figure legends
CDF4 promotes leaf senescence by regulating ABA and reactive oxygen species pathways in Arabidopsis (2020)
Figure 1

Figure 1. Molecular characterization of the CDF4 gene and effects of ABA, SA, H2O2, abiotic stresses and darkness on its expression in Arabidopsis leaves.

A The gene and protein structures of the Dof domain of CDF4. Cysteine residues conserved in the DOF domain are indicated in red, and the conserved DOF Zn-figure DNA-binding domain is found in CDF4.

B Trans-activation activity assays of CDF4 in Arabidopsis protoplasts. The full-length ORF of CDF4 and its four deletion constructs are described in the Methods section. Dof (gray boxes) represent the DNA-binding domains of CDF4. Negative control was the effector vector without gene inserts. The effector vector used is shown in the left panel. The GAL4 transient expression assays were performed using Arabidopsis protoplasts, shown in the right panel. Deletion of the CDF4 N-terminal domain compromised the transcriptional repression by CDF4 in our assay. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.

C Localized expression of CDF4 in a senescing rosette leaf. The ~50% senesced rosette leaf was cut into three parts as indicated by the red lines. The level of CDF4 mRNA was determined in the three sections of the senescing leaf. Base; Middle; Tip. Three independent experiments were conducted. Values are given as mean ± SD, n=3.

D CDF4 expression level at various developmental stages of rosette leaves. YL, young leaf; NS, fully expanded, non-senescent leaf; ES, early-senescent leaf, with 1/4 leaf area turned yellow; LS, late-senescent leaf, with >50% leaf area turned yellow. Three independent experiments were conducted. Values are given as mean ± SD, n=3.

E Effect of plant growth hormones (1 mM SA, 1 μM ABA, 10 μM JA and 10 μM 1-aminocyclopropane -1-carboxylic acid (ACC) on CDF4 expression. Three independent experiments were conducted. Values are given as mean ± SD, n=3.

F Effects of abiotic stress treatments on CDF4 expression. Rosette leaves were treated with salt (100 mM NaCl), drought, H2O2 (2mM) or darkness. Three independent experiments were conducted. Values are given as mean ± SD, n=3.

G Effects of SA, H2O2, and dark treatment on CDF4 expression in the aba2-1 mutant background. AtACT2 was used as an internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3.