EMBO/soda-nel huggingface dataset:
H3K9 methylation extends across natural boundaries of heterochromatin in the absence of an HP1 protein. (2015)
Figure 2

<styled-content style="fixed-case">RNA</styled-content> decelerates Swi6 dynamics

A, B

Average relative intensities over time and corresponding fluorescence t
1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with cells expressing NLS‐Swi6‐EGFP (blue) or NLS‐Swi6‐KR25A‐EGFP (red).

C, D

Average relative intensities over time and corresponding fluorescence t
1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild‐type (brown) or cid14∆ (dark green) cells expressing Swi6‐EGFP.

E, F

Average relative intensities over time and corresponding fluorescence t
1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild‐type cells expressing heterochromatic NLS‐Swi6‐EGFP (blue) or NLS‐Swi6 KR25A‐EGFP (red), or cid14∆ cells expressing NLS‐Swi6‐EGFP (green) or NLS‐Swi6‐KR25A‐EGFP (yellow).

Data information: In (B), (D) and (F), the box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.
H3K9 methylation extends across natural boundaries of heterochromatin in the absence of an HP1 protein. (2015)
Figure 3

Swi6 dynamics remain the same throughout the cell cycle

Schematic of a chromosome indicating Swi6‐EGFP heterochromatic domains (green). Cnp1‐mCherry and Taz1‐mCherry (red) mark centromeres and telomeres, respectively. Representative images demonstrating Swi6‐EGFP co‐localization with centromeres (Cnp1‐mCherry) and telomeres (Taz1‐mCherry) are shown.

Fluorescence t
1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing NLS‐Swi6‐EGFP.

Fluorescence t
1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP.

Fluorescence t
1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing Swi6‐EGFP.

Fluorescence t
1/2 values of centromeric Swi6 obtained from FRAP experiments performed after release of cells expressing NLS‐Swi6‐EGFP (black dots) or the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP (red dots) from G1/S cell cycle arrest. Each dot represents one FRAP experiment at the respective time after release from cell cycle arrest. Swi6 is dispersed in M phase due to H3S10 phosphorylation, and M phase was therefore not included in the FRAP analysis.

Fluorescence t
1/2 values obtained from FRAP experiments performed on centromeric Swi6. A total of 40, 66, and 104 individual cells residing in the G1, S, and G2 phases of the cell cycle were analyzed, respectively. Representative bright‐field images of cells in G1, S, or G2 are shown. Scale bars = 2 μm.

Fluorescence t
1/2 values obtained from FRAP experiments performed on centromeric Swi6 of cells expressing NLS‐Swi6‐EGFP and NLS‐Swi6‐KR25A‐EGFP that reside in the G2 of the cell cycle.

Data information: In (B–D), (F) and (G), the box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.
H3K9 methylation extends across natural boundaries of heterochromatin in the absence of an HP1 protein. (2015)
Figure 4

Methylation of H3K9 spreads across natural boundaries and heterochromatin becomes derepressed in <italic>swi6∆</italic> cells

H3K9me2 ChIP‐seq enrichment profiles for swi6+ and swi6∆ cells on centromere 1. Centromeric repeat elements (dg/dh) are indicated. Inverted repeats (IRC1) constitute heterochromatin boundaries in wild‐type cells (Cam et al, 2005). The y‐axes represent log2 ChIP‐seq enrichments in 200‐bp genomically tiled windows calculated over clr4Δ cells. Two independent biological replicates for each genotype were performed (replicates 1 and 2). Positions of genomic elements on the plus and minus strands are indicated. Blue, protein‐coding genes; white, repeats; green, long terminal repeat (LTR).

H3K9me2 ChIP‐seq enrichment profiles for swi6+ and swi6∆ cells on the telomere of the right arm of chromosome 1. Two independent biological replicates for each genotype were performed (replicates 1 and 2).

Differential expression of transcripts coming from centromere 1 assessed by tiling microarray gene expression analysis. Expression data for swi6+ and swi6∆ cells were taken from a previous publication (Woolcock et al, 2012).
H3K9 methylation extends across natural boundaries of heterochromatin in the absence of an HP1 protein. (2015)
Figure 5

Swi6 dimerization and H3K9me2 binding are required for the prevention of H3K9me2 spreading

A

Domain architecture of full‐length Swi6. The indicated mutations in red disrupt the dimerization property of the CSD (L315E) or the ability of the CD to bind H3K9me2 (W104A).

B

Schematic of the IRC1R boundary region. IRC1R partially overlaps with the region that expresses the noncoding RNA borderline (Keller et al, 2013). Positions of the quantitative RT–PCR primers are indicated in red.

C–E

H3K9me2 levels on heterochromatin‐adjacent genes assessed by ChIP–PCR in swi6+, swi6∆, and swi6‐L315E (CSD dimerization mutant) (C), swi6‐W104A (H3K9me2 binding mutant) (D), and epe1∆ (E) cells. Enrichments over clr4∆ are normalized to adh1+. Average fold enrichment with s.d. is shown for three (C and E) or four (D) independent experiments. P‐values were generated by the Student's t‐test (two‐tailed distribution, two‐sample, unequal variance).
H3K9 methylation extends across natural boundaries of heterochromatin in the absence of an HP1 protein. (2015)
Figure 6

Tas3 self‐association is required for H3K9 methylation at centromeric repeats in the absence of Swi6

sRNA reads mapping to centromere 1 in swi6+ (green) and swi6∆ (blue) cells. The dashed red box highlights the loss of brdrRNAs at the IRC1R in the absence of Swi6. Centromeric repeat elements and the central core are indicated. Counts were normalized to the library size. Asterisks denote tRNA fragments. Note that tRNA genes flank IRC3 (Fig EV4) but not IRC1R.

Domain architecture of full‐length Tas3, adapted from Li et al (2009). The N‐terminal domain (NTD) and the Tas3‐homology domain (THD) are responsible for the interactions with Chp1 and Ago1, respectively. Tas3 self‐association is mediated through the TAM motif, which is disrupted by the point mutation L497E, indicated in red. The location of residue L479 (red) is highlighted in the tube representation of two Tas3 molecules (dark blue and gray) with one surface view (light blue). Created from structure 3D1D of the PDB database.

Schematic of the IRC1R boundary region. IRC1R partially overlaps with the region that expresses the noncoding RNA borderline (Keller et al, 2013). The positions of the primers used for quantitative RT–PCR are indicated in red.

H3K9me2 levels on heterochromatin‐adjacent genes assessed by ChIP–PCR in swi6+, swi6∆, tas3 L479E, and tas3 L479E swi6∆ cells. Enrichments over clr4∆ are normalized to adh1+. Average fold enrichment with s.d. is shown for three independent experiments. P‐values were generated by Student's t‐test (two‐tailed distribution, two‐sample, unequal variance).
Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation (2020)
Figure 2

Figure 2. Lysine acetylation is induced in E. coli extracts upon Arabidopsis GNAT expression.

(A) Anti-acetyllysine Western blot analyses and total protein stains of E. coli cell extracts. Cellular protein extracts before and after induction of His6-MBP-GNAT expression were separated on 12 % acrylamide gels and immunoblotted by using an anti-acetyllysine antibody or stained with Coomassie Dye. GNAT1, 2, 5, 6, 7 and 10 as well as His6-MBP control were expressed in E. coli BL21(DE3)pLysS. GNAT3 and 4 were expressed in Rosetta(DE3). In the total protein stains, recombinant GNAT protein constructs were highlighted by blue arrowheads. Protein expression before (ni) and after (i) induction with IPTG is indicated. The luminescence signal, indicating acetylated proteins, was usually recorded after 40-120 seconds. Since MBP-GNAT4 expression resulted in a saturated signal, the luminescence was additionally recorded for 10 seconds (indicated by an asterisk).

(B) Sequence logos of all unique lysine acetylation sites after GNAT expression in E.coli. Uniprot E.coli protein sequences were used as background population (sequence logos were generated using iceLogo (Maddelein et al, 2015)).
Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation (2020)
Figure 5

Figure 5. Inactivation of GNAT2 unveils dual KA and NTA activity in planta and distinct targets for both acetylations.

(A) Venn diagram representing the overlap between the N-termini sets identified in wild type and nsi mutant lines. N-termini from four replicates of Arabidopsis wild type and two independent nsi knockout lines (nsi-1 and nsi-2) were retrieved as previously reported (Koskela et al, 2018) and compared. More than 300 N-termini could be retrieved in all samples (Huber et al, 2019; Linster et al, 2015)

(B) Venn diagram of quantified NTAed proteins. Half of the retrieved N-termini could be quantified and 173 were common to all samples.

(C) Comparison of NTA yield of retrieved N-termini of proteins starting at position 1 or 2. The majority of these proteins, corresponding mostly to cytosolic components, undergoing or not to N-terminal methionine excision, were not affected by inactivation of GNAT2. For statistical analyses nsi-1 and nsi-2 were pooled and compared to the wild type. Two independent technical replicates of four biological replicates for each of the WT, nsi-1 and nsi-2 samples were analysed. Error bars are ± SD.

(D) Comparison of NTA yield of retrieved N-termini of proteins starting at positions > 2. Clear alteration of NTA yield was observed in nsi mutant lines in the pool of nuclear-encoded plastid proteins. nsi-1 and nsi-2 samples were treated as in D. Error bars are ± SD (see details of sampling in panel C).

(E) Comparison of NTA yields of retrieved N-termini in plastid proteins. Similar variation as in panel D was observed when NTA of only plastid proteins was analyzed. Error bars are ± SD (see details of sampling in panel C).

(F) Volcano plot representing NTA analyses of nsi knockout lines (treated together) and wild type. For this analysis, the p-value was calculated using Excel's two-tailed t-test function, for two-sample with equal variance. The most impacted proteins are shown in green. See Table 2 for correspondence. N is related to the number of quantified N-termini.

(G) IceLogo representation (Colaert et al, 2009) of the protein N-termini with modified NTA yield vs proteins with unmodified NTA yield. The color symbol associated to each residue is detailed in the legend to Fig. 4B. Black is aliphatic, green is small hydrophilic.
Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits (2020)
Figure 1

Figure 1. Development of a PP2A-B56 specific inhibitor.

A-B) Schematic of the B56 inhibitor series and affinities and stoichiometry's for B56α measured by ITC. Global direct fitting shown for one experiment (reverse). Each dot is the integrated heat per injection and the error bars represent uncertainty with this integrated value. The experiment was done in both direct (B56 in cell) and reverse (B56 in syringe) with similar results.

C) Time from Nuclear envelope breakdown (NEBD) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell. Only cells with similar expression levels of the various B56 inhibitor constructs were analysed. Median time is indicated by red line. ­A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. A Mann-Whitney U‐test was used for statistical analysis (ns: non‐significant, ***P ≤ 0.001).

D) Volcano plot representing mass spectrometry identified proteins co-purifying with B56 inhibitor versus control inhibitor from HeLa cells. PP2A-B56 subunits co-purifying with the B56 inhibitor are indicated.

E) and F) Competition assay in HeLa cells stably expressing RFP-tagged B56 inhibitor (LxxIxE) or control inhibitor (AxxAxA). YFP-B56α was transfected into and subsequently purified from these cell lines. Loss of binding of indicated proteins determined by either mass spectrometry (pink - B56 SLIM containing protein and known B56 interactor; blue - known B56 interactor, green - B56 SLIM containing protein) (E) or Western blotting (F).
Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits (2020)
Figure 2

Figure 2. Phosphorylation site preference of PP2A-B56

A-B) Schematic of synchronisation protocol for G1/S (A) or mitotic (B) arrested cells and accompanying volcano plot of phosphorylation sites quantified. The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light grey. Phosphorylation sites in a protein containing an LxxIxE motif are coloured pink.

C)-E) IceLogo representation of over - and underrepresented amino acid residues surrounding phosphorylation sites for the indicated experiments (letter coloring is standard iceologo color output).

F) -G) IceLogo representation of over - and underrepresented amino acid residues surrounding phosphorylation sites for the experiments indicated in Figure EV2F and EV2G respectively (letter coloring is standard iceLogo color output).

H) Distribution of phosphorylation site consensus motifs surrounding B56- or B55-dependent upregulated phosphorylation sites (top panel) in comparison to all phosphorylation sites (bottom panel) in the indicated experiment (proline-directed: pS/TP; basic: R/KxxpS/T, R/KxpS/T, R/KpS/T; acidic: D/E/NxpS/T, D/EpS/T, pS/TD/E, pS/TxD/E; x - any amino acid.
Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits (2020)
Figure 3

Figure 3. Differential phosphorylation site preference of PP2A-B56 and PP2A-B55.

A) Michaelis-Menten kinetic parameters of purified PP2A-B56α and PP2A-B55α holoenzymes were determined against the indicated phospho-peptides. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)

B-D) In vitro dephosphorylation by the PP2A-B55α and PP2A-B56α holoenzymes of panels of phosphorylated peptides as indicated. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)
Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits (2020)
Figure 4

Figure 4. Mechanistic basis for site specific dephosphorylation by PP2A-B56.

A) Schematics of engineered FoxO3 constructs. KD measurements of the LxxIxE motifs fused to the C-terminus of FoxO3 were performed previously (Hertz et al., 2016; Kruse et al., 2018).

B) The indicated YFP-FoxO3 constructs were transfected into HeLa cells, purified using YFP resin and PP2A-B56 binding determined by Western blotting. PP2A-A; scaffold subunit.

C) - D) YFP-FoxO3 constructs were transfected into HeLa cells and lysates (pS413) or YFP purifications (pS253) were subjected to Western Blotting and probed with the indicated phosphoantibodies. Quantifications arise from five (pS413) or three (pS253) independent experiments. Mean and standard deviation shown in plots as black bars.

E) Steady-state localization of the indicated YFP-FOXO3 variants in HeLa cells. Each data point represents quantification from a single cell. ­A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. Scale bar is 10 μm. Mann-Whitney test significance values; ∗p < 0.02, ∗∗∗p < 0.0002.

F) Dephosphorylation by the PP2A-B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted. Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. Mean and standard deviation from 3 experiments is shown.
Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits (2020)
Figure 5

Figure 5. PP2A-B56 is a regulator of ADAM17 phosphorylation status and shedding activity.

A) Domain organization of ADAM17 and conservation of the PP2A-B56 binding motif.

B) KD values obtained by ITC measurements with full-length recombinant B56α and indicated ADAM17 variant peptides.

C) The indicated full-length murine Myc-ADAM17 derivatives were transfected into HeLa cells stably expressing YFP-B56α. YFP-B56α was purified (IP) and ADAM17 binding determined by Western blotting. PP2A-C; catalytic subunit, PP2A-A; scaffold subunit.

D) Dephosphorylation by the PP2A-B56α holoenzyme complex of the indicated phosphorylated GST-ADAM17 (V724-C827) fragments. The GST-ADAM17 (V724-C827) substrates was phosphorylated with radioactive ATP using Protein Kinase A and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. The mean and Standard deviation of 4 independent experiments are shown.

E) Protein expression of ADAM17 variants in the DLD-1 Adam17-/- cell line, determined by Western blot. GAPDH was used as an internal loading control.

F) Amphiregulin (AREG) shedding measured by ELISA of conditioned media from untreated, H2O2 treated or irradiated with x-ray DLD-1 Adam17-/- cells (clone #1) expressing full-length ADAM17 variants (wt, I762A or LEE). Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01, ***p<0.001. Mean and standard deviation indicated from at least three independent experiments.

G) Exogenous wt and I762A full length ADAM17 was immuno-purified from Adam17-/- cells treated with H2O2 and subjected to label-free LC-MS/MS to determine differential phosphorylation status of T735 and S808 (S811 in murine ADAM17).
RANKL from Bone Marrow Adipose Lineage Cells Promotes Osteoclast Formation and Bone Loss (2021)
Figure 1

Figure 1. AdipoqCre targets BM adipose lineage cells.

(A) FACS analysis of tdTomato+ cells in the bone marrow cells (percentages represent average values).

(B) Immunofluorescence staining of aggrecan and DAPI of femur sections from AdipoqCre; R26tdTomato mice. Scale bar=200/100/50µm in left/ upper right/ bottom right images. CB: cortical bone, TB: trabecular bone.

(C) Immunofluorescence staining of RANKL and Hoechst in bone marrow cells from AdipoqCre; R26tdTomato mice. Scale bar=50 µm.

(D) qPCR results of Rankl in BMAT and peripheral adipose tissues, including epididymal (eWAT), inguinal (iWAT), and interscapular (iBAT) (n=3 independent biological replicates). Data were compared using one-way ANOVA (** indicates P < 0.01), error bars are standard deviations.

(E) Immunofluorescence staining of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre; ranklfl/fl mice (Rankl-/-) and ranklfl/fl mice (Rankl+/+). Scale bar=50 μm.

(F) Western blotting analyses of RANKL in BMSCs during in vitro BMSCs adipogenesis (n=3 independent biological replicates). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.
RANKL from Bone Marrow Adipose Lineage Cells Promotes Osteoclast Formation and Bone Loss (2021)
Figure 2

Figure 2. Cancellous bone mass increases in AdipoqCre; ranklfl/fl mice.

(A-B) Micro-CT and statistical analyses of BMD, BV/TV, Tb.N and Tb.Sp in trabecular bone from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 4 weeks, 8 weeks and 16 weeks (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t-test (** indicates P < 0.01), error bars are standard deviations.

(C) Micro-CT images of cortical bone from Rankl-/- and Rankl+/+ mice at 8 weeks. Images are representative of five independent biological replicates. Scale bar =1 mm.

(D) Micro-CT analyses and statistical analyses of cortical bone from Rankl-/- and Rankl+/+ mice at 8 weeks (n=5 independent biological replicates). Data were compared using an unpaired t‐test, error bars are standard deviations.
RANKL from Bone Marrow Adipose Lineage Cells Promotes Osteoclast Formation and Bone Loss (2021)
Figure 3

Figure 3. RANKL knockout from BM adipose lineage cells regulates trabecular bone remodeling.

Representative images of TRAP staining of femur from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 8 weeks. Scale bar=200 μm. Panel a1 and a4 indicate trabecular bone, a2 and a5 stand for growth plate, a3 and a6 denote cortical bone.

(B) Numbers of Trap+ cells on trabecular bone (a1 vs. a4, see (A)) (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.

(C) Numbers of Trap+ cells under the growth plate (a2 vs. a5, see (A)) (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test, error bars are standard deviations.

(D) Numbers of periosteal Trap+ cells (a3 vs. a6, see (A)) (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test, error bars are standard deviations.

(E) Immunofluorescence staining of osteocalcin from 8-week-old mice (n=6 mice in Rankl-/- group and 4 mice in Rankl+/+ group). Scale bar=50 μm. TB: trabecular bone.

(F) Statistical analyses of Ocn+ cells (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.

(G) Calcein staining of trabecular bone at 8 weeks. Scale bar=50 μm. TB: trabecular bone. Images are representative of 6 independent biological replicates in Rankl-/- and 5 replicates in Rankl+/+ group.

(H) Mineral apposition rate (MAR) of trabecular bone at 8 weeks.(n=6 mice in Rankl-/- group and 5 mice in Rankl+/+ group). Data were compared using an unpaired t‐test ( ** indicates P < 0.01), error bars are standard deviations.

(I-K) Serum CTX-1, OCN and bone marrow RANKL levels from Rankl-/- and Rankl+/+ mice at 4 weeks (n=5 and 5), 8 weeks (n=5 and 6) and 16 weeks (n=5 and 5). Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations..
RANKL from Bone Marrow Adipose Lineage Cells Promotes Osteoclast Formation and Bone Loss (2021)
Figure 5

Figure 5. RANKL knockout from BM adipose lineage cells contributes to excess osteoclast formation and bone resorption in OVX mice.

(A-B) Micro-CT analyses of sham and OVX model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.

(C-D) TRAP staining and statistical analyses of numbers of osteoclasts in Sham and OVX model (Images are representative of 5 independent biological replicates in (C), and n=5 independent microscopic vision fields in (D)). Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.

(E-F) Immunofluorescence staining of OCN and statistical analyses of the number of osteoblasts (Images are representative of 5 independent biological replicates in (E), and n=5 independent microscopic vision fields in (F)). Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.