G - I : GFP - MOSPD2 - expressing cells ( green ) were left untransfected ( G ) or transfected with Flag - STARD3 ( H ) and Flag - STARD3 S209A ( I ) , and labelled using anti - Flag ( magenta ) antibodies . The subpanels on the right are higher magnification ( 3 . 5 × ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Scale bars : 10 µm .

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G-I: GFP-MOSPD2-expressing cells (green) were left untransfected (G) or transfected with Flag-STARD3 (H) and Flag-STARD3 S209A (I), and labelled using anti-Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Scale bars: 10 µm.


J : Pearson ' s correlation coefficients between MOSPD2 ( WT or RD / LD ) and STARD3 ( WT , S209A or FA / YA ) staining are shown . Each dot represents a single cell ( number of cells : MOSPD2 - STARD3 : 22 ; MOSPD2 - STARD3 S209A : 21 ; MOSPD2 - STARD3 FA / YA : 20 ; MOSPD2 RD / LD - STARD3 : 17 , from at least three independent experiments ) . Means and error bars ( SD ) are shown . Kruskal - Wallis with Dunn ' s multiple comparison test ( * * * P < 0 . 001 ) .

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J: Pearson's correlation coefficients between MOSPD2 (WT or RD/LD) and STARD3 (WT, S209A or FA/YA) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-STARD3: 22; MOSPD2- STARD3 S209A: 21; MOSPD2-STARD3 FA/YA: 20; MOSPD2 RD/LD-STARD3: 17, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal-Wallis with Dunn's multiple comparison test (***P < 0.001).


Schematic of single ‐ plane acquisition FRAP on endogenously tagged Swi6 ‐ EGFP . Representative still images of a FRAP experiment are shown . See also Movie EV1 . Green sphere : yeast nucleus containing Swi6 ‐ EGFP ; green dots : heterochromatic Swi6 ‐ EGFP ; red circle : bleached area .

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Schematic of single‐plane acquisition FRAP on endogenously tagged Swi6‐EGFP. Representative still images of a FRAP experiment are shown. See also Movie EV1. Green sphere: yeast nucleus containing Swi6‐EGFP; green dots: heterochromatic Swi6‐EGFP; red circle: bleached area.


Normalized average intensities ( black dots ) of FRAP on Swi6 ‐ EGFP heterochromatic loci ( n = 31 ) fitted to a one ‐ component model ( blue line ) . The dashed lines indicate the final relative intensity that is set to 1 and the fluorescence half ‐ recovery time ( t 1 / 2 ) .

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Normalized average intensities (black dots) of FRAP on Swi6‐EGFP heterochromatic loci (n = 31) fitted to a one‐component model (blue line). The dashed lines indicate the final relative intensity that is set to 1 and the fluorescence half‐recovery time (t 1/2).


Fluorescence t 1 / 2 values of FRAP on Swi6 ‐ EGFP heterochromatic loci . The box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box .

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Fluorescence t 1/2 values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.


Normalized fluorescence recovery values of FRAP on Swi6 ‐ EGFP heterochromatic loci . The box bounds the interquartile range ( IQR ) divided by the median , and whiskers extend to a maximum of 1 . 5 × IQR beyond the box .

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Normalized fluorescence recovery values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.


FLIP experiment showing a bleached ( FRAP , red circle ) and unbleached , adjacent locus ( FLIP , blue circle ) in the same nucleus . The graph displays average relative fluorescence of FLIP ( blue ) , FRAP ( red ) , and control loci ( no bleach , green ) in other non ‐ bleached cells .

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FLIP experiment showing a bleached (FRAP, red circle) and unbleached, adjacent locus (FLIP, blue circle) in the same nucleus. The graph displays average relative fluorescence of FLIP (blue), FRAP (red), and control loci (no bleach, green) in other non‐bleached cells.


Schematic of a line ‐ scan FRAP experiment . Fluorescence intensities are measured along a line of a non ‐ bleached nucleus ( Ctrl nucleus ) and a bleached nucleus ( FRAP nucleus ) in Swi6 ‐ EGFP clr4 ∆ cells . Red box : bleached region . Right : raw data example .

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Schematic of a line‐scan FRAP experiment. Fluorescence intensities are measured along a line of a non‐bleached nucleus (Ctrl nucleus) and a bleached nucleus (FRAP nucleus) in Swi6‐EGFP clr4∆ cells. Red box: bleached region. Right: raw data example.


Average relative intensities of a line ‐ scan FRAP experiment on Swi6 ‐ EGFP in heterochromatin ( blue line ) , euchromatin ( green line ) , and clr4 ∆ ( red line ) . The curves have a gliding average of 40 .

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Average relative intensities of a line‐scan FRAP experiment on Swi6‐EGFP in heterochromatin (blue line), euchromatin (green line), and clr4∆ (red line). The curves have a gliding average of 40.


A , BAverage 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 ) .

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A, BAverage 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 , DAverage 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 .

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C, DAverage 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 , FAverage 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 .

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E, FAverage 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.


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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


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 .

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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.


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 ) .

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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 ) .

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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 ) .

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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).


C - EH3K9me2 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 ) .

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C-EH3K9me2 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).


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 .

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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.


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 ) .

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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).


B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young ( 2 months ) and old ( 22 months ) mice ( n = 3 mice per group ) . Scale bar 200 µm .

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B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young (2 months) and old (22 months) mice (n = 3 mice per group). Scale bar 200 µm.


C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young ( 20 - 35 years old ) and old ( 60 - 80 years old ) humans ( n = 4 - 7 individuals per group ) . Scale bar , 200 µm .

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C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young (20-35 years old) and old (60-80 years old) humans (n = 4-7 individuals per group). Scale bar, 200 µm.


D Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive and CD31 - positive endothelia in lungs from young and old mice ( n = 3 - 6 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs from young and old mice (n = 3-6 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


E Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1 - positive and CD31 - positive endothelia in lungs from young and old humans ( n = 4 - 7 individuals per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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E Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1-positive and CD31-positive endothelia in lungs from young and old humans (n = 4-7 individuals per group). Scale bar, 100 µm. a.u. = arbitrary units.


C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age - matched wild type and G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 200 µm .

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C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 200 µm.


D Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive and CD31 - positive endothelia in lungs of age - matched wild type and G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F / F at the indicated time points following 4OHT treatment and consequent TRF2 knockout . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

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A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F/F at the indicated time points following 4OHT treatment and consequent TRF2 knockout. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown . Scale bar , 25 µm . D RT - qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C ( n = 6 independent experiments ) .

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C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown. Scale bar, 25 µm. D RT-qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C (n = 6 independent experiments).


E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F / F mice treated with tamoxifen ( to induce TRF2 loss and telomere uncapping ) or vehicle . The animals as been injected also with PBS . Scale bar , 10 µm . F RT - qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E ( n = 5 - 7 mice per group ) .

O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O

E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce TRF2 loss and telomere uncapping) or vehicle. The animals as been injected also with PBS. Scale bar, 10 µm. F RT-qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E (n = 5-7 mice per group).


B Relative luciferase activity in HeLa shTRF2 following ionizing radiation ( 5 Gy ) or TRF2 knockdown upon doxycycline - induced shTRF2 expression ( n = 3 independent experiments ) . Error bars represent the s . e . m . * P < 0 . 05 . Two - way paired ANOVA .

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B Relative luciferase activity in HeLa shTRF2 following ionizing radiation (5 Gy) or TRF2 knockdown upon doxycycline-induced shTRF2 expression (n=3 independent experiments). Error bars represent the s.e.m. *P<0.05. Two-way paired ANOVA.


A Immunofluorescence showing gammaH2AX foci in Trf2F / F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O B-CELL_TYPE B-GENEPROD O O O O O O O O O O O O O O

A Immunofluorescence showing gammaH2AX foci in Trf2F/F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F / F mice treated with tamoxifen ( to induce telomere uncapping ) or vehicle and injected with the indicated ASOs or PBS as control . Scale bar , 10 µm . D RT - qPCR detection of Ace2 mRNA levels in livers of mice treated as in C ( n = 5 - 8 mice per group ) .

O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-TISSUE O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O

C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce telomere uncapping) or vehicle and injected with the indicated ASOs or PBS as control. Scale bar, 10 µm. D RT-qPCR detection of Ace2 mRNA levels in livers of mice treated as in C (n = 5-8 mice per group).


E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age - matched wild type and G3 Terc - / - mice , treated with the indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 200 µm .

O O B-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O

E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 200 µm.


F Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive cells in lungs of age - matched wild type and G3 Terc - / - mice , treated with the indicated ASOs ( n = 4 - 9 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

F Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive cells in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


A Atf7ip KO cells show increased expression of SETDB1 - regulated ERVs and the provirus reporter , MSCV - GFP as evidenced by RT - qPCR analysis . RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells . Data are mean ± SD ; n = 3 , technical replicates .

O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O B-ORGANISM O O O O O B-ORGANISM O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

A Atf7ip KO cells show increased expression of SETDB1-regulated ERVs and the provirus reporter, MSCV-GFP as evidenced by RT-qPCR analysis. RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells. Data are mean ± SD; n=3, technical replicates.


B Atf7ip KO mESCs ( TT # 2 - 12 ) show decreased H3K9me3 at the LTR of the SETDB1 - regulated reporter and the ERVs , as evidenced by Native ChIP followed by qPCR analysis . Gapdh was used as a negative control . Data are mean ± SEM ; n = 5 , independent experiments . * P < 0 . 05 and * * P < 0 . 01 by paired t - test .

O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Atf7ip KO mESCs (TT#2-12) show decreased H3K9me3 at the LTR of the SETDB1-regulated reporter and the ERVs, as evidenced by Native ChIP followed by qPCR analysis. Gapdh was used as a negative control. Data are mean ± SEM; n=5, independent experiments. *P < 0.05 and **P < 0.01 by paired t-test.


C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs . Endogenous SETDB1 - ATF7IP interaction is validated by anti - ATF7IP antibody co - IP experiment with anti - ATF7IP antibody .

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C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs. Endogenous SETDB1-ATF7IP interaction is validated by anti-ATF7IP antibody co-IP experiment with anti-ATF7IP antibody.


A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs . Representative projected images are shown , and the quantitative analyses are shown in B - D . Scale bar : 10 µm . B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 100 cells were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - test . C , D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated . Violin plot for SETDB1 ' s foci numbers from all the cells analysed in three independent experiments is shown in c . The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D . Over 100 cells were analysed per sample per experiment . * P < 0 . 05 by unpaired Student ' s t - test .

O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown, and the quantitative analyses are shown in B-D. Scale bar: 10 µm. B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test. C, D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated. Violin plot for SETDB1's foci numbers from all the cells analysed in three independent experiments is shown in c. The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D. Over 100 cells were analysed per sample per experiment. *P < 0.05 by unpaired Student's t-test.


E , IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs . Representative projected images are shown . Arrows indicate the region used for line - plot analysis . Cells were treated with 10 ng / mL LMB or mock for 5 h before analysis . Scale bar : 10 µm . F The line - plot analysis for E . Relative intensities of SETDB1 signal were measured by Image J . G The quantitative analysis for E is shown . SETDB1 signal in the nucleus that was determined by DAPI staining was calculated . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 100 cells were analysed per sample per experiment . NS : P > 0 . 05 , * * P < 0 . 01 versus " Mock , WT " group by Dunnett ' s test .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown. Arrows indicate the region used for line-plot analysis. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis. Scale bar: 10 µm. F The line-plot analysis for E. Relative intensities of SETDB1 signal were measured by Image J. G The quantitative analysis for E is shown. SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. NS: P > 0.05, **P < 0.01 versus "Mock, WT" group by Dunnett's test.


A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype . Endogenous SETDB1 in HEK293T cells shows cytoplasm + nuclear localization profile . Scale bar : 10 µm .

O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O

A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype. Endogenous SETDB1 in HEK293T cells shows cytoplasm+nuclear localization profile. Scale bar: 10 µm.


B Over - production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells . The representative data of two independent experiments is shown . Scale bar : 10 µm .

O O O O O O B-ORGANISM O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O

B Over-production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells. The representative data of two independent experiments is shown. Scale bar: 10 µm.


C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells , but no significant impact for ATF7IP KO HEK293T cells . Cells were treated with 10ng / ml of LMB for 5 h and then analysed with immunofluorescent staining . The representative data of three independent experiments is shown . Scale bar : 10 µm . D SETDB1 signal in the nucleus shown in C was calculated . The mean from three independent experiments is shown as a bar - graph ± SEM ; n = 3 . Over 50 cells were analysed per sample per experiment . * * P < 0 . 01 by Student ' s t - test ( two - sided test ) .

O B-SMALL_MOLECULE O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O B-CELL_LINE O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells, but no significant impact for ATF7IP KO HEK293T cells. Cells were treated with 10ng/ml of LMB for 5 h and then analysed with immunofluorescent staining. The representative data of three independent experiments is shown. Scale bar: 10 µm. D SETDB1 signal in the nucleus shown in C was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. **P < 0.01 by Student's t-test (two-sided test).


E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells . The representative data of three independent experiments is shown . Scale bar : 10 µm . F SETDB1 signal in the nucleus shown in E was calculated . The mean from three independent experiments is shown as a bar - graph ± SEM ; n = 3 . Over 50 cells were analysed per sample per experiment . Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells . * P < 0 . 05 and * * P < 0 . 01 by Dunnett ' s method .

O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells. The representative data of three independent experiments is shown. Scale bar: 10 µm. F SETDB1 signal in the nucleus shown in E was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells. *P < 0.05 and **P < 0.01 by Dunnett's method.


A HEK293T cells were transfected with V5 - SETDB1 and either empty or 3xF - ATF7IP expressing vector . Over 50 transfected cells analysed per sample were analyzed . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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A HEK293T cells were transfected with V5-SETDB1 and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells analysed per sample were analyzed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


B HEK293T cells were transfected with V5 - SETDB1 d1 mutant and either empty or 3xF - ATF7IP expressing vector . Over 50 transfected cells per sample were analysed . The representative data of two independent experiments was shown . Scale bar : 10 µm . The yellow arrowheads indicate the cells that show nuclear ( and cytoplasmic ) - localization of introduced exogenous mSETDB1 .

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B HEK293T cells were transfected with V5-SETDB1 d1 mutant and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments was shown. Scale bar: 10 µm. The yellow arrowheads indicate the cells that show nuclear (and cytoplasmic)-localization of introduced exogenous mSETDB1.


A Co - transfection with ATF7IP augments the K885 ubiquitination of SETDB1 . HEK293T cells were transfected After the transfection , V5 - SETDB1 was immune - purified with anti - V5 antibody and used for WB analysis .

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A Co-transfection with ATF7IP augments the K885 ubiquitination of SETDB1. HEK293T cells were transfected After the transfection, V5-SETDB1 was immune-purified with anti-V5 antibody and used for WB analysis.


B ATF7IP regulates the ubiquitination of SETDB1 in mESCs . Endogenous SETDB1 was immune - purified from WT or Atf7ip KO mESCs cells and used for WB analysis .

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B ATF7IP regulates the ubiquitination of SETDB1 in mESCs. Endogenous SETDB1 was immune-purified from WT or Atf7ip KO mESCs cells and used for WB analysis.


C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs . Cells were treated with 10 ng / mL LMB or mock for 5 h before analysis .

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C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis.


D HEK293T cells were transfected with either 3xF - or 3xF - NLS - SETDB1 . Over 50 transfected cells per sample were analysed . The representative data of two independent experiments is shown . Scale bar : 10 µm .

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D HEK293T cells were transfected with either 3xF- or 3xF-NLS-SETDB1. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


E Nuclear localization itself promotes the ubiquitination of SETDB1 . HEK293T cells were transfected with 3xFLAG - or 3xFLAG - NLS - SETDB1 . After FLAG - IP , the purified SETDB1s were analyzed by WB .

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E Nuclear localization itself promotes the ubiquitination of SETDB1. HEK293T cells were transfected with 3xFLAG- or 3xFLAG-NLS-SETDB1. After FLAG-IP, the purified SETDB1s were analyzed by WB.


F , G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF - or 3xF - NLS - SETDB1 . Representative images are shown in F , and the quantitative analysis is shown in G . Scale bar : 10 µm . Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs . The mean from three independent experiments is shown as a bar - graph with jittered points indicating the average % intensity of each experiment . Over 50 cells were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF- or 3xF-NLS-SETDB1. Representative images are shown in F, and the quantitative analysis is shown in G. Scale bar: 10 µm. Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 50 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test.


H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs . Atf7ip KO mESCs expressing 3xF - or 3xF - NLS - SETDB were used for FLAG - IP , and the purified SETDB1s were analyzed by WB .

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H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs. Atf7ip KO mESCs expressing 3xF- or 3xF-NLS-SETDB were used for FLAG-IP, and the purified SETDB1s were analyzed by WB.


A ) Double fluorescence in situ hybridization using Nrg3 ( green ) and Gad1 ( red ) specific probes ; DAPI was used as a counter stain . Nrg3 is present in Gad1 - positive ( filled arrowheads ) and - negative neurons . ( A ' ) and ( A ' ' ) show higher magnifications of boxed areas indicated in ( A ) . Nrg3 - negative neurons are indicated by open arrowheads ; note that many but not all neurons express Nrg3 .

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A) Double fluorescence in situ hybridization using Nrg3 (green) and Gad1 (red) specific probes; DAPI was used as a counter stain. Nrg3 is present in Gad1-positive (filled arrowheads) and -negative neurons. (A') and (A'') show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3.


B ) Comparison of mRNA levels of Nrg3 , Nrg1β type III ( Nrg1β − III ) , Nrg2β , Nrg1β type II ( Nrg1β − II ) , Nrg2α and Nrg1β type I ( Nrg1β − I ) in the cortex ( Cx ) , hippocampus ( HC ) and striatum ( Str ) of juvenile mice ( P30 - 60 ) by quantitative RT - PCR . Data represent means ± SD of 4 biological replicates . 2 - way - ANOVA with Bonferroni ' s multiple comparisons test was performed to assess statistical significance ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , ns = not significant ) .

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B) Comparison of mRNA levels of Nrg3, Nrg1β type III (Nrg1β−III), Nrg2β, Nrg1β type II (Nrg1β−II), Nrg2α and Nrg1β type I (Nrg1β−I) in the cortex (Cx), hippocampus (HC) and striatum (Str) of juvenile mice (P30-60) by quantitative RT-PCR. Data represent means ± SD of 4 biological replicates. 2-way-ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (****P<0.0001, ***P<0.001, ns=not significant).


D ) Detection of Nrg3 by western blot analysis in fractions from brain lysates : S1 , crude lysate ; S2 , cytosol and light membranes ; S2 ' , cytosol ; LM , light membranes ; P2 ' , crude synaptosomes ; S3 ' , synaptosomal cytoplasm ; SV , synaptic vesicles ; P3 , synaptosomal membranes ; SPM , synaptic plasma membranes .

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D) Detection of Nrg3 by western blot analysis in fractions from brain lysates: S1, crude lysate; S2, cytosol and light membranes; S2', cytosol; LM, light membranes; P2', crude synaptosomes; S3', synaptosomal cytoplasm; SV, synaptic vesicles; P3, synaptosomal membranes; SPM, synaptic plasma membranes.


E , F ) Immunohistochemical analysis in the hippocampus of wildtype ( E , E ' , E ' ' ) and ErbB4 mutant mice ( F ) at 2 month of age using Nrg3 - , ErbB4 - and parvalbumin ( Parv ) - specific antibodies . The white box in ( E ) is displayed magnified in ( E ' , E ' ' ) . In control mice , Nrg3 is enriched on dendrites of ErbB4 + PV interneurons , compared to the surrounding neuropil ( E ' , E ' ' ) . In ErbB4 mutants , the Nrg3 enrichment on PV interneurons is not apparent ( F ) .

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E, F) Immunohistochemical analysis in the hippocampus of wildtype (E,E',E'') and ErbB4 mutant mice (F) at 2 month of age using Nrg3-, ErbB4- and parvalbumin (Parv)-specific antibodies. The white box in (E) is displayed magnified in (E',E''). In control mice, Nrg3 is enriched on dendrites of ErbB4+ PV interneurons, compared to the surrounding neuropil (E',E''). In ErbB4 mutants, the Nrg3 enrichment on PV interneurons is not apparent (F).


G ) Nrg3 , ErbB4 and GluA4 were identified by immunohistology and co - localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1 . Note that ( G ) displays Nrg3 / ErbB4 , ( G ' ) Nrg3 / GluA4 , and ( G ' ' ) ErbB4 / GluA4 signals of the same triple - stained image ; false colors were assigned for better signal visualization .

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G) Nrg3, ErbB4 and GluA4 were identified by immunohistology and co-localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1. Note that (G) displays Nrg3/ErbB4, (G') Nrg3/GluA4, and (G'') ErbB4/GluA4 signals of the same triple-stained image; false colors were assigned for better signal visualization.


H ) Quantification of GluA4 puncta present on ErbB4 + dendrites in the CA1 stratum radiatum of adult wildtype ( wt ) and Nrg3 - / - mice ( age P90 - 120 ) . Data are presented as box plots with Tukey ' s whiskers and outliers ; means are indicated by a Plus symbols . n = 65 ( wt ) and n = 62 ( Nrg3 - / - ) from 5 animals each . Unpaired t - test ( 2 - tailed ) with Welch ' s correction was performed to assess statistical significance ( * * * * P < 0 . 0001 ) .

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H) Quantification of GluA4 puncta present on ErbB4+ dendrites in the CA1 stratum radiatum of adult wildtype (wt) and Nrg3-/- mice (age P90-120). Data are presented as box plots with Tukey's whiskers and outliers; means are indicated by a Plus symbols. n=65 (wt) and n=62 (Nrg3-/-) from 5 animals each. Unpaired t-test (2-tailed) with Welch's correction was performed to assess statistical significance (****P<0.0001).


A ) His6 - tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations . Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho - tyrosine ( pY ) and ErbB4 , and of whole lysates using antibodies against pErk1 / 2 and pAkt

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A) His6-tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho-tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt


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C , D ) Neurons from Nrg3 - / - ; ErbB4 - / - ; vGAT - Cre mice were transduced with a lentivirus expressing either ( C ) ErbB4wt or ( D ) ErbB4kd ( kinase dead ErbB4 ) in a Cre - dependent manner , and with a second virus at a low titer expressing Nrg3 / SypCFP . Immunocytochemical analysis of ErbB4 and SypCFP ; ( C , C ' ) and ( D , D ' ) each show the same image , ( C ' , D ' ) show the SypCFP signal

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C, D) Neurons from Nrg3-/-;ErbB4-/-;vGAT-Cre mice were transduced with a lentivirus expressing either (C) ErbB4wt or (D) ErbB4kd (kinase dead ErbB4) in a Cre-dependent manner, and with a second virus at a low titer expressing Nrg3/SypCFP. Immunocytochemical analysis of ErbB4 and SypCFP; (C,C') and (D,D') each show the same image, (C',D') show the SypCFP signal


E - G ) Quantification of Synapse preference towards ErbB4 + neurons ( E ) , ErbB4 enrichment ( F ) and SypCFP enrichment ( G ) in synapses on neurons expressing wildtype ErbB4 ( wt ) or ErbB4kd ( kd )

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E-G) Quantification of Synapse preference towards ErbB4+ neurons (E), ErbB4 enrichment (F) and SypCFP enrichment (G) in synapses on neurons expressing wildtype ErbB4 (wt) or ErbB4kd (kd)


H ) Nrg3 - / - neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4 , GFP and GluA ; ( H ) shows the triple stained image , while ( H ' ) and ( H ' ' ) only show ErbB4 and GluA signals , respectively

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H) Nrg3-/- neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4, GFP and GluA; (H) shows the triple stained image, while (H') and (H'') only show ErbB4 and GluA signals, respectively


I ) Co - cultures of Nrg3 - / - neurons with HEK293 cells expressing Nrg3 . Cultures were stained with antibodies against Nrg3 , ErbB4 and GluA . Note that Nrg3 , ErbB4 and GluA are enriched at the contact sites between Nrg3 - expressing HEK293 cells and ErbB4 + neurons

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I) Co-cultures of Nrg3-/- neurons with HEK293 cells expressing Nrg3. Cultures were stained with antibodies against Nrg3, ErbB4 and GluA. Note that Nrg3, ErbB4 and GluA are enriched at the contact sites between Nrg3-expressing HEK293 cells and ErbB4+ neurons


C ) Example traces of evoked excitatory postsynaptic currents ( eEPSCs ) averaged over 50 sweeps . D , E ) Quantification of the peak amplitude of the first eEPSC ( D ) and of the paired pulse ratio ( E )

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C) Example traces of evoked excitatory postsynaptic currents (eEPSCs) averaged over 50 sweeps. D, E) Quantification of the peak amplitude of the first eEPSC (D) and of the paired pulse ratio (E)


F - I ) sEPSC recordings from CA1 pyramidal ( F , H ) and PV neurons ( G , I ) in acute slices . sEPSC frequencies ( F , G ) and amplitudes ( H , I ) are

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F-I) sEPSC recordings from CA1 pyramidal (F,H) and PV neurons (G, I) in acute slices. sEPSC frequencies (F,G) and amplitudes (H,I) are shown


( B - C ) Fluorescent dye release from the mitochondria - mimic liposomes by the indicated wild - type ( wt ) or mutant Bax in the presence ( B ) or absence ( C ) of tBid or by tBid alone ( C ) was measured by quenching the fluorescence of the released dyes by the dye - specific antibodies outside the liposomes during a time course . The fraction of dye release was normalized to that by detergent . The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes .

O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O

(B-C) Fluorescent dye release from the mitochondria-mimic liposomes by the indicated wild-type (wt) or mutant Bax in the presence (B) or absence (C) of tBid or by tBid alone (C) was measured by quenching the fluorescence of the released dyes by the dye-specific antibodies outside the liposomes during a time course. The fraction of dye release was normalized to that by detergent. The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes.


( D ) Digitonin permeabilized BMK Bax - / - / Bak - / - cells expressing SMAC - mCherry in the mitochondria intermembrane space ( 50 μL , 500 , 000 total cells ) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37 ° C in a 96 - well plate . The samples were centrifuged for 10 min and separated into supernatant and pellet fractions . SMAC - mCherry release was calculated as the fraction of total ( supernatant + pellet ) mCherry fluorescence coming from the supernatant fraction . The data was normalized to the percent SMAC - mCherry release of wt Bax and cBid at 60 min . Each symbol represents the normalized SMAC - mCherry release for one single replicate ( n = 3 or more independent replicates ) . Where applicable the data was fit with the [ Agonist ] vs . response - Variable slope equation in Graphpad Prism 8 . 0 . 1 . The dotted lines represent the 95 % confidence interval of the fit .

O O O B-SMALL_MOLECULE O B-CELL_TYPE B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Digitonin permeabilized BMK Bax-/-/Bak-/- cells expressing SMAC-mCherry in the mitochondria intermembrane space (50 μL, 500,000 total cells) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37°C in a 96-well plate. The samples were centrifuged for 10 min and separated into supernatant and pellet fractions. SMAC-mCherry release was calculated as the fraction of total (supernatant + pellet) mCherry fluorescence coming from the supernatant fraction. The data was normalized to the percent SMAC-mCherry release of wt Bax and cBid at 60 min. Each symbol represents the normalized SMAC-mCherry release for one single replicate (n = 3 or more independent replicates). Where applicable the data was fit with the [Agonist] vs. response - Variable slope equation in Graphpad Prism 8.0.1. The dotted lines represent the 95% confidence interval of the fit.


Fig . 2 Identification of protein complexes and communities in the cellular extracts . ( A ) Elution of selected protein complexes as a function of their retention times ( see Appendix Fig . S4 for their corresponding subunit elutions ) .

O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

Fig. 2 Identification of protein complexes and communities in the cellular extracts. (A) Elution of selected protein complexes as a function of their retention times (see Appendix Fig. S4 for their corresponding subunit elutions).


( B ) Scatter plot indicating discrepancies in the expected and measured molecular weights of 102 protein complexes that elute as a single peak ; 50 % of protein complexes are observed to have higher molecular weights than structurally characterized , indicating that they are organized in higher - order assemblies .

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(B) Scatter plot indicating discrepancies in the expected and measured molecular weights of 102 protein complexes that elute as a single peak; 50% of protein complexes are observed to have higher molecular weights than structurally characterized, indicating that they are organized in higher-order assemblies.


Fig . 4 Higher - order assemblies identified by proteome - wide cross - linking mass spectrometry , biomolecular modeling and negative - stain electron microscopy . ( A ) Distance distributions of identified cross - links on top of the modeled protein complexes and identification of novel interactions . Satisfied distances are shown in blue and over - length cross - links are shown in red .

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Fig. 4 Higher-order assemblies identified by proteome-wide cross-linking mass spectrometry, biomolecular modeling and negative-stain electron microscopy. (A) Distance distributions of identified cross-links on top of the modeled protein complexes and identification of novel interactions. Satisfied distances are shown in blue and over-length cross-links are shown in red.


( B ) Negatively stained electron micrographs of fractions 3 - 7 and 27 directly derived from size exclusion chromatography showing the structural signatures and their structural integrity within the fractions . Decreasing molecular weight correlates with increased protein concentration as a function of protein complex elution is highlighted .

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(B) Negatively stained electron micrographs of fractions 3-7 and 27 directly derived from size exclusion chromatography showing the structural signatures and their structural integrity within the fractions. Decreasing molecular weight correlates with increased protein concentration as a function of protein complex elution is highlighted.


( C ) Abundance profiles as determined by quantitative mass spectrometry correlate with the number of observed single particles of the corresponding structural signature within the negative - staining electron micrographs ; shown for fatty acid synthase , 20S proteasome , 60S and 40S ribosome ( the number of particles per image per fraction is indicated below the class averages ) .

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(C) Abundance profiles as determined by quantitative mass spectrometry correlate with the number of observed single particles of the corresponding structural signature within the negative-staining electron micrographs; shown for fatty acid synthase, 20S proteasome, 60S and 40S ribosome (the number of particles per image per fraction is indicated below the class averages).


( D ) Simplification of lysate ( collecting only the flow - through from anion exchange chromatography ) prior to SEC separation allows class - averaging of structural signatures from complex fractions that were previously too low abundant . Proteins identified in this simplified fraction are found in Table S4 .

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(D) Simplification of lysate (collecting only the flow-through from anion exchange chromatography) prior to SEC separation allows class-averaging of structural signatures from complex fractions that were previously too low abundant. Proteins identified in this simplified fraction are found in Table S4.


Fig . 5 Visualization of transient interactions in fatty acid synthesis . ( A ) Communities in fatty acid metabolism and the quantification of intra - community distances within cryo - electron micrographs . Fatty acid synthase ( FAS ) frequently interacts with other sizeable protein complexes in a linear ' pearl - string - like ' arrangement and usually localizes at the edges of the community . Scale bars correspond to 25 nm . FAS particles ( circles ) and their nearest neighbors ( arrow heads ) are indicated .

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Fig. 5 Visualization of transient interactions in fatty acid synthesis. (A) Communities in fatty acid metabolism and the quantification of intra-community distances within cryo-electron micrographs. Fatty acid synthase (FAS) frequently interacts with other sizeable protein complexes in a linear 'pearl-string-like' arrangement and usually localizes at the edges of the community. Scale bars correspond to 25 nm. FAS particles (circles) and their nearest neighbors (arrow heads) are indicated.


( C ) Related to ( A ) . Calculation of minimum distances between pairs of FAS molecules as well as FAS molecules and their closest non - FAS neighbors in comparison to random distributions . Whereas FAS molecules are randomly distributed , their binders are not , confirming physical interactions . Supervised picking means that all single - particles were manually picked from the images . Randomized distance means that these manually picked particles were assigned random coordinates in each image ( randomization of x , y coordinates considering image borders ) and then their distance is calculated .

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(C) Related to (A). Calculation of minimum distances between pairs of FAS molecules as well as FAS molecules and their closest non-FAS neighbors in comparison to random distributions. Whereas FAS molecules are randomly distributed, their binders are not, confirming physical interactions. Supervised picking means that all single-particles were manually picked from the images. Randomized distance means that these manually picked particles were assigned random coordinates in each image (randomization of x,y coordinates considering image borders) and then their distance is calculated.


( D ) Cross - linking mass spectrometry data show that the binder is a carboxylase that is bound to the malonyl transacylase domain and acyl carrier protein ( ACP ) is in the vicinity , considering cross - link length and the positions of the lysine on the ctFAS structure . Cross - links come from both affinity - purified and fractionated cell extracts .

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(D) Cross-linking mass spectrometry data show that the binder is a carboxylase that is bound to the malonyl transacylase domain and acyl carrier protein (ACP) is in the vicinity, considering cross-link length and the positions of the lysine on the ctFAS structure. Cross-links come from both affinity-purified and fractionated cell extracts.


Fig . 6 CryoEM structure of fatty acid synthase resolved to 4 . 7 Å as obtained from cryo electron micrographs of fraction numbers 7 - 9 . ( A ) The cryoEM of C . thermophilum fatty acid synthase ( ctFAS ) map is shown isosurface rendered and superimposed with the fitted X - ray structure of yeast FAS ( Jenni et al , 2007 ) . The domes and the cap show the unambiguous fit of α - helices and β - strands .

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Fig. 6 CryoEM structure of fatty acid synthase resolved to 4.7 Å as obtained from cryo electron micrographs of fraction numbers 7-9. (A) The cryoEM of C. thermophilum fatty acid synthase (ctFAS) map is shown isosurface rendered and superimposed with the fitted X-ray structure of yeast FAS (Jenni et al, 2007). The domes and the cap show the unambiguous fit of α-helices and β-strands.


( B ) A slice through the central wheel of fungal FAS . The pitch of α - helices is resolved .

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(B) A slice through the central wheel of fungal FAS. The pitch of α-helices is resolved.


( C ) As for ( B ) but sliced through the dome structure .

(C) As for (B) but sliced through the dome structure.


( D ) Location of acyl carrier protein ( ACP ) within the cryoEM map of ctFAS and the position of additional density outside the dome .

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(D) Location of acyl carrier protein (ACP) within the cryoEM map of ctFAS and the position of additional density outside the dome.


A Whole mount immunofluorescence of a P8 Syne4 - / - organ of Corti showing intact hair cells , as labelled with myosin VIIa . B Whole mount immunofluorescence of WT and Syne4 - / - organ of Corti from the 8 , 16 , and 32 kHz regions at P8 and P14 .

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A Whole mount immunofluorescence of a P8 Syne4-/- organ of Corti showing intact hair cells, as labelled with myosin VIIa. B Whole mount immunofluorescence of WT and Syne4-/- organ of Corti from the 8, 16, and 32 kHz regions at P8 and P14.


C Inner and outer hair cell counts of Syne4 - / - organ of Corti at P8 , P10 , P12 , and P14 .

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C Inner and outer hair cell counts of Syne4-/- organ of Corti at P8, P10, P12, and P14.


D FM1 - 43 uptake performed on P8 + 1 DIV ( days - in - vitro ) WT and Syne4 - / - organ of Corti . Top shows OHC plane and bottom shows IHC plane .

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D FM1-43 uptake performed on P8+1 DIV (days-in-vitro) WT and Syne4-/- organ of Corti. Top shows OHC plane and bottom shows IHC plane.


B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV . GFP at P1 showing complete transduction of both inner and outer HC . Myosin VIIa was used to label the hair cells . C Examples of 8 , 16 , and 32 kHz regions of an organ of Corti of a mouse injected with AAV . GFP . Top shows OHC plane , bottom shows IHC plane , right shows YZ orthogonal projection . Black asterisks show bright Deiters cells . D Quantification of GFP intensity of inner and outer HC from 3 injected mice , normalized to the average intensity of HC in a control , un - injected mouse .

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B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV.GFP at P1 showing complete transduction of both inner and outer HC. Myosin VIIa was used to label the hair cells. C Examples of 8, 16, and 32 kHz regions of an organ of Corti of a mouse injected with AAV.GFP. Top shows OHC plane, bottom shows IHC plane, right shows YZ orthogonal projection. Black asterisks show bright Deiters cells. D Quantification of GFP intensity of inner and outer HC from 3 injected mice, normalized to the average intensity of HC in a control, un-injected mouse.


E Transduction rates of AAV9 - PHP . B at 8 , 16 , and 32kHz regions based on GFP fluorescence . A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate .

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E Transduction rates of AAV9-PHP.B at 8, 16, and 32kHz regions based on GFP fluorescence. A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate.


F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV . Syne4 .

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F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4.


G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope . Eight OHC were measured .

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G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHC were measured.


H 3D surface projection of two adjacent OHC from a mouse injected with AAV . Syne4 . In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining .

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H 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining.


A Whole - mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT , Syne4 - / - , and Syne4 - / - mice injected with AAV . Syne4 . B 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT , Syne4 - / - mice and Syne4 - / - mice injected with AAV . Syne4 . Open arrows denote basal end , white arrows denote cuticular plate .

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A Whole-mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT, Syne4-/-, and Syne4-/- mice injected with AAV.Syne4. B 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT, Syne4-/- mice and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate.


C Image analysis of nucleus position of IHC and OHC . Images show an arc fitted through the apical surface , nucleus , and basal end of the cell in 3D . X , Y , and Z axes show position in μm .

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C Image analysis of nucleus position of IHC and OHC. Images show an arc fitted through the apical surface, nucleus, and basal end of the cell in 3D. X, Y, and Z axes show position in μm.


D Quantification of nuclear distance from the cuticular plate . A total of 45 IHC and 135 OHC from 3 WT mice , 45 IHC and 81 OHC from 3 Syne4 - / - mice , 30 IHC and 90 OHC from 2 Syne4 - / - mice injected with AAV . Syne4 were measured .

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D Quantification of nuclear distance from the cuticular plate. A total of 45 IHC and 135 OHC from 3 WT mice, 45 IHC and 81 OHC from 3 Syne4-/- mice, 30 IHC and 90 OHC from 2 Syne4-/- mice injected with AAV.Syne4 were measured.


A Representative example of ABR traces obtained at 4w from a WT , Syne4 - / - mouse , and Syne4 - / - mouse injected with AAV . Syne4 in response to an 18 kHz stimulus .

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A Representative example of ABR traces obtained at 4w from a WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4 in response to an 18 kHz stimulus.


B ABR thresholds at 4w of WT , Syne4 - / - , Syne4 - / - mice injected with AAV . Syne4 , and Syne4 - / - mice injected with AAV . GFP , n = 7 for WT , n = 8 for Syne4 - / - , n = 20 for Syne4 - / - + AAV . Syne4 , n = 6 for Syne4 - / - + AAV . GFP . C Quantification of P1 - N1 amplitude delta from ( B ) , n = 4 for WT , n = 4 for Syne4 - / - , n = 7 for Syne4 - / - + AAV . Syne4 . D Quantification of P1 latency from ( B ) , n = 4 for WT , n = 2 for Syne4 - / - , n = 7 for Syne4 - / - + AAV . Syne4 .

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B ABR thresholds at 4w of WT, Syne4-/-, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=7 for WT, n=8 for Syne4-/-, n=20 for Syne4-/- + AAV.Syne4, n=6 for Syne4-/- + AAV.GFP. C Quantification of P1-N1 amplitude delta from (B), n=4 for WT, n=4 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4. D Quantification of P1 latency from (B), n=4 for WT, n=2 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4.


E DPOAE thresholds at 4w , n = 6 for WT , n = 5 for Syne4 - / - , n = 6 for Syne4 - / - + AAV . Syne4 . F ABR threshold at 12w , n = 6 for WT , n = 8 for Syne4 - / - , n = 16 for Syne4 - / - + AAV . Syne4 , n = 5 for Syne4 - / - + AAV . GFP .

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E DPOAE thresholds at 4w, n=6 for WT, n=5 for Syne4-/-, n=6 for Syne4-/- + AAV.Syne4. F ABR threshold at 12w, n=6 for WT, n=8 for Syne4-/-, n=16 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP.


A Whole mount immunofluorescence at 12w of the organ of Corti of a Syne4 - / - mouse injected with AAV . Syne4 mouse injected with AAV . Syne4 . B Whole mount immunofluorescence at 12w of the 12 kHz region of WT , Syne4 - / - mouse , and Syne4 - / - mouse injected with AAV . Syne4 .

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A Whole mount immunofluorescence at 12w of the organ of Corti of a Syne4-/- mouse injected with AAV.Syne4 mouse injected with AAV.Syne4. B Whole mount immunofluorescence at 12w of the 12 kHz region of WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4.


C IHC and OHC counts , n = 3 for WT , n = 4 for Mut , n = 6 for Mut + AAV . Syne4 . D Correlation between HC count and ABR threshold at 12kHz .

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C IHC and OHC counts, n=3 for WT, n=4 for Mut, n=6 for Mut + AAV.Syne4. D Correlation between HC count and ABR threshold at 12kHz.


E 3D surface projection of an OHC at 12w from the 12kHz region of WT and Syne4 - / - mice injected with AAV . Syne4 . Open arrows denote basal end , white arrows denote cuticular plate . F Image analysis of nucleus position of OHC . G Quantification of nuclear distance from the cuticular plate . A total of 30 OHC from 3 WT mice and 38 OHC from 4 Syne4 - / - mice injected with AAV . Syne4 were measured .

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E 3D surface projection of an OHC at 12w from the 12kHz region of WT and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate. F Image analysis of nucleus position of OHC. G Quantification of nuclear distance from the cuticular plate. A total of 30 OHC from 3 WT mice and 38 OHC from 4 Syne4-/- mice injected with AAV.Syne4 were measured.


B , C Quantification of activity level and cumulative freezing duration at 12w during seconds 90 - 210 of day 2 in WT , Syne4 - / - mice , Syne4 - / - mice injected with AAV . Syne4 , and Syne4 - / - mice injected with AAV . GFP , n = 14 for WT , n = 16 for Syne4 - / - , n = 9 for Syne4 - / - + AAV . Syne4 , n = 5 for Syne4 - / - + AAV . GFP . ( C ) Activity level . ( B ) Cumulative freezing duration .

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B, C Quantification of activity level and cumulative freezing duration at 12w during seconds 90-210 of day 2 in WT, Syne4-/- mice, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=14 for WT, n=16 for Syne4-/-, n=9 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP. (C) Activity level. (B) Cumulative freezing duration.


Distribution of 25 - 35 mer reads mapping to the mouse genome . Proportion of 30 - 35 nt genomic reads matching mouse tRNA sequences . The sequencing was done in duplicates . Error bars represent SEM , significance was calculated using unpaired t - test .

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Distribution of 25-35 mer reads mapping to the mouse genome. Proportion of 30-35 nt genomic reads matching mouse tRNA sequences. The sequencing was done in duplicates. Error bars represent SEM, significance was calculated using unpaired t-test.


Perbase sequence coverage of 30 - 35 nt reads across parental tRNA sequence . Anticodon positions are demarcated by discontinuous lines ( Blue = LIF - treated , Grey = Wnt3 + LIF - treated , Red = RA - treated mESCs ) . Perbase coverage of 30 - 35 nt reads across the 468 individual parental tRNAs plotted as heatmaps .

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Perbase sequence coverage of 30-35 nt reads across parental tRNA sequence. Anticodon positions are demarcated by discontinuous lines (Blue = LIF-treated, Grey = Wnt3+LIF- treated, Red = RA- treated mESCs). Perbase coverage of 30-35 nt reads across the 468 individual parental tRNAs plotted as heatmaps.


Northern blot of candidate tRNAs and tsRNAs tsGlnCTG , tsGlyGCC , tsGluTTC , tsValCAC and tsLysTTT . The black arrows represent the tRNAs and the red arrows mark the tsRNAs .

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Northern blot of candidate tRNAs and tsRNAs tsGlnCTG, tsGlyGCC, tsGluTTC, tsValCAC and tsLysTTT. The black arrows represent the tRNAs and the red arrows mark the tsRNAs.


Northern Blot of 5 ' - tsRNAs ; tsGlyGCC , tsGlnCTG , tsGluTTC , tsLysTTT , tsValCAC at various timepoints of RA - induced mESCs differentiation showing dynamic expression of these 5 ' - tsRNAs . Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates . The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs .

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Northern Blot of 5'-tsRNAs; tsGlyGCC, tsGlnCTG, tsGluTTC, tsLysTTT, tsValCAC at various timepoints of RA-induced mESCs differentiation showing dynamic expression of these 5'-tsRNAs. Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates. The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs.


Effect of ASO - mediated inhibition of 5 ' - tsRNAs on alkaline phosphatase activity in RA - treated mESCs . ( n = 3 ) , Error bars represent SEM , significance was calculated using unpaired t - test .

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Effect of ASO-mediated inhibition of 5'-tsRNAs on alkaline phosphatase activity in RA-treated mESCs. (n = 3), Error bars represent SEM, significance was calculated using unpaired t-test.


Relative expression of stemness markers in RA - induced differentiation mESCs blocked for 5 ' - tsRNA function with ASOs . ( n = 2 ) Error bars represent SEM , significance was calculated using unpaired t - test .

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Relative expression of stemness markers in RA-induced differentiation mESCs blocked for 5'-tsRNA function with ASOs. (n = 2) Error bars represent SEM, significance was calculated using unpaired t-test.


Volcano plot of the transcriptome depicting the overexpressed transcripts ( in green ) and downregulated transcripts ( in pink ) in ASO treated - differentiating cells .

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Volcano plot of the transcriptome depicting the overexpressed transcripts (in green) and downregulated transcripts (in pink) in ASO treated-differentiating cells.


QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5 ' - tsRNA knockdowns using ASOs . ( n = 2 ) Error bars represent SEM , significance was calculated using unpaired t - test .

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QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5'-tsRNA knockdowns using ASOs. (n =2) Error bars represent SEM, significance was calculated using unpaired t-test.


Scatter plot showing Log2 fold - change enrichment of protein interactome between LIF - and RA - treated mESCs . All peptides identified by LC - MS / MS had < 1 % FDR ( Fig EV4E ) . LC - MS / MS was conducted twice ( R2 = 0 . 85 between LIF duplicates and R2 = 0 . 82 between RA - treated mESCs duplicates ) .

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Scatter plot showing Log2 fold-change enrichment of protein interactome between LIF- and RA-treated mESCs. All peptides identified by LC-MS/MS had < 1% FDR (Fig EV4E). LC-MS/MS was conducted twice (R2 = 0.85 between LIF duplicates and R2 = 0.82 between RA-treated mESCs duplicates).


Scatter plot showing Log2 fold - change enrichment of mRNAs associated with tsGlnCTG over Scramble ( Scr1 ) in LIF - versus RA - treated mESCs .

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Scatter plot showing Log2 fold-change enrichment of mRNAs associated with tsGlnCTG over Scramble (Scr1) in LIF- versus RA-treated mESCs.


Bar plots and representative gene lists depicting association of tsGlnCTG with ' pluripotency - associated ' ( D ) and ' differentiation - responsive ' ( D ' ) genes in LIF - or RA - treated mESCs .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_TYPE O

Bar plots and representative gene lists depicting association of tsGlnCTG with 'pluripotency-associated' (D) and 'differentiation-responsive' (D') genes in LIF- or RA-treated mESCs.


In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo ( ASO ) against tsGlnCTG . ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1 . ( n = 2 ) , Error bars represent S . E . M .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo (ASO) against tsGlnCTG. ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1. (n = 2), Error bars represent S.E.M.


Quantification of the Igf2bp1 bound c - Myc mRNA between LIF - versus RA - treated mESCs ( n = 2 ; see Fig . EV5F for duplicate data ) .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O

Quantification of the Igf2bp1 bound c-Myc mRNA between LIF- versus RA-treated mESCs (n = 2; see Fig. EV5F for duplicate data).


Association of c - Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition . ( n = 2 ) , Error bars represent SD , significance was calculated by one - tailed unpaired t - test .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

Association of c-Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition. (n = 2), Error bars represent SD, significance was calculated by one-tailed unpaired t-test.


Relative enrichment of c - Myc mRNA in translating ( 80S and Polysome ) and non - translating ( mRNPs ) pools ( fractionated from polysome profiling ) between ASO - treated and Mock - treated RA - induced differentiating mESCs . ( n = 2 ) Error bars represent SD , significance was calculated using one - tailed unpaired t - test .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O

Relative enrichment of c-Myc mRNA in translating (80S and Polysome) and non-translating (mRNPs) pools (fractionated from polysome profiling) between ASO-treated and Mock-treated RA-induced differentiating mESCs. (n =2) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.


Relative levels of c - Myc mRNA in ASO - treated and Mock - treated RA - induced differentiating mESCs compared to LIF treated mESCs . ( n = 3 ) Error bars represent SD , significance was calculated using one - tailed unpaired t - test .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O

Relative levels of c-Myc mRNA in ASO-treated and Mock-treated RA-induced differentiating mESCs compared to LIF treated mESCs. (n = 3) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.


Epistatic analysis of 5 ' - tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity . ( n = 3 ) , Error bars represent SD , significance was calculated using unpaired t - test .

O O O O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

Epistatic analysis of 5'-tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity. (n = 3), Error bars represent SD, significance was calculated using unpaired t-test.