Figure 4 A IL - 17 and IFN - γ production by ELISA on supernatants of spleen cells from mice with EAE ( EAE ) co - cultured for 3 days with MOG - specific Tr1 cells ( from mice immunized twice with MOG , RA and IL - 2 and amplified in vitro with MOG , RA and IL - 2 for 8 days ) or control T cells from LNs of mice injected with PBS + DMSO ( Veh ) . Bars are mean + SD of one experiment . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by unpaired two - tailed t test . B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG - specific Tr1 cells amplified in vitro with MOG , RA and IL - 2 . Results are mean + SEM ( n = 4 or 6 ) . * p < 0 . 05 and * * p < 0 . 01 by two - way ANOVA with Bonferroni post hoc test . C Representative FACS plots of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Cells were gated as live single CD45 . 2 + CD3 + CD4 + cells . D Mean absolute numbers of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Results are mean + SEM ( n = 6 ) . * p < 0 . 05 by unpaired two - tailed t test . E Representative FACS plots of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Cells were gated as live single CD45 . 1 + CD3 + CD4 + . F Mean percentages of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Results are mean + SEM ( n = 6 ) . * * p < 0 . 01 by unpaired two - tailed t test . G EAE clinical scores of WT and IL - 10 - / - mice immunized s . c . with MOG + RA + IL - 2 , or PBS + DMSO ( Veh ) , 7 and 21 days before induction of EAE . Results are mean + SEM ( n = 6 or 7 ) . * * * p < 0 . 001 , WT MOG + IL - 2 + RA vs WT Veh , # # p < 0 . 01 , # # # p < 0 . 001 , IL - 10 - / - MOG + IL - 2 + RA vs IL - 10 - / - Veh by two - way ANOVA and Tukey post hoc test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 A IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice with EAE (EAE) co-cultured for 3 days with MOG-specific Tr1 cells (from mice immunized twice with MOG, RA and IL-2 and amplified in vitro with MOG, RA and IL-2 for 8 days) or control T cells from LNs of mice injected with PBS + DMSO (Veh). Bars are mean + SD of one experiment. *p<0.05, **p<0.01 and ***p<0.001 by unpaired two-tailed t test.  B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG-specific Tr1 cells amplified in vitro with MOG, RA and IL-2. Results are mean + SEM (n=4 or 6). *p<0.05 and **p<0.01 by two-way ANOVA with Bonferroni post hoc test.  C Representative FACS plots of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Cells were gated as live single CD45.2+CD3+CD4+ cells. D Mean absolute numbers of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Results are mean + SEM (n=6). *p<0.05 by unpaired two-tailed t test.  E Representative FACS plots of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Cells were gated as live single CD45.1+CD3+CD4+. F Mean percentages of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Results are mean + SEM (n=6). **p<0.01 by unpaired two-tailed t test.  G EAE clinical scores of WT and IL-10-/- mice immunized s.c. with MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=6 or 7). ***p <0.001, WT MOG + IL-2 + RA vs WT Veh, ##p<0.01, ###p<0.001, IL-10-/- MOG + IL-2 + RA vs IL-10-/- Veh by two-way ANOVA and Tukey post hoc test.


Figure 5 Retinal inflammatory scores of mice immunized s . c . with PBS + DMSO ( Veh ) , IRBP + DMSO ( IRBP ) or IRBP + RA + IL - 2 21 and 7 days before induction of EAU by s . c . injection of IRBP emulsified in CFA . Bars are mean + SEM ( n = 6 ) , * p < 0 . 05 and * * * p < 0 . 001 , IRBP + IL - 2 + RA vs Veh , # # # p < 0 . 001 IRBP + IL - 2 + RA vs IRBP by two - way ANOVA with Tukey post hoc test . Representative fundus images of each group of mice 35 days after induction of EAU . Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze , where the image appears blurred . Retinal inflammation scores in HEL - TCR transgenic mice were s . c . injected with PBS + DMSO ( Veh ) or HEL + RA + IL - 2 on pn 18 ( 1x ) or on pn 18 and 25 ( 2x ) . Results are mean + SEM ( n = 3 , 4 or 5 ) . * p < 0 . 05 and * * * p < 0 . 001 , HEL + IL - 2 + RA 1x vs Veh , # # p < 0 . 01 HEL + IL - 2 + RA 2x vs Veh by two - way ANOVA with Tukey post hoc test . Representative photographs of each group of retinal fundus of HEL - TCR transgenic mice at pn 35 . Arrowheads indicate representative areas with retinal vasculitis . E Representative FACS plots of IL - 17 + CD4 or IL - 10 + CD4 T cells in the retina on day 36 of HEL - TCR transgenic mice . Cells were gated on live single CD3 + CD4 + and CD3 + CD4 + LAG - 3 + Foxp3 - respectively . F Mean absolute number of IL - 17 + CD4 T cells in the retina ( upper panels ) or mean percentages of IL - 10 + LAG - 3 + Foxp3 - CD4 T cells in the spleens of of HEL - TCR transgenic mice with vasculitis ( lower panels ) . Results are mean + SEM ( n = 3 , 4 or 5 ) . * * p < 0 . 01 and * * * p < 0 . 001 by one way ANOVA with Bonferroni post hoc test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 Retinal inflammatory scores of mice immunized s.c. with PBS + DMSO (Veh), IRBP + DMSO (IRBP) or IRBP + RA + IL-2 21 and 7 days before induction of EAU by s.c. injection of IRBP emulsified in CFA. Bars are mean + SEM (n=6), *p<0.05 and ***p<0.001, IRBP + IL-2 + RA vs Veh, ###p<0.001 IRBP + IL-2 + RA vs IRBP by two-way ANOVA with Tukey post hoc test.  Representative fundus images of each group of mice 35 days after induction of EAU. Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze, where the image appears blurred.  Retinal inflammation scores in HEL-TCR transgenic mice were s.c. injected with PBS + DMSO (Veh) or HEL + RA + IL-2 on pn 18 (1x) or on pn 18 and 25 (2x). Results are mean + SEM (n=3, 4 or 5). *p<0.05 and ***p<0.001, HEL + IL-2 + RA 1x vs Veh, ##p<0.01 HEL + IL-2 + RA 2x vs Veh by two-way ANOVA with Tukey post hoc test.  Representative photographs of each group of retinal fundus of HEL-TCR transgenic mice at pn 35. Arrowheads indicate representative areas with retinal vasculitis.  E Representative FACS plots of IL-17+ CD4 or IL-10+ CD4 T cells in the retina on day 36 of HEL-TCR transgenic mice. Cells were gated on live single CD3+CD4+ and CD3+CD4+LAG-3+Foxp3- respectively.  F Mean absolute number of IL-17+ CD4 T cells in the retina (upper panels) or mean percentages of IL-10+ LAG-3+ Foxp3- CD4 T cells in the spleens of of HEL-TCR transgenic mice with vasculitis (lower panels). Results are mean + SEM (n=3, 4 or 5). **p <0.01 and ***p <0.001 by one way ANOVA with Bonferroni post hoc test.


Figure 1A Domain architecture of S . pombe bromodomain AAA - ATPases . The D1 and D2 ATPase domains ( AAA ) are shaded blue and orange , respectively , and the bromodomain ( BD ) is grey . The percentage similarity to each other and the indicated proteins was determined by FASTA sequence comparison [ 74 ] using the scoring matrix BLOSUM50 . B Venn diagrams showing overlap between genes up - regulated ( 1 . 5 fold ) in abo1Δ mutants with genes up - regulated under the indicated condition , along with the significance of the overlaps ( based on hypergeometric distribution ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Domain architecture of S. pombe bromodomain AAA-ATPases. The D1 and D2 ATPase domains (AAA) are shaded blue and orange, respectively, and the bromodomain (BD) is grey. The percentage similarity to each other and the indicated proteins was determined by FASTA sequence comparison [74] using the scoring matrix BLOSUM50.B Venn diagrams showing overlap between genes up-regulated (1.5 fold) in abo1Δ mutants with genes up-regulated under the indicated condition, along with the significance of the overlaps (based on hypergeometric distribution).


Figure 2A Normalised cumulative nucleosome ( 150 ± 30 bp size class ) position frequency profiles for 4013 S . pombe genes aligned at the transcription start site ( TSS ) plotted from low MNase ( biorep1 ) and high MNase ( biorep 2 ) datasets . P - values ( calculated using a two - tailed unpaired t - test ) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ( " - 4 " nucleosome and " + 4 " nucleosome ) are shown . B Nucleosome position frequency values for the coding regions of 4013 S . pombe genes were k - means clustered ( k = 9 ) using the abo1Δ data from biorep 1 ( low MNase ) and displayed with positive values coloured yellow and other values coloured blue ( left - hand panel ) . The cluster order was then used to display the equivalent wild type frequency values in the right - hand panel . C Level of histone gene mRNAs was determined by qRT - PCR . Data are the mean of four independent biological repeats and error bars are ± SEM . Two - tailed unpaired t - tests showed no significant differences ( P > 0 . 05 ) between wild type and abo1Δ cells . D Whole cell extracts were subjected to western blotting with histone H3 ( Abcam ) , histone H2A ( Abcam ) and tubulin antibodies . Examples of the primary data are shown ( left ) along with a quantification of histone H2A and H3 levels normalized to tubulin ( right ) . Data are the mean of at least three independent repeats and error bars represent ± SEM . P - values were calculated using a two - tailed unpaired t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Normalised cumulative nucleosome (150 ± 30 bp size class) position frequency profiles for 4013 S. pombe genes aligned at the transcription start site (TSS) plotted from low MNase (biorep1) and high MNase (biorep 2) datasets. P-values (calculated using a two-tailed unpaired t-test) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ("-4" nucleosome and "+4" nucleosome) are shown.B Nucleosome position frequency values for the coding regions of 4013 S. pombe genes were k-means clustered (k = 9) using the abo1Δ data from biorep 1 (low MNase) and displayed with positive values coloured yellow and other values coloured blue (left-hand panel). The cluster order was then used to display the equivalent wild type frequency values in the right-hand panel.C Level of histone gene mRNAs was determined by qRT-PCR. Data are the mean of four independent biological repeats and error bars are ±SEM. Two-tailed unpaired t-tests showed no significant differences (P > 0.05) between wild type and abo1Δ cells.D Whole cell extracts were subjected to western blotting with histone H3 (Abcam), histone H2A (Abcam) and tubulin antibodies. Examples of the primary data are shown (left) along with a quantification of histone H2A and H3 levels normalized to tubulin (right). Data are the mean of at least three independent repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test.


Figure 3A Whole cell extracts ( WCE ) were prepared from the indicated strains , immunoprecipitated ( IP ) with anti - GFP antibody and subjected to western blotting with anti - V5 - Pk ( Serotec ) and anti - GFP ( Life Technologies ) antibodies . Data are representative of three independent biological repeats . B As for A . C RNA purified from wild type , abo1Δ and spt16 - 18 cells was analysed by northern blotting using a probe to the 3 ′ end of spbc19c7 . 11 ( top panel ) . RNA ( 5 µg ) used for northern blotting was analysed on an ethidium bromide stained 1 % TAE agarose gel ( bottom panel ) . Data are representative of two independent biological repeats . D RNA purified from wild type , and abo1Δ cells was analysed by strand specific RT - PCR . RNA from hip1Δ cells was analysed as a control . One primer , complementary to either the forward or reverse transcripts , was included during the reverse transcription step the second primer was then added during PCR amplification . Control reactions omitting the reverse transcription step ( - RT ) were included to demonstrate the absence of contaminating genomic DNA . Data are representative of two independent biological repeats . E The indicated strains were subjected to ChIP analysis with anti - GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus . Data are the mean of three independent biological repeats and error bars represent ± SEM . P - values were calculated using a two - tailed unpaired t - test . Significant ( P < 0 . 05 ) values are indicated . F ChIP analysis of histone H3 levels at act1 + was determined by qPCR . Data are the mean of three independent biological repeats and error bars represent ± SEM . P - value was calculated using a two - tailed unpaired t - test . G The indicated strains were subjected to ChIP analysis as described for E . H Log phase cells were subjected to five - fold serial dilution and spotted onto rich ( YE5S ) agar or agar supplemented with MMS ( 0 . 006 % ) . Plates were incubated for 3 - 5 days at 30 ° C or 7 days at 20 ° C . Images are representative of three independent biological repeats .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Whole cell extracts (WCE) were prepared from the indicated strains, immunoprecipitated (IP) with anti-GFP antibody and subjected to western blotting with anti-V5-Pk (Serotec) and anti-GFP (Life Technologies) antibodies. Data are representative of three independent biological repeats.B As for A.C RNA purified from wild type, abo1Δ and spt16-18 cells was analysed by northern blotting using a probe to the 3′ end of spbc19c7.11 (top panel). RNA (5 µg) used for northern blotting was analysed on an ethidium bromide stained 1% TAE agarose gel (bottom panel). Data are representative of two independent biological repeats.D RNA purified from wild type, and abo1Δ cells was analysed by strand specific RT-PCR. RNA from hip1Δ cells was analysed as a control. One primer, complementary to either the forward or reverse transcripts, was included during the reverse transcription step the second primer was then added during PCR amplification. Control reactions omitting the reverse transcription step (-RT) were included to demonstrate the absence of contaminating genomic DNA. Data are representative of two independent biological repeats.E The indicated strains were subjected to ChIP analysis with anti-GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test. Significant (P < 0.05) values are indicated.F ChIP analysis of histone H3 levels at act1+ was determined by qPCR. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-value was calculated using a two-tailed unpaired t-test.G The indicated strains were subjected to ChIP analysis as described for E.H Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with MMS (0.006%). Plates were incubated for 3-5 days at 30°C or 7 days at 20°C. Images are representative of three independent biological repeats.


Figure 4A The position of ura4 + reporter alleles in centromere 1 is shown in the top panel . Strains containing the imr : : ura4 + allele were grown to log phase in YE5S medium , subjected to 5 - fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5 - FOA ( 1 mg / ml ) . Data are representative of three independent biological repeats . B Strains carrying the otr : : ura4 + were analysed as described for A . C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT - PCR . Data is the mean of three independent repeats and error bars indicate ± SEM . P - value was calculated using a two - tailed unpaired t - test . D RNA was purified from the indicated strains and subjected to qRT - PCR for the subtelomeric gene , tlh1 + . Data is the mean of three biological repeats and error bars indicate ± SEM . P - value was calculated using a two - tailed unpaired Student ' s t - test . E The position of insertion of ade6 + reporter allele in the silent mat locus is shown in the top panel . Log phase cells containing the mat3 - M : : ade6 + allele were subjected to five - fold serial dilution and spotted onto YE5S agar plates lacking adenine ( Low Ade ) were incubated for 4 days at 30 ° C . Data are representative of at least three independent biological repeats . Note , microarray analysis indicates that deletion of abo1 + does not influence the expression of ade6 + ( or ura4 + ) when these genes are present at their normal genomic loci .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A The position of ura4+ reporter alleles in centromere 1 is shown in the top panel. Strains containing the imr::ura4+ allele were grown to log phase in YE5S medium, subjected to 5-fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5-FOA (1 mg/ml). Data are representative of three independent biological repeats.B Strains carrying the otr::ura4+ were analysed as described for A.C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT-PCR. Data is the mean of three independent repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired t-test.D RNA was purified from the indicated strains and subjected to qRT-PCR for the subtelomeric gene, tlh1+. Data is the mean of three biological repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired Student's t-test.E The position of insertion of ade6+ reporter allele in the silent mat locus is shown in the top panel. Log phase cells containing the mat3-M::ade6+ allele were subjected to five-fold serial dilution and spotted onto YE5S agar plates lacking adenine (Low Ade) were incubated for 4 days at 30°C. Data are representative of at least three independent biological repeats. Note, microarray analysis indicates that deletion of abo1+ does not influence the expression of ade6+ (or ura4+) when these genes are present at their normal genomic loci.


Figure 5A ChIP analysis was performed on wild type ( untagged ) and abo1 - GFP cells and the resulting DNA analysed by qPCR for centromeric ( dh , dg and imr ) repeat sequences . Data are the mean of four independent biological repeats and error bars represent ± SEM . P - values calculated using a two - tailed unpaired t - test indicates that all loci are significantly enriched ( P < 0 . 05 ) relative the untagged control . B The indicated strains expressing GFP - Swi6 were subjected to ChIP analysis . The level of centromeric ( dg ) repeat sequences relative to the euchromatic control locus , adh1 + was determined by qPCR and scaled to a clr4Δ ( - H3 K9me control ) mutant . Data are the mean of two independent ChIP experiments and error bars represent the range of the data . C ChIP analysis of histone H3 levels at centromeric ( dg ) repeat sequences was determined by qPCR . Data are the mean of three independent ChIP experiments and error bars are ± SEM . P - values were calculated using a two - tailed unpaired t - test . D . ChIP analysis of Abo1 - GFP at centromeric ( dh and imr ) repeat sequences regions in wild type and pob3Δ cells . Data are the mean of three independent experiments and error bars represent ± SEM . P - values , calculated using a two - tailed unpaired t - test , indicated no significant difference ( P > 0 . 05 ) for wild type and pob3Δ cells . E ChIP analysis of Abo1 - GFP at centromeric ( dh and imr ) repeat sequences in wild type and pob3Δ cells . Data are the mean of duplicate experiments and error bars represent the range of the data . F ChIP analysis of Abo1 - GFP over the dh and imr regions in wild type and swi6Δ cells . Data are the mean of three independent experiments and error bars represent ± SEM . P - values , calculated using a two - tailed unpaired t - test , indicated no significant differences ( P > 0 . 05 ) for wild type and swi6Δ cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A ChIP analysis was performed on wild type (untagged) and abo1-GFP cells and the resulting DNA analysed by qPCR for centromeric (dh, dg and imr) repeat sequences. Data are the mean of four independent biological repeats and error bars represent ±SEM. P-values calculated using a two-tailed unpaired t-test indicates that all loci are significantly enriched (P < 0.05) relative the untagged control.B The indicated strains expressing GFP-Swi6 were subjected to ChIP analysis. The level of centromeric (dg) repeat sequences relative to the euchromatic control locus, adh1+ was determined by qPCR and scaled to a clr4Δ (- H3 K9me control) mutant. Data are the mean of two independent ChIP experiments and error bars represent the range of the data.C ChIP analysis of histone H3 levels at centromeric (dg) repeat sequences was determined by qPCR. Data are the mean of three independent ChIP experiments and error bars are ±SEM. P-values were calculated using a two-tailed unpaired t-test.D. ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences regions in wild type and pob3Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant difference (P > 0.05) for wild type and pob3Δ cells.E ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences in wild type and pob3Δ cells. Data are the mean of duplicate experiments and error bars represent the range of the data.F ChIP analysis of Abo1-GFP over the dh and imr regions in wild type and swi6Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant differences (P > 0.05) for wild type and swi6Δ cells.


Figure 6A RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT - PCR , normalised to act1 + mRNA and scaled relative to the wild type level . Data are the mean of three independent biological repeats and error bars represent ± SEM . B Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β - galactosidase assays . Data are the mean of three independent biological repeats and error bars represent ± SEM . C As for BD ChIP DNA samples from wild type ( untagged ) and abo1 - GFP cells were analysed by qPCR for Tf2 LTR . Data are the mean of four independent biological repeats and error bars represent ± SEM . E Normalised cumulative nucleosome ( 150 ± 30 bp ) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase ( biorep1 ) and high MNase ( biorep 2 ) datasets . P - values were calculated using a two - tailed unpaired t - test .

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Figure 6A RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT-PCR, normalised to act1+ mRNA and scaled relative to the wild type level. Data are the mean of three independent biological repeats and error bars represent ±SEM.B Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β-galactosidase assays. Data are the mean of three independent biological repeats and error bars represent ±SEM.C As for BD ChIP DNA samples from wild type (untagged) and abo1-GFP cells were analysed by qPCR for Tf2 LTR. Data are the mean of four independent biological repeats and error bars represent ±SEM.E Normalised cumulative nucleosome (150 ± 30 bp) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase (biorep1) and high MNase (biorep 2) datasets.P-values were calculated using a two-tailed unpaired t-test.


Figure 7A Log phase cells were subjected to five - fold serial dilution and spotted onto rich ( YE5S ) agar or agar supplemented with TBZ ( 15 μg / ml ) and incubated for 3 - 5 days 30 ° C . Data are representative of at three independent biological repeats . B The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored ( red - white ) colonies . At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis . C Heterothallic ( h90 ) strains were cultured on nitrogen limiting ( EMMG ) medium for 3 days at 25 ° C to induce mating and meiosis . The resulting asci were stained with DAPI and visualised by fluorescence microscopy . Data are representative of three biological repeatsD Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4 - 5 days at 30 ° C . Data are the mean of three independent biological repeats and error bars are ± SEM . P - value was calculated using a two - tailed unpaired t - test .

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Figure 7A Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with TBZ (15 μg/ml) and incubated for 3-5 days 30°C. Data are representative of at three independent biological repeats.B The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored (red-white) colonies. At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis.C Heterothallic (h90) strains were cultured on nitrogen limiting (EMMG) medium for 3 days at 25°C to induce mating and meiosis. The resulting asci were stained with DAPI and visualised by fluorescence microscopy. Data are representative of three biological repeatsD Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4-5 days at 30°C. Data are the mean of three independent biological repeats and error bars are ±SEM. P-value was calculated using a two-tailed unpaired t-test.


Figure 1A ) Murine small intestines immunostained for the proliferation marker Ki67 , apoptosis marker cleaved caspase 3 ( Cas3 ) , or YAP from 4 control ( Yapfl / fl Tazfl / fl ) and 4 YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to a moderate increase in apoptosis ( Cas3 + ) in crypt base stem cells , quantified on the right , but no overall effect on the morphology of the small intestine . Arrows point to apoptotic cells . B ) Murine large intestines immunostained for proliferation marker Ki67 , apoptosis marker cleaved caspase 3 ( Cas3 ) , or YAP from control ( Yapfl / fl Tazfl / fl ) and YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to an increased number of apoptotic ( Cas3 + ) cells within the transverse folds of the ascending colon . C ) Murine small intestines immunostained for YAP or cleaved caspase 3 ( Cas3 ) from control ( Villin - CreERt ) and YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to a mild increase in apoptosis ( Cas3 + ) in crypt base stem cells , but no overall effect on the morphology of the small intestine . Arrows point to stem cells ( YAP nuclear staining ) , while arrows point to apoptotic cells ( Cas3 staining ) . N > 10 animals per genotype . D ) Murine large intestines immunostained for YAP or cleaved caspase 3 ( Cas3 ) from control ( Villin - CreERt ) and YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) animals treated with tamoxifen . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to increased apopotosis ( Cas3 + ) and severe morphological defects in the transverse folds of the ascending colon , but does not affect morphology of the remaining colon regions . Quantitatively , 75 % of n = 4 animals showed this severe phenotype .

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Figure 1A) Murine small intestines immunostained for the proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from 4 control (Yapfl/fl Tazfl/fl) and 4 YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a moderate increase in apoptosis (Cas3+) in crypt base stem cells, quantified on the right, but no overall effect on the morphology of the small intestine. Arrows point to apoptotic cells. B) Murine large intestines immunostained for proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from control (Yapfl/fl Tazfl/fl) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to an increased number of apoptotic (Cas3+) cells within the transverse folds of the ascending colon.C) Murine small intestines immunostained for YAP or cleaved caspase 3 (Cas3) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a mild increase in apoptosis (Cas3+) in crypt base stem cells, but no overall effect on the morphology of the small intestine. Arrows point to stem cells (YAP nuclear staining), while arrows point to apoptotic cells (Cas3 staining). N > 10 animals per genotype. D) Murine large intestines immunostained for YAP or cleaved caspase 3 (Cas3 ) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to increased apopotosis (Cas3+) and severe morphological defects in the transverse folds of the ascending colon, but does not affect morphology of the remaining colon regions. Quantitatively, 75% of n = 4 animals showed this severe phenotype.


Figure 2A ) Murine small ( top ) and large ( bottom ) intestines isolated from control ( Cre negative ) Lats1flox / flox Lats2flox / flox animals and Villin - CreERt Lats1flox / flox Lats2flox / flox animals treated with tamoxifen to induce homozygous deletion of Lats1 / 2 ( dKO ) . Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt - villus axis in controls , with nuclear YAP and Ki67 positive cells restricted to the crypt base ( representative images from n = 5 mice ) . Tamoxifen treated ( 3 days i . p . ) Villin - CreERt Lats1flox / flox Lats2flox / flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells . Note the gradient of YAP expression levels is maintained along the crypt - villus axis ( representative images from n = 5 mice for each genotype ) . ( Right ) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base . ( Bottom right ) Q - PCR analysis of Wnt pathway target genes reveals that Lats1 / 2 dKO causes a mild reduction in Lgr5 expression , with complete loss of Olfm4 . B ) Axin2 mRNA expression was measured by RNAscope ( red ) and found to be increased in both intensity and uniformity of staining along the crypt - villus axis in Lats1 / 2 dKO after 3 days post i . p . injection ( dpi ) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2 . At 7dpi , Axin2 mRNA levels remain uniform along the crypt - villus axis in Lats1 / 2 dKO animals , although their total level has declined compared to 3dpi . D ) Q - PCR analysis of YAP - TEAD and their target genes Ctgf and Cyr61 reveals that Lats1 / 2 dKO causes a strong induction of Ctgf and Cyr61 expression , as expected , as well as a strong induction of the common Wnt and YAP target gene CyclinD1 .

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Figure 2A) Murine small (top) and large (bottom) intestines isolated from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt-villus axis in controls, with nuclear YAP and Ki67 positive cells restricted to the crypt base (representative images from n=5 mice). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells. Note the gradient of YAP expression levels is maintained along the crypt-villus axis (representative images from n=5 mice for each genotype). (Right) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base. (Bottom right) Q-PCR analysis of Wnt pathway target genes reveals that Lats1/2 dKO causes a mild reduction in Lgr5 expression, with complete loss of Olfm4.B) Axin2 mRNA expression was measured by RNAscope (red) and found to be increased in both intensity and uniformity of staining along the crypt-villus axis in Lats1/2 dKO after 3 days post i.p. injection (dpi) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2. At 7dpi, Axin2 mRNA levels remain uniform along the crypt-villus axis in Lats1/2 dKO animals, although their total level has declined compared to 3dpi.D) Q-PCR analysis of YAP-TEAD and their target genes Ctgf and Cyr61 reveals that Lats1/2 dKO causes a strong induction of Ctgf and Cyr61 expression, as expected, as well as a strong induction of the common Wnt and YAP target gene CyclinD1.


Figure 3B ) RNAscope in situ hybridisation analysis of TEAD1 / 2 / 4 mRNA expression from control ( Cre negative ) Lats1flox / flox Lats2flox / flox animals and Villin - CreERt Lats1flox / flox Lats2flox / flox animals treated with tamoxifen to induce homozygous deletion of Lats1 / 2 ( dKO ) . Tamoxifen treated ( 3 days i . p . ) Villin - CreERt Lats1flox / flox Lats2flox / flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine ( top ) and colon ( bottom ) . Tissues were harvested and analyzed at day 7 after first tamoxifen injection . ( n = 5 animals for each genotype ) C ) Q - PCR analysis reveals a progressive increase in the YAP - TEAD target gene Ctgf in Lats1 / 2 dKO intestines at 3 - days and 7 - days post i . p . injection with tamoxifen , as well as similarly increased TEAD1 and TEAD4 expression , confirming that TEAD1 and TEAD4 are YAP - responsive genes . Notably , Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1 / 2 deletion , consistent with Axin2 being a known Wnt - specific target gene , and with Wnt signalling remaining active in Lats1 / 2 dKO intestines .

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Figure 3B) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine (top) and colon (bottom). Tissues were harvested and analyzed at day 7 after first tamoxifen injection. (n=5 animals for each genotype)C) Q-PCR analysis reveals a progressive increase in the YAP-TEAD target gene Ctgf in Lats1/2 dKO intestines at 3-days and 7-days post i.p. injection with tamoxifen, as well as similarly increased TEAD1 and TEAD4 expression, confirming that TEAD1 and TEAD4 are YAP-responsive genes. Notably, Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1/2 deletion, consistent with Axin2 being a known Wnt-specific target gene, and with Wnt signalling remaining active in Lats1/2 dKO intestines.


Figure 4A ) RNAscope in situ hybridisation analysis of TEAD1 / 2 / 4 mRNA expression from control ( wild - type ) and ApcMin mutant tumours from the small intestine ( top ) and colon ( bottom ) . All three genes were strongly induced within the ApcMin mutant tumours , with TEAD2 being most strongly induced in colon ApcMin tumours ( n = 3 animals with multiple tumours within the small and large intestines ) . C ) Antibody staining for TEAD4 reveals a gradient of expression along the crypt - villus axis in both the small and large intestine , with uniformly high expression in colorectal adenomas , similar to SOX9 .

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Figure 4A) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (wild-type) and ApcMin mutant tumours from the small intestine (top) and colon (bottom). All three genes were strongly induced within the ApcMin mutant tumours, with TEAD2 being most strongly induced in colon ApcMin tumours (n=3 animals with multiple tumours within the small and large intestines).C) Antibody staining for TEAD4 reveals a gradient of expression along the crypt-villus axis in both the small and large intestine, with uniformly high expression in colorectal adenomas, similar to SOX9.


Figure 5B ) YAP immunostaining of 3 - day old wild type organoids treated with DMSO ( Control ) or Porcupine inhibitor LGK974 at 5µM . Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP levels without affecting YAP subcellular localisation . Quantification of YAP protein levels is shown below , with statistical significance in an unpaired 2 - tailed t - test . C ) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid morphology from budding structures to cystic spheres and increases the YAP protein level . Scale bar is 250µm . n > 10 organoids in each experiment . Quantification of YAP protein levels from crypt and villus regions of control organoids as well as from the regions bounded by rectangles in Apc5 organoids are shown on the right , with statistical significance in an unpaired 2 - tailed t - test .

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Figure 5B) YAP immunostaining of 3-day old wild type organoids treated with DMSO (Control) or Porcupine inhibitor LGK974 at 5µM. Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP levels without affecting YAP subcellular localisation. Quantification of YAP protein levels is shown below, with statistical significance in an unpaired 2-tailed t-test.C) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid morphology from budding structures to cystic spheres and increases the YAP protein level. Scale bar is 250µm. n > 10 organoids in each experiment. Quantification of YAP protein levels from crypt and villus regions of control organoids as well as from the regions bounded by rectangles in Apc5 organoids are shown on the right, with statistical significance in an unpaired 2-tailed t-test.


Figure 6A ) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d . n > 10 organoids in each experiment . B ) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel ( A ) , error bars show 1 SD ( * * * * p < 0 . 0001 ) . n > 10 organoids per condition ( top ) . Quantification of the nuclear / cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel ( C ) . n > 10 organoids per condition ( bottom ) . Statistical analysis was performed with an unpaired student ' s t - test . C C ) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm . n > 10 organoids in each experiment . Q - PCR analysis of the YAP - TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 ( relative expression , error bars = 1 SD ) . Statistical analysis was performed with an unpaired student ' s t - test .

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Figure 6A) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d. n > 10 organoids in each experiment. B) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel (A), error bars show 1 SD (**** p <0.0001). n > 10 organoids per condition (top). Quantification of the nuclear/cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel (C). n > 10 organoids per condition (bottom). Statistical analysis was performed with an unpaired student's t-test. C C) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm. n > 10 organoids in each experiment. Q-PCR analysis of the YAP-TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 (relative expression, error bars = 1 SD). Statistical analysis was performed with an unpaired student's t-test.


Figure 7A ) Mouse intestinal organoids immunostained for YAP ( green ) . Note the mostly cytoplasmic localisation of YAP at 48h in the organoid . Irradiation with 4Gy of X - rays drives strong nuclear localisation of YAP in many cells , which is reversible by treatment with the Src family kinase inhibitor Dasatinib . The Dasatinib treated organoids exhibit increased cell death after irradiation , as indicated by pyknotic nuclei ( white arrow ) . n > 10 organoids in each experiment . This experiment employs the same control batch of 2 - 3d organoids also shown in Figure 6A . B ) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation . Irradiation with 14Gy of X - rays drives strong nuclear localisation of YAP along the entire crypt - villus axis . Deletion of Src within VillinCreERt Srcflox / flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation . Arrows point to villar cells with nuclear YAP localisation . Lower panels show high - mag views of a different example than those shown in upper panels .

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Figure 7A) Mouse intestinal organoids immunostained for YAP (green). Note the mostly cytoplasmic localisation of YAP at 48h in the organoid. Irradiation with 4Gy of X-rays drives strong nuclear localisation of YAP in many cells, which is reversible by treatment with the Src family kinase inhibitor Dasatinib. The Dasatinib treated organoids exhibit increased cell death after irradiation, as indicated by pyknotic nuclei (white arrow). n > 10 organoids in each experiment. This experiment employs the same control batch of 2-3d organoids also shown in Figure 6A.B) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation. Irradiation with 14Gy of X-rays drives strong nuclear localisation of YAP along the entire crypt-villus axis. Deletion of Src within VillinCreERt Srcflox/flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation. Arrows point to villar cells with nuclear YAP localisation. Lower panels show high-mag views of a different example than those shown in upper panels.


Figure 8A ) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA ( black arrows point to mRNA granules in the control ) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes . Nuclear localisation of beta - catenin is readily detectable after irradiation . n = 3 samples per condition , quantification shown in the insets , error bars = 1 SD . Statistical analysis was performed with an unpaired student ' s t - test . Similar results were obtained at 3dpi and 5dpi . B ) Q - PCR analysis of YAP , TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy . Note the strong upregulation of TEAD4 , Ctgf , and Cyr61 . In contrast , Axin2 , TEAD2 and Sox9 mRNA expression levels are only moderately altered , confirming that Wnt signalling remains active upon irradiation - induced YAP - TEAD activation . n = 4 biological replicates per time point . Statistical analysis was performed with unpaired t - test compared to the control 0Gy condition , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C ) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation . Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 ( n = 4 animals in each condition ) . D ) Q - PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation . Note the inhibition of Axin2 , Sox9 , YAP , Ctgf , Cyr61 , and TEAD family expression upon treatment with Porcupine inhibitor LGK974 . The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation ( 1dpi ) becomes more pronounced at 3 days after irradiation ( 3dpi ) . n = 4 biological replicates per condition , statistical analysis was performed with Student ' s 2 - tailed t - test , error bars = 1 SD , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Figure 8A) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA (black arrows point to mRNA granules in the control) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes. Nuclear localisation of beta-catenin is readily detectable after irradiation. n = 3 samples per condition, quantification shown in the insets, error bars = 1 SD. Statistical analysis was performed with an unpaired student's t-test. Similar results were obtained at 3dpi and 5dpi.B) Q-PCR analysis of YAP, TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy. Note the strong upregulation of TEAD4, Ctgf, and Cyr61. In contrast, Axin2, TEAD2 and Sox9 mRNA expression levels are only moderately altered, confirming that Wnt signalling remains active upon irradiation-induced YAP-TEAD activation. n = 4 biological replicates per time point. Statistical analysis was performed with unpaired t-test compared to the control 0Gy condition, * p<0.05, ** p<0.01, ***p<0.001.C) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation. Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 (n= 4 animals in each condition).D) Q-PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation. Note the inhibition of Axin2, Sox9, YAP, Ctgf, Cyr61, and TEAD family expression upon treatment with Porcupine inhibitor LGK974. The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation (1dpi) becomes more pronounced at 3 days after irradiation (3dpi). n = 4 biological replicates per condition, statistical analysis was performed with Student's 2-tailed t-test, error bars = 1 SD, * p<0.05, ** p<0.01, ***p<0.001.


Figure 1Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3 - / - naïve mESCs . DNA counterstain is Hoechst . Dashed white lines indicate the embryonic Gata6 : : mCherry negative part . Scale bar : 10 μm .

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Figure 1Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3-/- naïve mESCs. DNA counterstain is Hoechst. Dashed white lines indicate the embryonic Gata6::mCherry negative part. Scale bar: 10 μm.


Figure 2Nanog > GFP and Gata6 : : mCherry fluorescence intensities of WT and Hdac3 - / - Fraction of Gata6 : : mCherry positive cells in indicated genotypes and conditions after 4d Jak ( i ) blocks LIF , LDN19 BMP4 and PD03 FGF signaling . Average and SD of at least two independent clones . Quantification of spatial lineage segregation in spheroids . Representative spheroid segmentations of indicated genotypes Scale bar : 25 μm .

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Figure 2Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3-/- Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 4d Jak(i) blocks LIF, LDN19 BMP4 and PD03 FGF signaling. Average and SD of at least two independent clones.Quantification of spatial lineage segregation in spheroids. Representative spheroid segmentations of indicated genotypes Scale bar: 25 μm.


Figure 3Scatter plots of log2 fold change ( FC ) gene expression changes ( ∆ mRNA ) between naïve Hdac3 - / - or Dax1 - / - mutants and WT mESCs ( left ) , between Nanog > GFP - expressing Hdac3 - / - or Dax1 - / - mutants and WT cells ( middle ) , and between Gata6 : : mCherry - expressing Hdac3 - / - or Dax1 - / - mutants and Nanog > GFP - expressing WT cells ( right ) . Cluster 5 genes are colored and selected PrE TFs labeled .

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Figure 3Scatter plots of log2 fold change (FC) gene expression changes (∆mRNA) between naïve Hdac3-/- or Dax1-/- mutants and WT mESCs (left), between Nanog>GFP-expressing Hdac3-/- or Dax1-/- mutants and WT cells (middle), and between Gata6::mCherry-expressing Hdac3-/- or Dax1-/- mutants and Nanog>GFP-expressing WT cells (right). Cluster 5 genes are colored and selected PrE TFs labeled.


Figure 4Expression of PrE markers relative to Dax1 - / - cells ( B ) , and fraction of Gata6 : : mCherry positive cells after 3d in SLRA ( C ) in indicated genotypes . Average and SD of three independent clones .

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Figure 4Expression of PrE markers relative to Dax1-/- cells (B), and fraction of Gata6::mCherry positive cells after 3d in SLRA (C) in indicated genotypes. Average and SD of three independent clones.


Figure 5Genome - browser view of the Gata6 locus showing accessibility and chromatin mark changes in indicated mutants , and of ChIPseq signals of indicated TFs and H3K27ac in E6 . 5 VE and EPI . The grey area indicates enh - 45 . FC of PrE and naïve pluripotency markers relative to WT cells in 2iL of indicated genotypes after 3d in SLRA ( D ) and in 2iL ( E ) . Average and SD of at least three independent clones .

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Figure 5Genome-browser view of the Gata6 locus showing accessibility and chromatin mark changes in indicated mutants, and of ChIPseq signals of indicated TFs and H3K27ac in E6.5 VE and EPI. The grey area indicates enh-45.FC of PrE and naïve pluripotency markers relative to WT cells in 2iL of indicated genotypes after 3d in SLRA (D) and in 2iL (E). Average and SD of at least three independent clones.


Figure 1C - E . Logistic statistical model based on the ( C ) single cell sequencing , ( D ) ImageStream ( E ) analysis or automated FISH of RPE - 1 cells in untreated condition ( blue circles ) or treated with Auxin for 48 h ( red squares ) . Error bars represent the SEM based on the number of cells analyzed Dashed lines indicate the means of aneuploidy rates in untreated ( blue line ) or Auxin - treated ( red line ) condition . Red asterisks ( IAA ) and Blue ( Untreated ) indicate significance over the respective mean using a binomial test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . F . Table summarizing whole - chromosome aneuploidy ( fold over the mean ) using the indicated methods to measure chromosome mis - segregation rate . Bold numbers represent a statistically significant difference from the mean for each method . Orange to dark red gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly higher rate compared to the mean level ( weak chromosomes ) . Light green to dark green gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly lower rate compared to the mean ( stronger chromosomes ) . Data obtained with only one method were excluded . * = centromere 4 probe with image stream was reported to lead to non - specific signal ( Worrall et al , 2018 ) . Im . St . = image stream ; Seq = single cell sequencing .

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Figure 1C-E. Logistic statistical model based on the (C) single cell sequencing, (D) ImageStream (E) analysis or automated FISH of RPE-1 cells in untreated condition (blue circles) or treated with Auxin for 48 h (red squares). Error bars represent the SEM based on the number of cells analyzed Dashed lines indicate the means of aneuploidy rates in untreated (blue line) or Auxin-treated (red line) condition. Red asterisks (IAA) and Blue (Untreated) indicate significance over the respective mean using a binomial test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.F. Table summarizing whole-chromosome aneuploidy (fold over the mean) using the indicated methods to measure chromosome mis-segregation rate. Bold numbers represent a statistically significant difference from the mean for each method. Orange to dark red gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly higher rate compared to the mean level (weak chromosomes). Light green to dark green gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly lower rate compared to the mean (stronger chromosomes). Data obtained with only one method were excluded. * = centromere 4 probe with image stream was reported to lead to non-specific signal (Worrall et al, 2018). Im. St. = image stream; Seq = single cell sequencing.


Figure 2B - C . ( B ) Micronuclei frequency formation by live cell imaging in untreated ( NT ) and Auxin - treated ( IAA ) condition . ( C ) Bar graph represents the type of chromosome mis - segregation ( independently of the dCas9 signal ) observed in the indicated conditions , with ( IAA ) or without ( NT ) auxin , in the cell lines expressing dCas9 mScarlet - I and sgRNA targeting chromosome 1 or 3 . Error bars represent the SEM of four independent experiments in which cells labeled for chromosome 1 and 3 were analyzed together ( n = 45 - 149 cells ) . D . Representative live cell imaging of RPE - 1 cells dCas9 mScarlet - I with sgRNA targeting chromosome 1 ( red dots ) starting from metaphase and showing example of correct ( untreated ) or mis - aligned chromosome ( auxin ) leading to the formation of a micronucleus . Yellow arrows mark mis - aligned - and micronucleus - containing chromosome 1 . Cells were imaged every five minutes . The numbers indicate the time point at which the presented images were taken . Cells were fixed at the end of the movie to detect Cas9 with an antibody . Scale bar represents 10 µm . E . Bar graph representing the proportion of chromosome mis - segregation observed in both cell lines after auxin addition . Only movies in which the dCas9 mScarlet - I dots were clearly visible during the whole division were taken into consideration for the analysis ( N = 6 - 10 cells ) . F . Representative images of mitotic errors leading to aneuploidy in fixed cells after CENP - A depletion ( 28 h auxin ) . Chromosomes are stained using whole ( X ) or centromeric ( 6 ) FISH probes . Yellow arrows mark lagging chromosomes . Scale bar represents 5 µm . G - H . Bars represent the ( G ) proportion of chromosome mis - segregation and ( H ) the type of the different chromosome mis - segregations observed . Error bars represent the SEM of two replicates . n > 66 mis - segregation events . Paired t test lagging chromosomes vs mis - alignment chromosomes , * p = 0 . 0275 .

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Figure 2B-C. (B) Micronuclei frequency formation by live cell imaging in untreated (NT) and Auxin-treated (IAA) condition. (C) Bar graph represents the type of chromosome mis-segregation (independently of the dCas9 signal) observed in the indicated conditions, with (IAA) or without (NT) auxin, in the cell lines expressing dCas9 mScarlet-I and sgRNA targeting chromosome 1 or 3. Error bars represent the SEM of four independent experiments in which cells labeled for chromosome 1 and 3 were analyzed together (n = 45 - 149 cells).D. Representative live cell imaging of RPE-1 cells dCas9 mScarlet-I with sgRNA targeting chromosome 1 (red dots) starting from metaphase and showing example of correct (untreated) or mis-aligned chromosome (auxin) leading to the formation of a micronucleus. Yellow arrows mark mis-aligned- and micronucleus-containing chromosome 1. Cells were imaged every five minutes. The numbers indicate the time point at which the presented images were taken. Cells were fixed at the end of the movie to detect Cas9 with an antibody. Scale bar represents 10 µm.E. Bar graph representing the proportion of chromosome mis-segregation observed in both cell lines after auxin addition. Only movies in which the dCas9 mScarlet-I dots were clearly visible during the whole division were taken into consideration for the analysis (N = 6 - 10 cells).F. Representative images of mitotic errors leading to aneuploidy in fixed cells after CENP-A depletion (28 h auxin). Chromosomes are stained using whole (X) or centromeric (6) FISH probes. Yellow arrows mark lagging chromosomes. Scale bar represents 5 µm. G-H. Bars represent the (G) proportion of chromosome mis-segregation and (H) the type of the different chromosome mis-segregations observed. Error bars represent the SEM of two replicates. n > 66 mis-segregation events. Paired t test lagging chromosomes vs mis-alignment chromosomes, *p = 0.0275.


Figure 3Bar plot showing the mean of ( B ) centromere length ( n = 4 ) as determined by whole genome sequencing . Error bars represent the SEM of four independent experiments . Acrocentric chromosomes 13 , 14 , 21 and 22 were marked by a line as we could not assign the respective reads . Dashed lines indicate the mean . Bars were labeled with asterisks according to the significance of their difference from the mean ( t - test ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Bars represent the sum of the lengthBar plot showing the mean of ( C ) CENP - B boxes counts ( n = 4 ) as determined by whole genome sequencing . Error bars represent the SEM of four independent experiments . Acrocentric chromosomes 13 , 14 , 21 and 22 were marked by a line as we could not assign the respective reads . Dashed lines indicate the mean . Bars were labeled with asterisks according to the significance of their difference from the mean ( t - test ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Bars represent the sum of counts of different HOR arrays CENP - B boxes counts are normalized to the average number of mapped reads in each replicate . D . Schematic using representative images of the mFISH labeling followed by CENP - B box FISH method used to identify and quantify centromere specific CENP - B boxes signal in ( E ) . Scale bar represents 10 µm . E . Box and whisker plots of normalized CENP - B boxes intensity over the mean on metaphase spread from three independent experiments ( n > 50 cells ) using the Tukey plot . One - way ANOVA with post - hoc Tukey ' s multiple comparison test shows high diversity between chromosomes . t - test against the mean was used to estimate the statistical significance for each chromosome . * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . . Scatter plot showing a non - significant or significant negative correlation between the mean of ( F ) centromere length ( n = 4 ) and mis - segregation rate ( from Fig . 1F ) ( r = Spearman rank coefficient ) . Lines represent the linear regression with a 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis . Scatter plot showing a non - significant or significant negative correlation between the mean of ( G ) centromere CENP - B boxes FISH signal ( n > 50 cells ) and mis - segregation rate , respectively ( r = Spearman rank coefficient ) . Lines represent the linear regression with a 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis .

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Figure 3Bar plot showing the mean of (B) centromere length (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of the lengthBar plot showing the mean of (C) CENP-B boxes counts (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of counts of different HOR arrays CENP-B boxes counts are normalized to the average number of mapped reads in each replicate.D. Schematic using representative images of the mFISH labeling followed by CENP-B box FISH method used to identify and quantify centromere specific CENP-B boxes signal in (E). Scale bar represents 10 µm.E. Box and whisker plots of normalized CENP-B boxes intensity over the mean on metaphase spread from three independent experiments (n>50 cells) using the Tukey plot. One-way ANOVA with post-hoc Tukey's multiple comparison test shows high diversity between chromosomes. t-test against the mean was used to estimate the statistical significance for each chromosome. **p < 0.01; ***p < 0.001; ****p < 0.0001..Scatter plot showing a non-significant or significant negative correlation between the mean of (F) centromere length (n = 4) and mis-segregation rate (from Fig. 1F) (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis.Scatter plot showing a non-significant or significant negative correlation between the mean of (G) centromere CENP-B boxes FISH signal (n>50 cells) and mis-segregation rate, respectively (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis.


Figure 4B . Representative immunofluorescence FISH ( IF - FISH ) images to measure CENP - B intensity at specific chromosome in untreated cells . Scale bar represents 5 µm . C . Bar plot showing the normalized CENP - B fluorescence intensity at every chromosome over the mean on each metaphase spread ( n > 37 per chromosome ) ± SEM . Acrocentric chromosomes 13 , 14 , 21 and 22 were not analyzed and are marked by a line . One - way ANOVA with post - hoc Tukey ' s multiple comparison test shows high diversity between chromosomes . t - test against the mean was used to estimate the statistical significance for each chromosome . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . D . Scatter plot showing a significant negative correlation between the mean of CENP - B IF - FISH signal ( n > 37 ) and the mis - segregation rate ( r = Spearman rank coefficient ) . The lines represent linear regression with 95 % confidence band . E - F . Bar graphs report the sum of the normalized reads counts of different HOR arrays representing CENP - C binding following CUT & RUN , sequencing and centromere mapping . Cells were treated for 6 h with IAA to deplete CENP - A . Error bars represent the SEM of three independent experiments and the dashed line represents the mean . Acrocentric chromosomes 13 - 14 , 21 and 22 were marked by a line . t - test against the mean was used to estimate the statistical significance for each chromosome . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . G . Scatter plot showing a significant positive correlation between the mean of CENP - C reads ( n = 3 ) and CENP - B reads ( n = 3 ) ( r = Spearman rank coefficient ) . Data from chr 1 , 5 and 19 were excluded from the analysis in addition to chr 13 , 14 , 21 and 22 to better assess correlation without the FISH correction The lines represent linear regression with a 95 % confidence interval . H . Scatter plot showing a significant negative correlation between the mean of CENP - C reads ( n = 3 ) and the mis - segregation rate ( r = Spearman rank coefficient ) . The lines represent linear regression with 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis . The lines represent linear regression with a 95 % confidence interval .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. Representative immunofluorescence FISH (IF-FISH) images to measure CENP-B intensity at specific chromosome in untreated cells. Scale bar represents 5 µm.C. Bar plot showing the normalized CENP-B fluorescence intensity at every chromosome over the mean on each metaphase spread (n > 37 per chromosome) ± SEM. Acrocentric chromosomes 13, 14, 21 and 22 were not analyzed and are marked by a line. One-way ANOVA with post-hoc Tukey's multiple comparison test shows high diversity between chromosomes. t-test against the mean was used to estimate the statistical significance for each chromosome. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.D. Scatter plot showing a significant negative correlation between the mean of CENP-B IF-FISH signal (n > 37) and the mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence band.E-F. Bar graphs report the sum of the normalized reads counts of different HOR arrays representing CENP-C binding following CUT&RUN, sequencing and centromere mapping. Cells were treated for 6 h with IAA to deplete CENP-A. Error bars represent the SEM of three independent experiments and the dashed line represents the mean. Acrocentric chromosomes 13-14, 21 and 22 were marked by a line. t-test against the mean was used to estimate the statistical significance for each chromosome. *p < 0.05; **p < 0.01; ***p < 0.001.G. Scatter plot showing a significant positive correlation between the mean of CENP-C reads (n = 3) and CENP-B reads (n = 3) (r = Spearman rank coefficient). Data from chr 1, 5 and 19 were excluded from the analysis in addition to chr 13, 14, 21 and 22 to better assess correlation without the FISH correction The lines represent linear regression with a 95% confidence interval.H. Scatter plot showing a significant negative correlation between the mean of CENP-C reads (n = 3) and the mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis. The lines represent linear regression with a 95% confidence interval.


Figure 5B . Representative image of centromere 11 ( red ) and centromere X ( green ) FISH on RPE - 1 CENP - AAID CENP - B KO mutant in control ( top ) or IAA treated condition ( bottom ) . Scale bar represents 5 µm . Bar graphs show automatic FISH quantification of chromosome mis - segregation in the indicated cell lines with and without IAA addition for 24 h . Error bars represent the SEM of three independent experiments ( n > 200 cells per experiment ) . Dunnett ' s multiple comparisons test was used to compare conditions . Grey lines separate independent experiments . Fold changes between grey and red bars are also indicated . C . Schematic of the centromere strength model with the microtubule destabilizer BAL27862 compound . D . Representative image of centromere 17 ( red ) and centromere X ( green ) FISH on RPE - 1 cells treated with the BAL27862 compound . Scale bar represents 5 µm . E . Bars represent the frequency of chromosome mis - segregation into micronuclei after 28 h treatment with the BAL27862 drug at indicated concentration . Error bars represent the SEM of three independent experiments , dashed line represents the mean . n > 40 micronuclei were analyzed . Dunnett ' s multiple comparisons test , * : p < 0 . 05 . DMSO was used at the same concentration of BAL27862 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B. Representative image of centromere 11 (red) and centromere X (green) FISH on RPE-1 CENP-AAID CENP-B KO mutant in control (top) or IAA treated condition (bottom). Scale bar represents 5 µm. Bar graphs show automatic FISH quantification of chromosome mis-segregation in the indicated cell lines with and without IAA addition for 24 h. Error bars represent the SEM of three independent experiments (n> 200 cells per experiment). Dunnett's multiple comparisons test was used to compare conditions. Grey lines separate independent experiments. Fold changes between grey and red bars are also indicated.C. Schematic of the centromere strength model with the microtubule destabilizer BAL27862 compound. D. Representative image of centromere 17 (red) and centromere X (green) FISH on RPE-1 cells treated with the BAL27862 compound. Scale bar represents 5 µm. E. Bars represent the frequency of chromosome mis-segregation into micronuclei after 28 h treatment with the BAL27862 drug at indicated concentration. Error bars represent the SEM of three independent experiments, dashed line represents the mean. n > 40 micronuclei were analyzed. Dunnett's multiple comparisons test, *: p<0.05. DMSO was used at the same concentration of BAL27862.


Figure 6A . Representative image of CENP - B box FISH and mFISH stained RPE - 1 metaphase spread cells . Chromosome 3 homologs are indicated by white arrows with single CENP - B box chromosomes signal magnified in insets . Scale bar represents 5 µm . B . Representative image of CENP - B box ( red ) and centromere 3 ( green ) FISH on RPE - 1 interphase nucleus after 48 h IAA treatment . CENP - B box signal at individual homologs are magnified in insets . Scale bar represents 5 µm . C . Bar plots represent the quantification of the CENP - B box FISH intensity at the chromosome 3 homolog contained in the main nucleus versus the homolog present in the micronucleus Error bars represent the SEM of three independent experiments . n = 69 cells . Unpaired t test , * * * * p < 0 . 0001 . E . Immunoblot shows expression of the ( ΔN ) CENP - B - dCas9 after doxycycline induction . F . Representative images show a micronucleus or the main nucleus containing the Y chromosome ( red ) detected by FISH in no - doxycycline or doxycycline treated cells respectively . Chromosome X is shown in green . Scale bar represents 5 µm . G . Bar plot shows the mean ( n ≥ 3 ) frequency of micronuclei containing the chromosome Y or chromosome X ± doxycycline treatment for 72 h and IAA for 48 h ± SEM . n > 200 cells with a micronucleus . Mann - Whitney test . * p = 0 . 0173 .

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Figure 6A. Representative image of CENP-B box FISH and mFISH stained RPE-1 metaphase spread cells. Chromosome 3 homologs are indicated by white arrows with single CENP-B box chromosomes signal magnified in insets. Scale bar represents 5 µm.B. Representative image of CENP-B box (red) and centromere 3 (green) FISH on RPE-1 interphase nucleus after 48 h IAA treatment. CENP-B box signal at individual homologs are magnified in insets. Scale bar represents 5 µm.C. Bar plots represent the quantification of the CENP-B box FISH intensity at the chromosome 3 homolog contained in the main nucleus versus the homolog present in the micronucleus Error bars represent the SEM of three independent experiments. n = 69 cells. Unpaired t test, ****p < 0.0001.E. Immunoblot shows expression of the (ΔN)CENP-B-dCas9 after doxycycline induction.F. Representative images show a micronucleus or the main nucleus containing the Y chromosome (red) detected by FISH in no-doxycycline or doxycycline treated cells respectively. Chromosome X is shown in green. Scale bar represents 5 µm.G. Bar plot shows the mean (n ≥ 3) frequency of micronuclei containing the chromosome Y or chromosome X ± doxycycline treatment for 72 h and IAA for 48 h ± SEM. n > 200 cells with a micronucleus. Mann-Whitney test. *p=0.0173.


Figure 7B . Table summarizing whole - chromosome aneuploidy ( fold over the mean ) using the indicated methods to measure chromosome mis - segregation rate . Bold numbers represent significant statistical difference from the mean for each method . Orange to dark red gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly higher rate compared to the mean level ( weak chromosomes ) . Light green to dark green gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly lower rate compared to the mean ( stronger chromosomes ) . Data obtained with only one method were excluded . Im . St . = image stream . C . Plot shows the karyotype distribution of DLD - 1 and RPE - 1 cells in untreated or Auxin - treated conditionE . Representative image of a chromosome spreads in DLD - 1 cells following mFISH and subsequently CENP - B boxes FISH . Inset shows the supernumerary ( SN ) chromosome . Scale bar represents 10 µm . F . Bar plot shows the mean variation in CENP - B boxes in DLD - 1 cells from data from E ( n = 10 cells ) and whole - genome sequencing and centromere mapping ± SD . G - H . Scatter plot showing a significant positive correlation between the indicated features in DLD - 1 cells ( n = 1 ) . The lines represent linear regression with 95 % confidence band . Data from chr 13 , 14 , 21 , 22 , Y and SN were excluded from the analysis . J . Bars show frequency of micronuclei with or without siRNA treatment . Error bars represent the SEM of three independent experiments ( n > 170 cells per replicate ) . K . ( Left ) Representative image of CENP - B box ( red ) staining after siRNA against CENP - A / HJURP for 48 h . CENP - B box signals at individual micronuclei are magnified in insets . Scale bar represent 5 µm . ( Right ) Bar graph represents the mean of the CENP - B box FISH intensity contained in the main nucleus versus the one present in the micronucleus ± SEM . Color dots represent independent experiments . ( n > 43 micronuclei and 840 main nucleus ) Mann - Whitney U test : * * * p = 0 . 0001 .

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Figure 7B. Table summarizing whole-chromosome aneuploidy (fold over the mean) using the indicated methods to measure chromosome mis-segregation rate. Bold numbers represent significant statistical difference from the mean for each method. Orange to dark red gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly higher rate compared to the mean level (weak chromosomes). Light green to dark green gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly lower rate compared to the mean (stronger chromosomes). Data obtained with only one method were excluded. Im. St. = image stream.C. Plot shows the karyotype distribution of DLD-1 and RPE-1 cells in untreated or Auxin-treated conditionE. Representative image of a chromosome spreads in DLD-1 cells following mFISH and subsequently CENP-B boxes FISH. Inset shows the supernumerary (SN) chromosome. Scale bar represents 10 µm. F. Bar plot shows the mean variation in CENP-B boxes in DLD-1 cells from data from E (n = 10 cells) and whole-genome sequencing and centromere mapping ± SD. G-H. Scatter plot showing a significant positive correlation between the indicated features in DLD-1 cells (n = 1). The lines represent linear regression with 95% confidence band. Data from chr 13, 14, 21, 22, Y and SN were excluded from the analysis.J. Bars show frequency of micronuclei with or without siRNA treatment. Error bars represent the SEM of three independent experiments (n > 170 cells per replicate).K. (Left) Representative image of CENP-B box (red) staining after siRNA against CENP-A/HJURP for 48 h. CENP-B box signals at individual micronuclei are magnified in insets. Scale bar represent 5 µm. (Right) Bar graph represents the mean of the CENP-B box FISH intensity contained in the main nucleus versus the one present in the micronucleus ± SEM. Color dots represent independent experiments. (n > 43 micronuclei and 840 main nucleus) Mann-Whitney U test: ***p = 0.0001.


Figure 8A - B . Scatter plot showing ( A ) a significant positive or ( B ) a negative correlation between chromosome length and the mis - segregation rate or CENP - C ( n = 3 ) reads over chromosome length and mis - segregation rate ( r = Spearman rank coefficient ) . The lines represent linear regression with 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis . Numbers indicate chromosomes with low or high mis - segregation frequency .

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Figure 8A-B. Scatter plot showing (A) a significant positive or (B) a negative correlation between chromosome length and the mis-segregation rate or CENP-C (n = 3) reads over chromosome length and mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis. Numbers indicate chromosomes with low or high mis-segregation frequency.


Figure 1A - F ( A , B , D - F ) RNA levels determined by RT - qPCR . Results are presented as mean ± SD , n = 10 . P - values are indicated on the top of each plot . ( C ) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner . Spearman correlation coefficient , P - values , and sample numbers are marked on the top left of the plot .

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Figure 1A-F (A, B, D-F) RNA levels determined by RT-qPCR. Results are presented as mean ± SD,n = 10. P-values are indicated on the top of each plot. (C) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner. Spearman correlation coefficient, P-values, and sample numbers are marked on the top left of the plot.


Figure 2A - D Endometrial stromal cells from patients # 166 and # 80 were each transfected with a mixture of siCon ( control siRNA ) and microRNA inhibitor control ( iCon ) , siH19 and iCon , or siH19 and iLet7 ( let - 7 - specific inhibitor ) . RNA and proteins were analyzed at 48 h post - transfection . Combined results from the two patient cells are presented . A - D Endometrial stromal cells from patients # 166 and # 80 were each transfected with a mixture of siCon ( control siRNA ) and microRNA inhibitor control ( iCon ) , siH19 and iCon , or siH19 and iLet7 ( let - 7 - specific inhibitor ) . Western blot gels from # 166 cells are shown in ( C ) . Quantitation of Western blots combining both patient cells are shown in ( D ) . E - H Endometrial stromal cells from patients # 98 and # 212S were each transfected with empty vector or pH19 . RNA and proteins were analyzed 48 h post - transfection . Combined results from the two patient cells are presented . E - H Endometrial stromal cells from patients # 98 and # 212S were each transfected with empty vector or pH19 . RNA and proteins were analyzed 48 h post - transfection . Combined results from the two patient cells are presented . Western blot gels from # 98 cells are shown in ( G ) . Quantitation of Western blots combining both patient cells are shown in ( H ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). RNA and proteins were analyzed at 48 h post-transfection. Combined results from the two patient cells are presented.A-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). Western blot gels from #166 cells are shown in (C). Quantitation of Western blots combining both patient cells are shown in (D).E-H Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented.E-H Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented. Western blot gels from #98 cells are shown in (G). Quantitation of Western blots combining both patient cells are shown in (H).