Figure 1Sites that bound either HIF - 1α ( A & B ) or HIF - 2α ( C & D ) in both replicates were identified by the MACS peak caller and ordered on the y - axis according to HIF - 1β signal intensity . Heatmaps show HIF ChIP - seq signal ( read counts per million mapped reads , CPM ; expressed as colour intensity , averaged across two independent ChIP - seq experiments ) at HIF binding sites and across the flanking ± 5kb regions ( x - axis ) . ( A ) HIF - 1β signal intensity and ( B ) HIF - 1α signal intensity at HIF - 1α binding sites and ( C ) HIF - 1β signal intensity and ( D ) HIF - 2α signal intensity at HIF - 2α binding sites . HIF - 1β signal intensity above local background levels is observed at all HIF - 1α and HIF - 2α binding sites . ( E & F ) Line plots showing average ( n = 2 , independent ChIP - seq experiments ) HIF - 1β signal intensity ( solid blue lines ) within the MACS defined ( E ) HIF - 1α and ( F ) HIF - 2α binding sites compared to the average background HIF - 1β binding signal ( dashed black line ) at non - HIF - α - binding accessible sites ( defined by FAIRE - seq ) . Sites are ranked on the x - axis according to HIF - 1β signal . HIF - 1β signal intensity at both HIF - 1α and HIF - 2α binding sites was consistently above genome - wide background levels . ( G ) HIF - 1β signal intensity was plotted against total HIF - α ( HIF - 1α + HIF - 2α ) signal intensity for all sites that bound one or more HIF subunits . Strong correlation was observed between HIF - α and HIF - 1β signal intensities . ( H ) The ratio of total HIF - α signal to HIF - 1β signal ( x - axis ) was determined for each site and plotted as a frequency distribution ( y - axis ) showing a tight unimodal distribution . ( I ) The non - reproducibility of the total HIF - α / HIF - 1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Sites that bound either HIF-1α (A & B) or HIF-2α (C&D) in both replicates were identified by the MACS peak caller and ordered on the y-axis according to HIF-1β signal intensity. Heatmaps show HIF ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity, averaged across two independent ChIP-seq experiments) at HIF binding sites and across the flanking ±5kb regions (x-axis). (A) HIF-1β signal intensity and (B) HIF-1α signal intensity at HIF-1α binding sites and (C) HIF-1β signal intensity and (D) HIF-2α signal intensity at HIF-2α binding sites. HIF-1β signal intensity above local background levels is observed at all HIF-1α and HIF-2α binding sites. (E & F) Line plots showing average (n = 2, independent ChIP-seq experiments) HIF-1β signal intensity (solid blue lines) within the MACS defined (E) HIF-1α and (F) HIF-2α binding sites compared to the average background HIF-1β binding signal (dashed black line) at non-HIF-α-binding accessible sites (defined by FAIRE-seq). Sites are ranked on the x-axis according to HIF-1β signal. HIF-1β signal intensity at both HIF-1α and HIF-2α binding sites was consistently above genome-wide background levels. (G) HIF-1β signal intensity was plotted against total HIF-α (HIF-1α + HIF-2α) signal intensity for all sites that bound one or more HIF subunits. Strong correlation was observed between HIF-α and HIF-1β signal intensities. (H) The ratio of total HIF-α signal to HIF-1β signal (x-axis) was determined for each site and plotted as a frequency distribution (y-axis) showing a tight unimodal distribution. (I) The non-reproducibility of the total HIF-α/HIF-1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate 2


Figure 2For each cell line , genes were ranked according to hypoxic induction ( 0 . 5 % hypoxia for 16 hours ) in RNA - seq analyses ( n = 3 ) . Enrichment of genes ( TSS ) closest to each canonical HIF - α / β binding site ( i . e . identified in both HIF - 1α ( or HIF - 2α ) and both HIF - 1β ChIP - seq experiments ) was then examined using Gene Set Enrichment Analysis ( ES = enrichment score , NES = normalized enrichment score )

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

Figure 2For each cell line, genes were ranked according to hypoxic induction (0.5% hypoxia for 16 hours) in RNA-seq analyses (n = 3). Enrichment of genes (TSS) closest to each canonical HIF-α/β binding site (i.e. identified in both HIF-1α (or HIF-2α) and both HIF-1β ChIP-seq experiments) was then examined using Gene Set Enrichment Analysis (ES = enrichment score, NES = normalized enrichment score)


Figure 3Immunoblots show progressive induction of HIF - 1α and HIF - 2α protein levels with increasing severity of hypoxiaHIF - 1α ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for all canonical HIF - binding sites that bound at one oxygen concentration or the other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen and no new sites were generated ( C ) HIF - 2α and ( D ) HIF - 1β ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for all canonical HIF - binding sites that bound at one oxygen concentration or the other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen and no new sites were generatedFrequency distribution of ChIP - seq signals at 0 . 5 % oxygen compared to 3 % oxygen . The ratio of total HIF - α signal was unimodally distributed ( purple line ) . Upper tertile HIF - α sites ( solid red line ) had a significantly ( p = 10 - 7 , Wilcox rank sum test ) higher ratio of HIF - 1β signal ( dotted red line ) and vice versa ( blue lines ) The ratio of HIF - 1α to HIF - 2α ChIP - seq signal for all canonical HIF - binding sites at 0 . 5 % oxygen was plotted against that at 3 % oxygen . Strong correlation in the HIF - 1α to HIF - 2α ratio was observed between the two oxygen

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

Figure 3Immunoblots show progressive induction of HIF-1α and HIF-2α protein levels with increasing severity of hypoxiaHIF-1α ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated(C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generatedFrequency distribution of ChIP-seq signals at 0.5% oxygen compared to 3% oxygen. The ratio of total HIF-α signal was unimodally distributed (purple line). Upper tertile HIF-α sites (solid red line) had a significantly (p = 10-7, Wilcox rank sum test) higher ratio of HIF-1β signal (dotted red line) and vice versa (blue lines)The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at 0.5% oxygen was plotted against that at 3% oxygen. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the two oxygen concentrations


Figure 4Immunoblots show induction of HIF - 1α and HIF - 2α protein level , which is highest at 6 hours and falls by 48 hoursHIF - 1α , ( C ) HIF - 2α and ( D ) HIF - 1β ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 16 hours is plotted against that at 6 hours for all canonical HIF - binding sites that bound at any of the three timepoints . ChIP - seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia , but correlated well between the two timepoints and no novel sites were observed at either timepoint . ( E - F ) The same plots comparing ( E ) HIF - 1α , ( F ) HIF - 2α and ( G ) HIF - 1β ChIP - seq signals at 48 hours and 6 hours hypoxia . ChIP - seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either timepointFrequency distribution of ChIP - seq signals at 48 hours compared to 6 hours hypoxia . The ratio of total HIF - α signal had a unimodal distribution ( purple line ) . However , upper tertile HIF - α sites ( solid red line ) had a comparable ratio of HIF - 1β signal ( dotted red line ) to the ratio of HIF - 1β signal in the lower tertile HIF - α sites ( blue lines ) suggesting that differences in HIF - α signal resulted from random variation rather than a true biological differenceThe ratio of HIF - 1α to HIF - 2α ChIP - seq signal for all canonical HIF - binding sites at ( I ) 16 hours hypoxia and at ( J ) 48 hours hypoxia was plotted against that at 6 hours hypoxia . Strong correlation in the HIF - 1α to HIF - 2α ratio was observed between the various

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 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 O O O O O O O O O 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 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

Figure 4Immunoblots show induction of HIF-1α and HIF-2α protein level, which is highest at 6 hours and falls by 48 hoursHIF-1α, (C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 16 hours is plotted against that at 6 hours for all canonical HIF-binding sites that bound at any of the three timepoints. ChIP-seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia, but correlated well between the two timepoints and no novel sites were observed at either timepoint. (E-F) The same plots comparing (E) HIF-1α, (F) HIF-2α and (G) HIF-1β ChIP-seq signals at 48 hours and 6 hours hypoxia. ChIP-seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either timepointFrequency distribution of ChIP-seq signals at 48 hours compared to 6 hours hypoxia. The ratio of total HIF-α signal had a unimodal distribution (purple line). However, upper tertile HIF-α sites (solid red line) had a comparable ratio of HIF-1β signal (dotted red line) to the ratio of HIF-1β signal in the lower tertile HIF-α sites (blue lines) suggesting that differences in HIF-α signal resulted from random variation rather than a true biological differenceThe ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at (I) 16 hours hypoxia and at (J) 48 hours hypoxia was plotted against that at 6 hours hypoxia. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the various timepoints


Figure 5Cells were incubated in 0 . 5 % ambient oxygen for 16 hours . Immunoblots show HIF - 1α , HIF - 2α and HIF - 1β protein levels in the wild type and single clones of CRISPR - Cas9 engineered cell linesHIF - 1α ChIP - seq signal in the HIF - 2α KO cells was plotted against that in the wild - type cells for all canonical HIF - 1 binding sites in either cell typeHIF - 2α ChIP - seq signal in the HIF - 1α KO cells was plotted against that in the wild - type cells for all canonical HIF - 2 binding sites in either cell type . No significant increase ( Wilcoxon rank sum test ) in binding of either isoform was observed following deletion of the otherBox - and - whisker plots showing : ( D ) The effect of HIF - 2α inactivation on HIF - 1α ChIP - seq signal at sites that specifically bound HIF - 2α , but not HIF - 1α in wild - type cells showing a slight , but significant ( p = 4 x 10 - 5 , Wilcoxon signed rank test ) decrease rather than increase ( compare red boxes ) consistent with the reduction in HIF - 1α protein observed in panel AThe effect of HIF - 1α inactivation on HIF - 2α ChIP - seq signal ( compare blue boxes ) at sites that specifically bound HIF - 1α , but not HIF - 2α in wild - type cells showing no significant effect ( p = 0 . 5 , Wilcoxon signed rank test ) The effect , at sites that bind both isoforms in wild - type cells , of ( F ) HIF - 2α inactivation on HIF - 1α binding intensity showing a similar small but significant reduction rather than increase ( p = 2 x 10 - 16 , Wilcoxon Signed rank test ) as abovThe effect , at sites that bind both isoforms in wild - type cells , o HIF - 1α inactivation on HIF - 2α binding intensity showing no significant effect ( p = 0 . 4 , Wilcoxon signed rank 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 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 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 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 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 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

Figure 5Cells were incubated in 0.5% ambient oxygen for 16 hours. Immunoblots show HIF-1α, HIF-2α and HIF-1β protein levels in the wild type and single clones of CRISPR-Cas9 engineered cell linesHIF-1α ChIP-seq signal in the HIF-2α KO cells was plotted against that in the wild-type cells for all canonical HIF-1 binding sites in either cell typeHIF-2α ChIP-seq signal in the HIF-1α KO cells was plotted against that in the wild-type cells for all canonical HIF-2 binding sites in either cell type. No significant increase (Wilcoxon rank sum test) in binding of either isoform was observed following deletion of the otherBox-and-whisker plots showing: (D) The effect of HIF-2α inactivation on HIF-1α ChIP-seq signal at sites that specifically bound HIF-2α, but not HIF-1α in wild-type cells showing a slight, but significant (p = 4 x 10-5, Wilcoxon signed rank test) decrease rather than increase (compare red boxes) consistent with the reduction in HIF-1α protein observed in panel AThe effect of HIF-1α inactivation on HIF-2α ChIP-seq signal (compare blue boxes) at sites that specifically bound HIF-1α, but not HIF-2α in wild-type cells showing no significant effect (p = 0.5, Wilcoxon signed rank test)The effect, at sites that bind both isoforms in wild-type cells, of (F) HIF-2α inactivation on HIF-1α binding intensity showing a similar small but significant reduction rather than increase (p = 2 x 10-16, Wilcoxon Signed rank test) as abovThe effect, at sites that bind both isoforms in wild-type cells, o HIF-1α inactivation on HIF-2α binding intensity showing no significant effect (p = 0.4, Wilcoxon signed rank test)


Figure 6Canonical HIF - 1 and HIF - 2 binding sites were determined by overlap of HIF - α and HIF - 1β binding sites identified by the MACS peak caller as for Figure 4 . The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for ( A ) HIF - 1 and ( B ) HIF - 2 sites in HKC - 8 cells , ( C ) HIF - 1 and ( D ) HIF - 2 sites in RCC4 cells , ( E ) HIF - 1 and ( F ) HIF - 2 sites in HepG2 cells . The distribution of HIF - 1 binding sites was consistently more promoter proximal than HIF - 2 sites despite the sites themselves differing between cell typesThe distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF - 1 ( p ~ 10 - 50 ) and HIF - 2 ( p ~ 10 - 80 ) binding sites ( Chi - squared 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 O O O O O O O O O O O O O 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

Figure 6Canonical HIF-1 and HIF-2 binding sites were determined by overlap of HIF-α and HIF-1β binding sites identified by the MACS peak caller as for Figure 4. The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for (A) HIF-1 and (B) HIF-2 sites in HKC-8 cells, (C) HIF-1 and (D) HIF-2 sites in RCC4 cells, (E) HIF-1 and (F) HIF-2 sites in HepG2 cells. The distribution of HIF-1 binding sites was consistently more promoter proximal than HIF-2 sites despite the sites themselves differing between cell typesThe distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF-1 (p~10-50) and HIF-2 (p~10-80) binding sites (Chi-squared test)


Figure 7Immunoblots showing HIF - 1α , HIF - 2α and HIF - 1β protein levels in HKC - 8 , HepG2 , RCC4 and MCF - 7 cell lines in normoxia and following 16 hours incubation at 0 . 5 % oxygenSites that bound ( B ) canonical HIF - 1 ( i . e . identified in both HIF - 1α and both HIF - 1β ChIP - seq experiments ) or ( C ) canonical HIF - 2 ( i . e . identified in both HIF - 2α and both HIF - 1β ChIP - seq experiments ) were identified from ChIP - seq datasets in HepG2 and HKC - 8 cells following 16 hours incubation in 0 . 5 % oxygen and from normoxic RCC4 cells and compared with previously published data from MCF - 7 cells . ( D ) The proportion of total HIF - 1 or HIF - 2 sites that were shared between 2 , 3 or 4 cell lines is plotted . For sites that are shared between ( E ) HepG2 and HKC - 8 cells , ( F ) HKC - 8 and RCC4 cells , or ( G ) RCC4 and HepG2 cells , the ratio of HIF - 1α / HIF - 2α signal for one cell line was plotted against the other . The ratio of HIF - 1α / HIF - 2α in one cell line correlates well with that in the other . ChIP - seq signal is averaged across two independent

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7Immunoblots showing HIF-1α, HIF-2α and HIF-1β protein levels in HKC-8, HepG2, RCC4 and MCF-7 cell lines in normoxia and following 16 hours incubation at 0.5% oxygenSites that bound (B) canonical HIF-1 (i.e. identified in both HIF-1α and both HIF-1β ChIP-seq experiments) or (C) canonical HIF-2 (i.e. identified in both HIF-2α and both HIF-1β ChIP-seq experiments) were identified from ChIP-seq datasets in HepG2 and HKC-8 cells following 16 hours incubation in 0.5% oxygen and from normoxic RCC4 cells and compared with previously published data from MCF-7 cells. (D) The proportion of total HIF-1 or HIF-2 sites that were shared between 2, 3 or 4 cell lines is plotted. For sites that are shared between (E) HepG2 and HKC-8 cells, (F) HKC-8 and RCC4 cells, or (G) RCC4 and HepG2 cells, the ratio of HIF-1α/HIF-2α signal for one cell line was plotted against the other. The ratio of HIF-1α/HIF-2α in one cell line correlates well with that in the other. ChIP-seq signal is averaged across two independent experiments


Figure 8Heatmaps showing average ( n = 2 , independent ChIP - seq experiments ) ChIP - seq signal ( read counts per million mapped reads , CPM ; expressed as colour intensity ) at canonical HIF - 1 and HIF - 2 binding sites and across the flanking ± 5kb regions ( x - axis ) in HepG2 cells incubated in 0 . 5 % oxygen for 16 hours . Sites are ordered on the y - axis according to HIF signal derived from HepG2 cells incubated in hypoxia . ChIP - seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxiaBar chart showing enrichment of transcription factor binding sites ( TFBS ) at canonical HIF - 1 and HIF - 2 sites in HepG2 cells ( identified as in Figure 4 ) . TFBSs for 61 additional DNA - binding proteins were determined from ENCODE ChIP - seq data in normoxic HepG2 cells . The significance of the overlap between each set of binding sites and canonical HIF - 1 and HIF - 2 sites , was determined using a hypergeometric test . Accessible sites determined from ENCODE DNAse - seq data in HepG2 cells was used as a negative

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 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

Figure 8Heatmaps showing average (n = 2, independent ChIP-seq experiments) ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity) at canonical HIF-1 and HIF-2 binding sites and across the flanking ±5kb regions (x-axis) in HepG2 cells incubated in 0.5% oxygen for 16 hours. Sites are ordered on the y-axis according to HIF signal derived from HepG2 cells incubated in hypoxia. ChIP-seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxiaBar chart showing enrichment of transcription factor binding sites (TFBS) at canonical HIF-1 and HIF-2 sites in HepG2 cells (identified as in Figure 4). TFBSs for 61 additional DNA-binding proteins were determined from ENCODE ChIP-seq data in normoxic HepG2 cells. The significance of the overlap between each set of binding sites and canonical HIF-1 and HIF-2 sites, was determined using a hypergeometric test. Accessible sites determined from ENCODE DNAse-seq data in HepG2 cells was used as a negative control


Figure 1Time lapse images of T6SS activity in tagged parental strain ( VipA - mCherry2 ) and ∆ tssA mutant background . The parental strain forms multiple dynamic structures per cell . ∆ tssA mutant predominantly forms dynamic sheath spots ( white arrow ) and few WT - like sheath structures ( blue arrow ) . Scale bars : 2 µm . H2 - T6SS dynamics in tagged parental strain ( ∆ retS TssB2 - mCherry2 , referred to as WT H2 ) and ΔtssA2PA strain . Blue arrow indicates extending and contracting T6SS sheath structures , white arrow points to dynamic sheath spots . The WT H2 strain harbors multiple dynamic sheath structures per cell , while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures . Scale bars : 2 µm .

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 O O O O O O

Figure 1Time lapse images of T6SS activity in tagged parental strain (VipA-mCherry2) and ∆tssA mutant background. The parental strain forms multiple dynamic structures per cell. ∆tssA mutant predominantly forms dynamic sheath spots (white arrow) and few WT-like sheath structures (blue arrow). Scale bars: 2 µm.H2-T6SS dynamics in tagged parental strain (∆retS TssB2-mCherry2, referred to as WT H2) and ΔtssA2PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. The WT H2 strain harbors multiple dynamic sheath structures per cell, while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures. Scale bars: 2 µm.


Figure 2Time lapse images of T6SS activity in tagged strain ( VipA - mCherry2 TssAVC - mNeonGreen ) . White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics . Scale bar : 1 µm . Kymographs of dynamics observed for TssAVC . Frequency of specific dynamics is indicated in the upper right corner of the merge image . ( B ) Sheath and TssAVC dynamics from A . TssAVC localizes to T6SS initiation site and to distal end of growing sheath ( white arrow ) . ( C ) Once sheath reaches cell periphery , TssAVC dissociates from distal end ( white arrow ) and sheath stays extended a prolonged period of time . ( D ) TssAVC dissociates from growing sheath structure and re - associates at a later time point ( white arrows ) to continue catalysis of polymerization . ( E ) TssAVC stays attached to sheath distal end after contraction ( white arrow ) . Fluorescence microscopy of T6SS dynamics in a double tagged strain ( ∆ retS TssB2 - mCherry2 TssA2PA - mNeonGreen ) . White arrow indicates full cycle of extending and contracting T6SS sheath . Scale bar : 1 µm . Kymographs of TssB2 and TssA2PA dynamics . TssA2PA localizes at sheath initiation site and on distal end during polymerization ( G ) , dissociates before contraction ( H ) , dissociates and re - associated during sheath extension ( I ) or stays attached to distal end after contraction ( J ) .

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 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 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 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

Figure 2Time lapse images of T6SS activity in tagged strain (VipA-mCherry2 TssAVC-mNeonGreen). White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics. Scale bar: 1 µm.Kymographs of dynamics observed for TssAVC. Frequency of specific dynamics is indicated in the upper right corner of the merge image. (B) Sheath and TssAVC dynamics from A. TssAVC localizes to T6SS initiation site and to distal end of growing sheath (white arrow). (C) Once sheath reaches cell periphery, TssAVC dissociates from distal end (white arrow) and sheath stays extended a prolonged period of time. (D) TssAVC dissociates from growing sheath structure and re-associates at a later time point (white arrows) to continue catalysis of polymerization. (E) TssAVC stays attached to sheath distal end after contraction (white arrow).Fluorescence microscopy of T6SS dynamics in a double tagged strain (∆retS TssB2-mCherry2 TssA2PA-mNeonGreen). White arrow indicates full cycle of extending and contracting T6SS sheath. Scale bar: 1 µm.Kymographs of TssB2 and TssA2PA dynamics. TssA2PA localizes at sheath initiation site and on distal end during polymerization (G), dissociates before contraction (H), dissociates and re-associated during sheath extension (I) or stays attached to distal end after contraction (J).


Figure 3Top view ( XY - plane ) and side view ( XZ - plane ) of the TssAVC Nt2 domain cryo - EM reconstruction ( pink , non transparent ) shown with fitted Nt2 - dimer model ( left ) . Partial Nt2 - CTD and Nt1 - Nt2 linker densities are highlighted with black arrows . Top and side views of ribbon diagram of Nt2 - dimer ( middle ) . Representative 2D class averages of TssAVC particles with visible connections between Nt2 - dimer and CTD ( top right ) , representative 2D class averages of Nt2 - dimer particles , Nt2 - CTD linker and Nt1 - Nt2 linker densities are highlighted with blue and yellow arrows ( bottom right ) .

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 O O O

Figure 3Top view (XY-plane) and side view (XZ-plane) of the TssAVC Nt2 domain cryo-EM reconstruction (pink, non transparent) shown with fitted Nt2-dimer model (left). Partial Nt2-CTD and Nt1-Nt2 linker densities are highlighted with black arrows. Top and side views of ribbon diagram of Nt2-dimer (middle). Representative 2D class averages of TssAVC particles with visible connections between Nt2-dimer and CTD (top right), representative 2D class averages of Nt2-dimer particles, Nt2-CTD linker and Nt1-Nt2 linker densities are highlighted with blue and yellow arrows (bottom right).


Figure 4The ∆ tagA mutant displays long and bent but still dynamic structures . Scale bar : 2 µm . Frames from time lapse movies showing sheath dynamics in the ∆ tagA mutant . Sheath polymerization does not stop when the distal end reaches opposing membrane ( white arrow ) . Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole , ultimately effecting bending of the whole structure . C . Sheath detaches from its membrane anchor ( orange arrow ) , keeps extending to maximum cell length and then contracts . Scale bars : 1 µm . Time lapse series of T6SS activity in tagged strain ( VipA - mCherry2 TagAVC - mNeonGreen ) . Orange arrow indicates sheath distal end , white arrow indicates initiation site of another T6SS sheath . Scale bar : 1 µm . Kymographs of dynamics observed for TagAVC . E . TagAVC localizes at the distal end when sheath reaches the cell periphery . Signal of TagAVC - mNeonGreen disappeared and reappeared on this site repeatedly . F . TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction . G . TagAVC briefly localizes at T6SS initiation site . H . TagAVC briefly localizes at T6SS initiation site and at distal end , where it stays attached after contraction .

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 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 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

Figure 4The ∆tagA mutant displays long and bent but still dynamic structures. Scale bar: 2 µm. Frames from time lapse movies showing sheath dynamics in the ∆tagA mutant. Sheath polymerization does not stop when the distal end reaches opposing membrane (white arrow). Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole, ultimately effecting bending of the whole structure. C. Sheath detaches from its membrane anchor (orange arrow), keeps extending to maximum cell length and then contracts. Scale bars: 1 µm.Time lapse series of T6SS activity in tagged strain (VipA-mCherry2 TagAVC-mNeonGreen). Orange arrow indicates sheath distal end, white arrow indicates initiation site of another T6SS sheath. Scale bar: 1 µm.Kymographs of dynamics observed for TagAVC. E. TagAVC localizes at the distal end when sheath reaches the cell periphery. Signal of TagAVC-mNeonGreen disappeared and reappeared on this site repeatedly. F. TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction. G. TagAVC briefly localizes at T6SS initiation site. H. TagAVC briefly localizes at T6SS initiation site and at distal end, where it stays attached after contraction.


Figure 5H1 - T6SS dynamics in tagged parental strain ( ∆ retS TssB1 - mCherry2 , referred to as WT H1 ) and ΔtssA1PA strain . Blue arrow indicates extending and contracting T6SS sheath structures , white arrow points to dynamic sheath spots . Scale bars : 2 µm . Fluorescence microscopy of T6SS dynamics in ∆ retS TssB1 - mCherry2 strain harboring pPSV35 plasmid with TssA1PA - mNeonGreen fusion . Scale bar : 2 µm . Kymographs of TssB1 and TssA1PA dynamics . TssA1PA localizes exclusively at sheath initiation site ( B - E ) , disappears after contraction ( C , D ) or remains in place for longer timeframe ( C , E ) . TssA1PA signal appears in neighboring cell shortly after T6SS attack ( B , C ) .

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 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

Figure 5H1-T6SS dynamics in tagged parental strain (∆retS TssB1-mCherry2, referred to as WT H1) and ΔtssA1PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. Scale bars: 2 µm.Fluorescence microscopy of T6SS dynamics in ∆retS TssB1-mCherry2 strain harboring pPSV35 plasmid with TssA1PA-mNeonGreen fusion. Scale bar: 2 µm.Kymographs of TssB1 and TssA1PA dynamics. TssA1PA localizes exclusively at sheath initiation site (B-E), disappears after contraction (C,D) or remains in place for longer timeframe (C,E). TssA1PA signal appears in neighboring cell shortly after T6SS attack (B,C).


Figure 1Representative confocal images of cell - type - specific promoter : : GFP : AGO1 translational fusions . Immunoblot analysis of FLAG : AGO1 IPed in each root - tip layer . Scale bars : 50μM , NT : Non - transformed . CB : Coomassie blue . Left : input and IPed AGO1 following post - lysis addition of a radiolabeled siRNA duplex ( siGFP ) . Right : northern analysis of AGO1 - bound sRNAs . Layer - specific distribution of all AGO1 - loaded miRNAs . Northern analysis of layer - specific AGO1 - loaded miRNAs using sRNAs IPed independently from that used for deep - sequencing . Bar - plots underneath each panel display the deep - sequencing results as normalized reads counts .

O B-PANEL_START 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 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 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

Figure 1Representative confocal images of cell-type-specific promoter::GFP:AGO1 translational fusions.Immunoblot analysis of FLAG:AGO1 IPed in each root-tip layer. Scale bars: 50μM, NT: Non-transformed. CB: Coomassie blue.Left: input and IPed AGO1 following post-lysis addition of a radiolabeled siRNA duplex (siGFP). Right: northern analysis of AGO1-bound sRNAs.Layer-specific distribution of all AGO1-loaded miRNAs.Northern analysis of layer-specific AGO1-loaded miRNAs using sRNAs IPed independently from that used for deep-sequencing. Bar-plots underneath each panel display the deep-sequencing results as normalized reads counts.


Figure 2Representative confocal images of cell - type - specific promoter : : GFP transcriptional fusions in hyl1 vs WT roots . Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots . qRT - PCR validation of three distinct classes of direct miRNA target transcripts from total root - , total root polysome - or epidermis - specific polysome - associated RNA . The AT2G37050 kinase is positively regulated by LAC17 . Scale bars : 50μM . All qPCR values were normalized to the internal control ACTIN2 . miRoot snapshots of whole - root ( left ) and layer - specific ( right ) LAC17 mRNA accumulation in WT versus hyl1 , as assessed by polysome profiling .

O B-PANEL_START 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 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 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

Figure 2Representative confocal images of cell-type-specific promoter::GFP transcriptional fusions in hyl1 vs WT roots.Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots.qRT-PCR validation of three distinct classes of direct miRNA target transcripts from total root-, total root polysome- or epidermis-specific polysome-associated RNA. The AT2G37050 kinase is positively regulated by LAC17. Scale bars: 50μM. All qPCR values were normalized to the internal control ACTIN2.miRoot snapshots of whole-root (left) and layer-specific (right) LAC17 mRNA accumulation in WT versus hyl1, as assessed by polysome profiling.


Figure 3miRoot snapshot of cell - type specific miRNAs showing , in this case , a sum group of the miR156 family . The right panel shows the gradient of expression revealed upon removal of the stele ( blue box ) . Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds . Removal of the stele ( blue box ) reveals subtleties of miR156 - mediated gene regulation in the other layers , resulting in a near - mirror image of ( A ) .

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 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

Figure 3miRoot snapshot of cell-type specific miRNAs showing, in this case, a sum group of the miR156 family. The right panel shows the gradient of expression revealed upon removal of the stele (blue box).Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds. Removal of the stele (blue box) reveals subtleties of miR156-mediated gene regulation in the other layers, resulting in a near-mirror image of (A).


Figure 4Confocal image of pmiR396a : : H2B : GFP . Normalized read counts from AGO1 - IP - Seq and independent northern analysis of AGO1 - loaded miR396 . Representative confocal image of the pGRF2 : : H2B : GFP translational fusion . Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella . Graph : GRF2 normalized read counts . Representative confocal images of the pGRF2 : : GRF2 : GFP translational fusion in WT or hyl1 backgrounds . Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella .

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 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 B-PANEL_START 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

Figure 4Confocal image of pmiR396a::H2B:GFP. Normalized read counts from AGO1-IP-Seq and independent northern analysis of AGO1-loaded miR396.Representative confocal image of the pGRF2::H2B:GFP translational fusion.Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella. Graph: GRF2 normalized read counts.Representative confocal images of the pGRF2::GRF2:GFP translational fusion in WT or hyl1 backgrounds.Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella.


Figure 5Layer - specific normalized read counts of miR169 paralogs ( separated into 4 different groups based on sequence identity : a , b - c , d - g , h - n ) from AGO1 - IP - deep - Seq and independent northern analysis , as in Fig . 1F . Cell - type specific AGO1 - IP signals for each miR169 sequence - group with ( upper ) or without ( lower panel ) the miRoot scale normalization option . Predicted base - pairing of indicated miR169 sequence - group to four NF - YA target transcripts . Pairing with expectation value greater than 3 was not considered . The color gradient ( pale : low , dark : strong ) qualitatively approximates the pairing contribution of each miR169 sequence - group according to the upper panel of ( B ) . miRoot snapshots of layer - specific translatome signals of four out - of - seven miR169 NF - YA targets in WT versus hyl1 backgrounds .

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 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

Figure 5Layer-specific normalized read counts of miR169 paralogs (separated into 4 different groups based on sequence identity: a, b-c, d-g, h-n) from AGO1-IP-deep-Seq and independent northern analysis, as in Fig.1F.Cell-type specific AGO1-IP signals for each miR169 sequence-group with (upper) or without (lower panel) the miRoot scale normalization option.Predicted base-pairing of indicated miR169 sequence-group to four NF-YA target transcripts. Pairing with expectation value greater than 3 was not considered. The color gradient (pale: low, dark: strong) qualitatively approximates the pairing contribution of each miR169 sequence-group according to the upper panel of (B). miRoot snapshots of layer-specific translatome signals of four out-of-seven miR169 NF-YA targets in WT versus hyl1 backgrounds.


Figure 6Browser representation and independent northern analysis and AGO1 - loaded miR165 / 166 . miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds . Representative confocal images of pmiR395 ( a / c / e ) : : H2B : GFP transcriptional fusions under SO4 - - deficient or SO4 - - proficient growth conditions . Northern analysis of AGO1 - loaded miR395 under the conditions in ( C ) . Representative confocal images of the pAPS4 : : APS4 : GFP translational fusion under the conditions in ( C ) .

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

Figure 6Browser representation and independent northern analysis and AGO1-loaded miR165/166.miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds.Representative confocal images of pmiR395(a/c/e)::H2B:GFP transcriptional fusions under SO4--deficient or SO4--proficient growth conditions. Northern analysis of AGO1-loaded miR395 under the conditions in (C).Representative confocal images of the pAPS4::APS4:GFP translational fusion under the conditions in (C).


Figure 7Representative confocal image of the pSHR : : GFP : P19 translational fusion and ( B ) principle of P19 - based miRNA duplex sequestration . Binding of miR395 by SHR : : HA : P19 under SO4 - - sufficient ( + ) and - deficient ( - ) growth conditions . qRT - PCR analysis of polysome - associated APS4 in the cortex or epidermis in the presence ( + ) or absence ( - ) of SHR : : HA : P19 . Signals are relative to SHR : : HA : P19 ( - ) lines given a value of 1 . Representative confocal image of the pmiR397b : : H2B : GFP transcriptional fusion ( left ) and the browser AGO1 - loaded signal for miR397b ( right ) . Cross section of Arabidopsis leaf showing cell - types isolated via Meselect . miR160 northern analysis in whole - leaf , vasculature and epidermis . U6 : loading control . Western analysis of the promoter : : H2B : GFP translational fusions shown in ( 5C ) in the cell types isolated in ( G ) . qRT - PCR analysis of ARF17 in whole - leaves or in separated cell types in WT versus hyl1 backgrounds .

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

Figure 7Representative confocal image of the pSHR::GFP:P19 translational fusion and (B) principle of P19-based miRNA duplex sequestration.Binding of miR395 by SHR::HA:P19 under SO4--sufficient (+) and -deficient (-) growth conditions.qRT-PCR analysis of polysome-associated APS4 in the cortex or epidermis in the presence (+) or absence (-) of SHR::HA:P19. Signals are relative to SHR::HA:P19 (-) lines given a value of 1.Representative confocal image of the pmiR397b::H2B:GFP transcriptional fusion (left) and the browser AGO1-loaded signal for miR397b (right).Cross section of Arabidopsis leaf showing cell-types isolated via Meselect.miR160 northern analysis in whole-leaf, vasculature and epidermis. U6: loading control.Western analysis of the promoter::H2B:GFP translational fusions shown in (5C) in the cell types isolated in (G).qRT-PCR analysis of ARF17 in whole-leaves or in separated cell types in WT versus hyl1 backgrounds.


f1a - c , RFA1 : : FLAG DDC2 : : MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease . After 30 min , the culture was split in two : + VPA and - VPA . a , Samples were processed for western blot using anti - Rad53 , Srs2 and Ddc2 antibodies . a - c , RFA1 : : FLAG DDC2 : : MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease . After 30 min , the culture was split in two : + VPA and − VPA . b , A schematic diagram showing probe locations with respect to the HO cut site . The resection rate was calculated as the rate of HO cut band disappearance . a - c , RFA1 : : FLAG DDC2 : : MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease . After 30 min , the culture was split in two : + VPA and - VPA . c , Fold enrichment of the 0 . 2 - kb fragment was calculated after ChIP of Rfa1 - Flag , Ddc2 - Myc . d , VPA effect on ectopic recombination ( rec . ) in wild - type ( WT ) cells . Error bars represent standard deviation ( s . d . ) calculated from four independent experiments .

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 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 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 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

f1a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. a, Samples were processed for western blot using anti-Rad53, Srs2 and Ddc2 antibodies.a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and −VPA. b, A schematic diagram showing probe locations with respect to the HO cut site. The resection rate was calculated as the rate of HO cut band disappearance.a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. c, Fold enrichment of the 0.2-kb fragment was calculated after ChIP of Rfa1-Flag, Ddc2-Myc.d, VPA effect on ectopic recombination (rec.) in wild-type (WT) cells. Error bars represent standard deviation (s.d.) calculated from four independent experiments.


f2a , MRE11 : : MYC cells were treated as in Fig . 1a . Cell samples were processed for ChIP analysis and the fold enrichment of the 0 . 2 - kb fragment after ChIP of Mre11 - Myc without ( - VPA ) or with ( + VPA ) VPA was calculated . b , EXO1 : : FLAG SAE2 : : PK MRE11 : : MYC cells were grown as in a . Cell samples were taken and processed for western blot analysis using anti - Flag , PK and Myc antibodies .

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

f2a, MRE11::MYC cells were treated as in Fig. 1a. Cell samples were processed for ChIP analysis and the fold enrichment of the 0.2-kb fragment after ChIP of Mre11-Myc without (-VPA) or with (+VPA) VPA was calculated.b, EXO1::FLAG SAE2::PK MRE11::MYC cells were grown as in a. Cell samples were taken and processed for western blot analysis using anti-Flag, PK and Myc antibodies.


f3a , Cherry : : APE1GFP : : ATG8 cells were grown and shifted to YPD , nitrogen starvation ( SD - N ) or YPD + VPA medium for 3 h . Samples were processed for microscopy . The table shows numbers corresponding to the experiment . Percentage of fluorescence signals is presented and error bars represent the s . d . obtained from three independent experiments . DIC , differential interference contrast . Scale bars , 3 µm . b , pho8Δ60 and pho8Δ60 atg1Δ ; cells were grown as in a . Pho8Δ60 activity was calculated by measuring alkaline phosphatase levels . Error bars represent s . d . calculated from five independent experiments . c , GFP : : ATG8 and GFP : : ATG8atg1Δ ; cells were grown as in a . Cell samples were processed for western blot using anti - GFP antibody . Quantification is presented in Supplementary Fig . 3 .

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 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 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

f3a, Cherry::APE1GFP::ATG8 cells were grown and shifted to YPD, nitrogen starvation (SD-N) or YPD+VPA medium for 3 h. Samples were processed for microscopy. The table shows numbers corresponding to the experiment. Percentage of fluorescence signals is presented and error bars represent the s.d. obtained from three independent experiments. DIC, differential interference contrast. Scale bars, 3 µm.b, pho8Δ60 and pho8Δ60 atg1Δ; cells were grown as in a. Pho8Δ60 activity was calculated by measuring alkaline phosphatase levels. Error bars represent s.d. calculated from five independent experiments.c, GFP::ATG8 and GFP::ATG8atg1Δ; cells were grown as in a. Cell samples were processed for western blot using anti-GFP antibody. Quantification is presented in Supplementary Fig. 3.


f4a , 4HA - Sae2 was immunoprecipitated ± VPA with anti - HA and subsequently ± anti - acetyl - Lysine . Eluate was analysed using anti - HA . Lane 1 : input Sae2 ; 2 : input Sae2 - HA - VPA ; 3 : as in 2 but + VPA ; 4 : 3 µl elution Sae2 - HA - VPA after anti - HAimmunoprecipitation ( IP ; input AcK - IP ) ; 5 : double amount of 4 ; 6 : IP anti - AcK elution from anti - HA IP of Sae2 - HA - VPA ; 7 : as in 6 but + VPA . b , SAE2 : : PKerg6Δ cells were treated as in Fig . 2a . After 30 min induction , VPA and PMSF were added or not . Samples were processed for western blot using anti - PK . c , Wild - type SAE2 : : PK , SAE2 : : PKatg1Δ and SAE2 : : PKatg19Δ cells were grown as in b . After 30 min induction , VPA was added or not and samples treated as in b . d , Wild - type SAE2 : : PK cells were grown as in b . After 120 min induction , rapamycin ( 200 ng ml - 1 ) was added or not and samples were treated as in b .

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 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 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

f4a, 4HA-Sae2 was immunoprecipitated ± VPA with anti-HA and subsequently ± anti-acetyl-Lysine. Eluate was analysed using anti-HA. Lane 1: input Sae2; 2: input Sae2-HA -VPA; 3: as in 2 but +VPA; 4: 3 µl elution Sae2-HA -VPA after anti-HAimmunoprecipitation (IP; input AcK-IP); 5: double amount of 4; 6: IP anti-AcK elution from anti-HA IP of Sae2-HA -VPA; 7: as in 6 but +VPA.b, SAE2::PKerg6Δ cells were treated as in Fig. 2a. After 30 min induction, VPA and PMSF were added or not. Samples were processed for western blot using anti-PK.c, Wild-type SAE2::PK, SAE2::PKatg1Δ and SAE2::PKatg19Δ cells were grown as in b. After 30 min induction, VPA was added or not and samples treated as in b.d, Wild-type SAE2::PK cells were grown as in b. After 120 min induction, rapamycin (200 ng ml-1) was added or not and samples were treated as in b.


f5a , Survival of wild - type , rpd3Δ , hda1Δ and rpd3Δ hda1Δ strains after 4NQO , MMS and HU treatment . Error bars represent s . d . calculated from seven independent experiments . b , HO was induced in G2 wild - type , hda1Δ , rpd3Δ and hda1Δ rpd3Δ cells and western and Southern blot analyses were performed . c , Wild - type SAE2 : : PK and hda1Δ rpd3Δ SAE2 : : PK cells were grown as in b and western blot was performed as in Fig . 4b . d , e , Wild - type SAE2 : : PK and gcn5Δ SAE2 : : PK strains were grown as in b and after 30 min of HO induction either VPA ( d ) or rapamycin ( e ) was added or not . Western blot was performed . f , Percentage of viability of wild - type , atg1Δ , rpd3Δ hda1Δ and atg1Δ rpd3Δ hda1Δ strains . Error bars represent s . d . calculated from four independent experiments .

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 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 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 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 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

f5a, Survival of wild-type, rpd3Δ, hda1Δ and rpd3Δ hda1Δ strains after 4NQO, MMS and HU treatment. Error bars represent s.d. calculated from seven independent experiments.b, HO was induced in G2 wild-type, hda1Δ, rpd3Δ and hda1Δ rpd3Δ cells and western and Southern blot analyses were performed.c, Wild-type SAE2::PK and hda1Δ rpd3Δ SAE2::PK cells were grown as in b and western blot was performed as in Fig. 4b.d, e, Wild-type SAE2::PK and gcn5Δ SAE2::PK strains were grown as in b and after 30 min of HO induction either VPA (d) or rapamycin (e) was added or not. Western blot was performed.f, Percentage of viability of wild-type, atg1Δ, rpd3Δ hda1Δ and atg1Δ rpd3Δ hda1Δ strains. Error bars represent s.d. calculated from four independent experiments.


Figure 1Immunofluorescence labeling of QSOX1 and a Golgi marker ( GM130 ) in epithelial cells isolated from murine colon . QSOX1 is green , GM130 is red , and DAPI staining of nuclei is blue in the merged image . Scale bar is 10 µm . Top , average colon length of WT and QSOX1 KO mice following indicated treatments . For untreated mice , colon lengths were calculated for 3 WT and 3 KO . For treated mice , lengths were calculated from 5 mice except for the DSS - treated WT , for which 3 mice were analyzed . Error bars are standard deviation . Statistical analysis was performed using Tukey multiple comparison of means ( * * p < 0 . 01 ) . Administration of QSOX1 inhibitory antibody MAb316 . 1 ( αQSOX1 ) , did not enhance the sensitivity of WT colons to DSS ( p > 0 . 4 for MAb316 . 1 - treated WT vs . WT control antibody ( IgG ) - treated ; p > 0 . 5 for MAb316 . 1 - treated WT vs . WT without antibody treatment ) . Bottom , representative images of colons following DSS treatment . Scale bar is 1 cm .

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 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 O O O O O O O O O O O O O O O O O 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 1Immunofluorescence labeling of QSOX1 and a Golgi marker (GM130) in epithelial cells isolated from murine colon. QSOX1 is green, GM130 is red, and DAPI staining of nuclei is blue in the merged image. Scale bar is 10 µm.Top, average colon length of WT and QSOX1 KO mice following indicated treatments. For untreated mice, colon lengths were calculated for 3 WT and 3 KO. For treated mice, lengths were calculated from 5 mice except for the DSS-treated WT, for which 3 mice were analyzed. Error bars are standard deviation. Statistical analysis was performed using Tukey multiple comparison of means (** p<0.01). Administration of QSOX1 inhibitory antibody MAb316.1 (αQSOX1), did not enhance the sensitivity of WT colons to DSS (p>0.4 for MAb316.1-treated WT vs. WT control antibody (IgG)-treated; p>0.5 for MAb316.1-treated WT vs. WT without antibody treatment). Bottom, representative images of colons following DSS treatment. Scale bar is 1 cm.


Figure 3Representative TEM micrographs of about a hundred collected from 3 mice of each genotype show large and well - packed goblet cell granules in WT and QSOX1 KO colons . Scale bar is 5 µm . Alcian blue and PAS staining of reduced guanidine - insoluble mucins from WT and QSOX1 KO mice , separated on a 6 % polyacrylamide gel . Data information : each lane corresponds to a sample from one mouse . Muc5b fluorescent immunoblot of WT and QSOX1 KO lung lavage samples separated on agarose gels . Data information : each lane corresponds to a sample from one mouse . Western blot analysis of blood Vwf from WT and QSOX1 KO mice separated on agarose gels . Data information : each lane corresponds to a sample from one mouse . QSOX1 immunofluorescence ( green ) in control ( siControl ) and QSOX1 knockdown ( siQSOX1 ) MDA - MB - 231 cells . Blue is DAPI staining of nuclei . Scale bar is 20 µm . Data information : The results described in are representative of 3 experiments that were performed . Western blot analysis of the MUC2 N - terminal region and indicated cysteine mutants in supernatants of transfected cell cultures . No differences in disulfide - mediated dimerization were observed for any MUC2 variant between siC ( siControl ) and siQ ( siQSOX1 ) . Data information : The results described in G are representative of 3 experiments that were performed .

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 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 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 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 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

Figure 3Representative TEM micrographs of about a hundred collected from 3 mice of each genotype show large and well-packed goblet cell granules in WT and QSOX1 KO colons. Scale bar is 5 µm.Alcian blue and PAS staining of reduced guanidine-insoluble mucins from WT and QSOX1 KO mice, separated on a 6% polyacrylamide gel. Data information: each lane corresponds to a sample from one mouse.Muc5b fluorescent immunoblot of WT and QSOX1 KO lung lavage samples separated on agarose gels. Data information: each lane corresponds to a sample from one mouse.Western blot analysis of blood Vwf from WT and QSOX1 KO mice separated on agarose gels. Data information: each lane corresponds to a sample from one mouse.QSOX1 immunofluorescence (green) in control (siControl) and QSOX1 knockdown (siQSOX1) MDA-MB-231 cells. Blue is DAPI staining of nuclei. Scale bar is 20 µm. Data information: The results described in are representative of 3 experiments that were performed.Western blot analysis of the MUC2 N-terminal region and indicated cysteine mutants in supernatants of transfected cell cultures. No differences in disulfide-mediated dimerization were observed for any MUC2 variant between siC (siControl) and siQ (siQSOX1). Data information: The results described in G are representative of 3 experiments that were performed.


Figure 4Western blot analysis of colon epithelial cell lysates from WT and QSOX1 KO mice . Lysates were treated with PEG - mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1 . As for panel A but using antibodies to St3gal1 . As for panel A but using antibodies to B3galt5 . Data information : For the experiments in panels A - C , equal amounts of total protein were applied to each gel lane , and no reducing agent was added . Experiments were repeated 3 times with fresh new lysates , and representative blots are shown . QSOX1 oxidizes St6gal1 in vitro . MAb492 . 1 is a monoclonal antibody that inhibits human QSOX1 St6gal1 with one pair of free cysteines is modified by two PEG - mal additions ( 2PEG ) , whereas protein with two pairs of free cysteines acquires four PEG - mal additions ( 4PEG ) . The change in migration per two PEG - mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs . protein QSOX1 oxidation of St6gal1 mutants . The C350 - C361 disulfide ( the redox - active disulfide present in the C139A - C403A mutant ) is oxidized rapidly , while the C142 - C406 disulfide ( present in the C350A - C361A mutant ) is oxidized slowly . Data information : Experiments were repeated 3 times with fresh new lysates , and representative blots are shown .

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 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 O O O O O O O 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 4Western blot analysis of colon epithelial cell lysates from WT and QSOX1 KO mice. Lysates were treated with PEG-mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1. As for panel A but using antibodies to St3gal1. As for panel A but using antibodies to B3galt5. Data information: For the experiments in panels A-C, equal amounts of total protein were applied to each gel lane, and no reducing agent was added. Experiments were repeated 3 times with fresh new lysates, and representative blots are shown.QSOX1 oxidizes St6gal1 in vitro. MAb492.1 is a monoclonal antibody that inhibits human QSOX1 St6gal1 with one pair of free cysteines is modified by two PEG-mal additions (2PEG), whereas protein with two pairs of free cysteines acquires four PEG-mal additions (4PEG). The change in migration per two PEG-mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs. protein QSOX1 oxidation of St6gal1 mutants. The C350-C361 disulfide (the redox-active disulfide present in the C139A-C403A mutant) is oxidized rapidly, while the C142-C406 disulfide (present in the C350A-C361A mutant) is oxidized slowly. Data information: Experiments were repeated 3 times with fresh new lysates, and representative blots are shown.


Figure 1 A ) Oocytes were fixed for chromosome spreads 8 hours after GVBD , and stained for PP2A - c ( red ) , Sgo2 ( green ) , CREST ( blue ) and DNA ( Hoechst , shown in grey ) . Spreads were classified into metaphase I and anaphase I , depending on whether chromosome segregation had taken place , or not . Arrowhead indicates fused sister kinetochores , arrows indicate separated sister kinetochores . B ) Example of a whole mount oocyte staining , in early anaphase I after cold - treatment to visualize only cold - stable microtubule fibers , stained with anti - tubulin antibody ( green ) . Kinetochores are stained with CREST serum ( red ) and chromosomes with Hoechst ( blue ) . Shown is an overlay of 20 z - sections . Arrows indicate separated sister kinetochores . C ) Metaphase I ( 6 hours after GVBD ) and metaphase II ( 16 - 20 hours after GVBD ) chromosome spreads were stained with H3K9me3 antibody and CREST serum to reveal pericentromeric heterochromatin ( pink ) and the centromere ( green ) , respectively . Chromosomes were stained with Hoechst ( blue ) . Below , schematic representation of a bivalent in metaphase I and a dyad in metaphase II . Enlargement shows the centromere region and a region that we call " chromatid junction " with the corresponding scheme . D ) Quantification of total Sgo2 signal at the centromere and the chromatid junction , normalized to CREST at centromeres , on chromosome spreads from oocytes at the indicated stages of meiotic maturation . Each dot shows mean per oocyte ; the number of oocytes analyzed is indicated . Below : scheme illustrating how Sgo2 signals ( in red ) were measured at the indicated meiotic stages . Ifluor stands for a mean fluorescence intensity of the area within the box ( dotted lines , see Material and Methods for quantification approach ) . E ) Chromosome spreads as in A ) , stained for Sgo2 ( red ) , CREST ( green ) , and DNA ( Hoechst , in blue ) . Spreads in early anaphase I and late anaphase I - telophase I were classified , depending on whether dyads of two chromosome sets had been found close to each other as one group , or were scattered apart into two groups . In early anaphase I , Sgo2 is found inbetween sister kinetochores that are already separated . F ) Quantification of E ) showing total Sgo2 signal in the centromere region relative to CREST per kinetochore pair ( dot plot on the left ) , and distance between sister kinetochores ( dot plot on the right ) at the indicated stages of meiosis I . Meta : metaphase , ana : anaphase , telo : telophase . Each dot represents one kinetochore pair ; the number of kinetochore pairs analysed is indicated . Below : scheme of Sgo2 signal ( in red ) measurements .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1 A) Oocytes were fixed for chromosome spreads 8 hours after GVBD, and stained for PP2A-c (red), Sgo2 (green), CREST (blue) and DNA (Hoechst, shown in grey). Spreads were classified into metaphase I and anaphase I, depending on whether chromosome segregation had taken place, or not. Arrowhead indicates fused sister kinetochores, arrows indicate separated sister kinetochores.  B) Example of a whole mount oocyte staining, in early anaphase I after cold-treatment to visualize only cold-stable microtubule fibers, stained with anti-tubulin antibody (green). Kinetochores are stained with CREST serum (red) and chromosomes with Hoechst (blue). Shown is an overlay of 20 z-sections. Arrows indicate separated sister kinetochores.  C) Metaphase I (6 hours after GVBD) and metaphase II (16-20 hours after GVBD) chromosome spreads were stained with H3K9me3 antibody and CREST serum to reveal pericentromeric heterochromatin (pink) and the centromere (green), respectively. Chromosomes were stained with Hoechst (blue). Below, schematic representation of a bivalent in metaphase I and a dyad in metaphase II. Enlargement shows the centromere region and a region that we call "chromatid junction" with the corresponding scheme. D) Quantification of total Sgo2 signal at the centromere and the chromatid junction, normalized to CREST at centromeres, on chromosome spreads from oocytes at the indicated stages of meiotic maturation. Each dot shows mean per oocyte; the number of oocytes analyzed is indicated. Below: scheme illustrating how Sgo2 signals (in red) were measured at the indicated meiotic stages. Ifluor stands for a mean fluorescence intensity of the area within the box (dotted lines, see Material and Methods for quantification approach). E) Chromosome spreads as in A), stained for Sgo2 (red), CREST (green), and DNA (Hoechst, in blue). Spreads in early anaphase I and late anaphase I - telophase I were classified, depending on whether dyads of two chromosome sets had been found close to each other as one group, or were scattered apart into two groups. In early anaphase I, Sgo2 is found inbetween sister kinetochores that are already separated. F) Quantification of E) showing total Sgo2 signal in the centromere region relative to CREST per kinetochore pair (dot plot on the left), and distance between sister kinetochores (dot plot on the right) at the indicated stages of meiosis I. Meta: metaphase, ana: anaphase, telo: telophase. Each dot represents one kinetochore pair; the number of kinetochore pairs analysed is indicated. Below: scheme of Sgo2 signal (in red) measurements.


Figure 2 A ) Chromosome spreads at the indicated stages of meiosis , stained for endogenous Sgo2 ( red ) , centromeres with CREST serum ( green ) and DNA with Hoechst ( blue ) . On the right , schemes illustrate the chromosome figures observed , and the corresponding Sgo2 staining ( in red ) . B ) Dyads of metaphase II chromosome spreads of control , Bub1KD , Mps1 ∆ N and Reversine - treated oocytes ( from GVBD onwards ) stained with Sgo2 antibody ( red ) , CREST serum ( green ) and Hoechst ( blue ) . C ) Dyads of metaphase II chromosome spreads of oocytes treated with Reversine from GVBD + 8 hours onwards , corresponding to the time of anaphase I onset . PBE : Polar Body Extrusion . D ) Chromosome spreads at the indicated stages of meiosis , stained for endogenous Mps1 ( red ) , centromeres with CREST serum ( green ) and DNA with Hoechst ( blue ) . For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later , respectively . E ) Quantification of D ) showing Mps1 signal relative to CREST per kinetochore pair ( per single kinetochore in anaphase II ) ( left dot plot ) , and Mps1 signal averaged per oocyte ( right dot plot ) at the indicated time points . The number of kinetochore pairs ( kinetochores in anaphase II ) and oocytes analyzed is indicated . The scheme of measurements is shown on the right . F ) Percentage of oocytes harbouring one or more single sister chromatids when analysed by chromosome spreads stained with CREST and Hoechst . Where indicated , oocytes were treated with Reversine from GVBD onwards , and treatment was renewed every 8 hours . Early CSF corresponds to 8 hours after GVBD ( Reversine treated oocytes undergo anaphase I on average 4 - 5 hours after GVBD ) , and late CSF corresponds to 36 hours after GVBD ( > 29 hours in CSF arrest ) . PSCS : precocious sister chromatid segregation . G ) Quantification of total Sgo2 signal on spreads in early or late CSF arrest ( 16 hours or 30 hours after GVBD , respectively ) relative to CREST per kinetochore pair ( per single kinetochore in anaphase II ) ( left dot plot ) , and Sgo2 signal averaged per oocyte ( right dot plot ) at the indicated stages . The number of kinetochore pairs and oocytes analyzed is indicated . The scheme of measurements is shown on the right . Meta : metaphase , ana : anaphase .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2 A) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Sgo2 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). On the right, schemes illustrate the chromosome figures observed, and the corresponding Sgo2 staining (in red).  B) Dyads of metaphase II chromosome spreads of control, Bub1KD, Mps1∆N and Reversine-treated oocytes (from GVBD onwards) stained with Sgo2 antibody (red), CREST serum (green) and Hoechst (blue).  C) Dyads of metaphase II chromosome spreads of oocytes treated with Reversine from GVBD + 8 hours onwards, corresponding to the time of anaphase I onset. PBE: Polar Body Extrusion.  D) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Mps1 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later, respectively. E) Quantification of D) showing Mps1 signal relative to CREST per kinetochore pair (per single kinetochore in anaphase II) (left dot plot), and Mps1 signal averaged per oocyte (right dot plot) at the indicated time points. The number of kinetochore pairs (kinetochores in anaphase II) and oocytes analyzed is indicated. The scheme of measurements is shown on the right.  F) Percentage of oocytes harbouring one or more single sister chromatids when analysed by chromosome spreads stained with CREST and Hoechst. Where indicated, oocytes were treated with Reversine from GVBD onwards, and treatment was renewed every 8 hours. Early CSF corresponds to 8 hours after GVBD (Reversine treated oocytes undergo anaphase I on average 4-5 hours after GVBD), and late CSF corresponds to 36 hours after GVBD (>29 hours in CSF arrest). PSCS: precocious sister chromatid segregation.  G) Quantification of total Sgo2 signal on spreads in early or late CSF arrest (16 hours or 30 hours after GVBD, respectively) relative to CREST per kinetochore pair (per single kinetochore in anaphase II) (left dot plot), and Sgo2 signal averaged per oocyte (right dot plot) at the indicated stages. The number of kinetochore pairs and oocytes analyzed is indicated. The scheme of measurements is shown on the right. Meta: metaphase, ana: anaphase.


Figure 3 ( A and D ) Oocytes in meiosis I ( 6 hours after GVBD ) or meiosis II ( left and right panels , respectively ) were treated with STLC for 2 . 5 hours and fixed for whole mount immunofluorescence . Cold stable microtubules were stained with an antibody against tubulin ( green ) , centromeres with CREST ( red ) and DNA with Hoechst ( blue ) . Scale bar indicates 5 µm in ( A and D ) , and 10 µm in ( B , C , E and F ) . In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed . ( B and E ) Chromosome spreads at metaphase and anaphase of meiosis I ( 6 and 9 hours after GVBD ) or meiosis II ( left and right panels , respectively ) , in the presence or absence of STLC ( for 2 . 5 hours ) and Reversine ( + Rev . , for 1 hour ) as indicated . Spreads were stained with CREST serum ( red ) and Hoechst ( grey ) . Single chromosomes are present in anaphase I and single sister chromatids in anaphase II . Scale bar indicates 5 µm in ( A and D ) , and 10 µm in ( B , C , E and F ) . In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed . ( C and F ) Chromosome spreads exactly as described in B and E ) , except that spreads were additionally stained with an antibody against Rec8 ( green ) . The CREST signal appears in red and DNA in blue . Rev : Reversine . Scale bar indicates 5 µm in ( A and D ) , and 10 µm in ( B , C , E and F ) . In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 (A and D) Oocytes in meiosis I (6 hours after GVBD) or meiosis II (left and right panels, respectively) were treated with STLC for 2.5 hours and fixed for whole mount immunofluorescence. Cold stable microtubules were stained with an antibody against tubulin (green), centromeres with CREST (red) and DNA with Hoechst (blue). Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.  (B and E) Chromosome spreads at metaphase and anaphase of meiosis I (6 and 9 hours after GVBD) or meiosis II (left and right panels, respectively), in the presence or absence of STLC (for 2.5 hours) and Reversine (+Rev., for 1 hour) as indicated. Spreads were stained with CREST serum (red) and Hoechst (grey). Single chromosomes are present in anaphase I and single sister chromatids in anaphase II. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.  (C and F) Chromosome spreads exactly as described in B and E), except that spreads were additionally stained with an antibody against Rec8 (green). The CREST signal appears in red and DNA in blue. Rev: Reversine. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.


Figure 4 A ) Metaphase II oocytes from mice carrying a conditional , oocyte - specific invalidation of separase ( Separaseflox / flox Zp3 Cre + , or Sep - / - ) and oocytes from litter mates ( Separaseflox / flox , or Sep + / + ) were obtained after superovulation . In vivo matured control ( Sep + / + ) and Sep - / - oocytes are found in oviducts at comparable numbers , enclosed by cumulus cells . Example of cumulus enclosed oocytes from the oviduct is shown below . In ( A ) , n indicates the number of mice and a mean number of oocytes per mouse of each genotype , error bars are + s . e . m . B ) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation . Microtubules were stained with anti - tubulin antibody ( green ) , centromeres with CREST serum ( red ) and DNA with Hoechst ( blue ) . Bivalents are attached in a monopolar fashion . Approximate number of z - sections used for overlays to visualize the whole spindle ( on the left ) or individual chromosomes ( magnifications on the right ) are indicated . C ) Same as B ) , except that oocytes of the indicated genotypes were obtained after in vitro maturation from GV onwards . In ( B and C ) n indicates number of oocytes analyzed . D ) Metaphase - to - anaphase transition of meiosis II in oocytes of the indicated genotype cultured in vitro and injected with the separase constructs as indicated , in GV . The schemes indicate the chromosome figures observed upon activation . Chromosome spreads are stained with CREST serum ( green ) , and Rec8 antibody ( red ) . DNA was stained with DAPI ( blue ) . In ( D ) n indicates the number of oocytes with the shown phenotype ( e . g . , upon successful activation ) and the total number of oocytes analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Metaphase II oocytes from mice carrying a conditional, oocyte-specific invalidation of separase (Separaseflox/flox Zp3 Cre+, or Sep-/-) and oocytes from litter mates (Separaseflox/flox, or Sep+/+) were obtained after superovulation. In vivo matured control (Sep+/+) and Sep-/- oocytes are found in oviducts at comparable numbers, enclosed by cumulus cells. Example of cumulus enclosed oocytes from the oviduct is shown below. In (A), n indicates the number of mice and a mean number of oocytes per mouse of each genotype, error bars are + s.e.m.  B) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation. Microtubules were stained with anti-tubulin antibody (green), centromeres with CREST serum (red) and DNA with Hoechst (blue). Bivalents are attached in a monopolar fashion. Approximate number of z-sections used for overlays to visualize the whole spindle (on the left) or individual chromosomes (magnifications on the right) are indicated. C) Same as B), except that oocytes of the indicated genotypes were obtained after in vitro maturation from GV onwards. In (B and C) n indicates number of oocytes analyzed. D) Metaphase-to-anaphase transition of meiosis II in oocytes of the indicated genotype cultured in vitro and injected with the separase constructs as indicated, in GV. The schemes indicate the chromosome figures observed upon activation. Chromosome spreads are stained with CREST serum (green), and Rec8 antibody (red). DNA was stained with DAPI (blue). In (D) n indicates the number of oocytes with the shown phenotype (e.g., upon successful activation) and the total number of oocytes analyzed.


Figure 5 A ) Sep + / + or Sep - / - oocytes were matured in vitro until metaphase II . Sep - / - oocytes were injected with wild - type separase or cleavage mutant separase encoding mRNAs in metaphase II . Oocytes were either fixed in metaphase II or activated and fixed in anaphase II . Spreads were stained with Rec8 antibody ( red ) , CREST serum ( green ) , and DAPI ( blue ) to label DNA . n indicates the number of oocytes with the shown phenotype ( e . g . in anaphase II , after successful activation ) and the total number of oocytes analysed B ) Representative image of kinetochore individualization in Sep - / - oocytes injected with wild - type separase in metaphase II and fixed after activation , from A ) . Shown is staining with CREST serum ( green ) , and DAPI ( blue ) to label DNA . Arrows indicate separated sister kinetochores . The scheme on the left illustrates the individualization of sister kinetochores ( green arrows ) at anaphase II . n indicates the number of oocytes with the shown phenotype ( e . g . in anaphase II , after successful activation ) and the total number of oocytes analysed Scale bars : 10 µm , except bottom panel in B ) , where the scale bar is 2 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A) Sep+/+ or Sep-/- oocytes were matured in vitro until metaphase II. Sep-/- oocytes were injected with wild-type separase or cleavage mutant separase encoding mRNAs in metaphase II. Oocytes were either fixed in metaphase II or activated and fixed in anaphase II. Spreads were stained with Rec8 antibody (red), CREST serum (green), and DAPI (blue) to label DNA. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed  B) Representative image of kinetochore individualization in Sep-/- oocytes injected with wild-type separase in metaphase II and fixed after activation, from A). Shown is staining with CREST serum (green), and DAPI (blue) to label DNA. Arrows indicate separated sister kinetochores. The scheme on the left illustrates the individualization of sister kinetochores (green arrows) at anaphase II. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed Scale bars: 10 µm, except bottom panel in B), where the scale bar is 2 μm.


Figure 6 A ) Live imaging movie of GFP - Sgo2 expressing oocytes ( from GV onwards ) undergoing meiosis I . The green channel to visualize GFP - Sgo2 localized to chromosomes is shown . Timepoints were taken every 10min , shown is a montage of selected frames such as indicated ( hours : time after GVBD ) at the metaphase - to - anaphase transition of meiosis I and in metaphase II . Each timepoint is comprised of an overlay of 11 z - sections of 3μm for GFP . The arrow heads indicate bivalents . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes analyzed B ) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP - Sgo2 expressing oocyte . Spreads were stained with Rec8 antibody ( pink ) , CREST serum ( green ) , Hoechst for DNA ( blue ) , GFP - Sgo2 was visualized by GFP fluorescence ( grey ) . The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes analyzed C ) Metaphase I ( 4hrs after GVBD ) and II spreads after GFP - Sgo2 expression ( from GV onwards ) . Spreads were stained with CREST serum ( red ) , Hoechst for DNA ( grey ) , GFP - Sgo2 was visualized by GFP fluorescence ( green ) . Arrowheads mark separated sister kinetochores . On the right , graph showing percentage of metaphase II spreads containing bivalent chromosomes . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes analyzed The number of analysed oocytes in C ) D ) Chromosome spreads of oocytes expressing GFP - Sgo2 ( from GV onwards ) in metaphase II , and upon activation . Spreads were labeled with Rec8 antibody ( shown in red , far - red secondary antibody was used ) , CREST serum ( pseudo - colored in green ) , and Hoechst ( blue ) . GFP direct fluorescence was imaged for GFP - Sgo2 detection . Below the corresponding chromosome figures are represented in the schemes . E ) Quantification of Rec8 signal found around and in between CREST signals that were close to each other . Below : scheme illustrating how Rec8 signals ( in red ) were measured at the indicated meiotic stages . Ifluor stands for a mean fluorescence intensity of the area within the box ( dotted lines ) . Meta : metaphase , ana : anaphase . D ) , the number of oocytes with the shown phenotype and the total number of oocytes analyzed . On each graph , mean is shown , error bars are + s . e . m . in C ) and and ± SD in E ) , asterisks indicate significant difference ( * * * * = p < 0 , 0001 ) according to Mann - Whitney U test . AU : arbitrary units . The number of analysed kinetochore pairs in E ) is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6 A) Live imaging movie of GFP-Sgo2 expressing oocytes (from GV onwards) undergoing meiosis I. The green channel to visualize GFP-Sgo2 localized to chromosomes is shown. Timepoints were taken every 10min, shown is a montage of selected frames such as indicated (hours: time after GVBD) at the metaphase-to-anaphase transition of meiosis I and in metaphase II. Each timepoint is comprised of an overlay of 11 z-sections of 3μm for GFP. The arrow heads indicate bivalents. In (A-C) n indicates the number of control non-injected and GFP-Sgo2-expressing oocytes analyzed  B) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP-Sgo2 expressing oocyte. Spreads were stained with Rec8 antibody (pink), CREST serum (green), Hoechst for DNA (blue), GFP-Sgo2 was visualized by GFP fluorescence (grey). The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms. In (A-C) n indicates the number of control non-injected and GFP-Sgo2-expressing oocytes analyzed  C) Metaphase I (4hrs after GVBD) and II spreads after GFP-Sgo2 expression (from GV onwards). Spreads were stained with CREST serum (red), Hoechst for DNA (grey), GFP-Sgo2 was visualized by GFP fluorescence (green). Arrowheads mark separated sister kinetochores. On the right, graph showing percentage of metaphase II spreads containing bivalent chromosomes. In (A-C) n indicates the number of control non-injected and GFP -Sgo2-expressing oocytes analyzed The number of analysed oocytes in C) D) Chromosome spreads of oocytes expressing GFP-Sgo2 (from GV onwards) in metaphase II, and upon activation. Spreads were labeled with Rec8 antibody (shown in red, far-red secondary antibody was used), CREST serum (pseudo-colored in green), and Hoechst (blue). GFP direct fluorescence was imaged for GFP-Sgo2 detection. Below the corresponding chromosome figures are represented in the schemes. E) Quantification of Rec8 signal found around and in between CREST signals that were close to each other. Below: scheme illustrating how Rec8 signals (in red) were measured at the indicated meiotic stages. Ifluor stands for a mean fluorescence intensity of the area within the box (dotted lines). Meta: metaphase, ana: anaphase. D), the number of oocytes with the shown phenotype and the total number of oocytes analyzed. On each graph, mean is shown, error bars are +s.e.m. in C) and and ± SD in E), asterisks indicate significant difference (**** = p<0,0001) according to Mann-Whitney U test. AU: arbitrary units. The number of analysed kinetochore pairs in E) is indicated.


Figure 1Giemsa staining of a femoral section from 8 - day - old mice showing endochondral ossification defects . Scale bar : 100 μm . Representative femur sections from 8 - day - old wild - type and Dnmt2 − / − mice . The staining of perivascular cells ( GLS , green ) is strongly reduced in Dnmt2 − / − bone marrow . Nuclei are counterstained with DAPI . Scale bar : 100 μm . Quantitative analysis of capillary numbers . GLS - positive capillaries were counted in wild - type ( wt ) and Dnmt2 − / − ( D2 - ) mice , in three independent high - power fields ( HPF ) per mouse ( n = 7 ) . Quantification of cell numbers in haematopoietic tissues of 8 - day - old mice ( n = 8 ) . Representative FACS plots of wild - type and Dnmt2 − / − BM stained for Lin − Sca1 + and cKit + cells . Quantification of bone marrow LSK cells in wild - type ( three independent experiments , each with pooled BM samples from 4 to 6 mice ) and Dnmt2 − / − bone marrow ( three independent experiments , each with pooled BM samples of 5 - 9 mice ) . Restoration of the LSK cell population in adult Dnmt2 − / − mice . Frequencies of LSK cells were normalized to the frequencies of LSK cells from the bone marrow of wild - type littermates at the indicated age ( 2 - 3 independent experiments , each with pooled bone marrow samples from 3 to 9 mice ) . Increased LSK CD34 − and reduced LSK CD34 + cell populations in 8 - day - and 1 - year - old Dnmt2 − / − mice compared to wild - type ( two independent experiments , each with pooled bone marrow samples from 3 - 9 mice ) .

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 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 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 B-PANEL_START 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 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 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

Figure 1Giemsa staining of a femoral section from 8-day-old mice showing endochondral ossification defects. Scale bar: 100 μm.Representative femur sections from 8-day-old wild-type and Dnmt2−/− mice. The staining of perivascular cells (GLS, green) is strongly reduced in Dnmt2−/− bone marrow. Nuclei are counterstained with DAPI. Scale bar: 100 μm.Quantitative analysis of capillary numbers. GLS-positive capillaries were counted in wild-type (wt) and Dnmt2−/− (D2-) mice, in three independent high-power fields (HPF) per mouse (n = 7).Quantification of cell numbers in haematopoietic tissues of 8-day-old mice (n = 8).Representative FACS plots of wild-type and Dnmt2−/− BM stained for Lin− Sca1+ and cKit+ cells.Quantification of bone marrow LSK cells in wild-type (three independent experiments, each with pooled BM samples from 4 to 6 mice) and Dnmt2−/−bone marrow (three independent experiments, each with pooled BM samples of 5-9 mice).Restoration of the LSK cell population in adult Dnmt2−/− mice. Frequencies of LSK cells were normalized to the frequencies of LSK cells from the bone marrow of wild-type littermates at the indicated age (2-3 independent experiments, each with pooled bone marrow samples from 3 to 9 mice).Increased LSK CD34− and reduced LSK CD34+cell populations in 8-day- and 1-year-old Dnmt2−/− mice compared to wild-type (two independent experiments, each with pooled bone marrow samples from 3-9 mice).