Journal: Cell Proliferation

Article Title: The effect of trisomic chromosomes on spatial genome organization and global transcription in embryonic stem cells

doi: 10.1111/cpr.13639

Figure Lengend Snippet: Compartments and TADs are unaffected by trisomic chromosomes. (A) Representative genomic tracks showing A/B compartments in WT and trisomic mESCs. (B) The proportions of genomic regions with switched A/B compartment status between WT and trisomic mESCs (left box) and between replicates of each cell line (right box). Two‐sided Wilcoxon rank‐sum test was used. p Value = 0.4252. NS: not significant. (C) The distribution of A/B compartment segregation scores in WT and trisomic mESCs. The segregation score of a chromosome is the ratio of the interactions of different types of compartments (A–B interaction) to the sum of the interactions of the same type of compartments (A–A interaction and B–B interaction). Each boxplot consists of the segregation scores of all individual chromosomes. Two‐sided Wilcoxon rank‐sum test was used. From left to right, p value = 0.7788, 0.6588, 0.6205 and 0.4945. NS: not significant. Representative chromatin interaction heatmaps, TAD domains and insulation scores (IS) in different mESC samples (bin resolution: 40 kb). (E) The distribution of TAD lengths and the number of TADs in different mESC samples and the E14 cell line. Two‐sided Wilcoxon rank‐sum test was used to compare each trisomic cell and E14 with WT, respectively. From left to right, p value = 0.54, 0.3093, 0.3164, 0.689 and 0.6493. NS: not significant. (F) The TAD boundaries are largely conserved (distance <1 bin) between WT and trisomic mESCs. More than 50% of the TAD boundaries in WT and trisomic mESCs were overlapped, and about 90% of the TAD boundaries were shifted within 80 kb. (G) The distributions of TAD compaction scores in WT and trisomic mESCs are overlapping. Two‐sided Wilcoxon rank‐sum test was used to compare each trisomic cell with WT, respectively. From Ts6, Ts8, Ts11 to Ts15, p value = 0.7785, 0.2301, 1.776e−06 and 0.642. *** p Value < 0.001, NS: not significant. mESC, mouse embryonic stem cell; WT, wild‐type.

Article Snippet: When mESCs reached 70%–80% confluence on the coverslips, Alexa 647‐5‐EU was added to the culture medium at the concentration of 1 mM for 1 h. After that, cells were fixed with a mixture of methanol and acetic acid (3:1) under −20°C for 20 min. Then the samples were labelled with commercial multicolour FISH probes for specific mouse chromosomes (XMP 8/9/12, MetaSystems) and proceeded following established FISH procedures.

Techniques: Insulation

Journal: Cell Proliferation

Article Title: The effect of trisomic chromosomes on spatial genome organization and global transcription in embryonic stem cells

doi: 10.1111/cpr.13639

Figure Lengend Snippet: Inter‐chromosomal interactions are non‐random and preserved in trisomic mESCs. (A) Genome‐wide inter‐chromosomal proximity score matrices of the mESCs and mouse cortical neurons (CNs). (B) Scatter plots of the inter‐chromosomal proximity scores of sample pairs. Pearson's correlation coefficients are displayed in the figures. (C) The relationship between the inter‐chromosomal proximity score of each chromosome with chromosome 11 ( y ‐axis) and the gene density (gene number/Mb) of each chromosome ( x ‐axis). The chromosomes are coloured by chromosome‐wide expression level, defined as the average FPKM (fragments per kilobase per million mapped reads) of all genes on each chromosome. Top: WT mESC; bottom: Ts11 mESC. (D) Scatter plot of inter‐chromosomal proximity scores between the indicated chromosomes with chromosome 11 in WT ( x ‐axis) and Ts11 ( y ‐axis) mESCs. Chromosomes whose scores change most between the two samples are labelled in red. (E) The distribution of gene expression changes between WT and Ts11 mESCs for genes on chromosomes 2 and 12 (increased proximity score with chromosome 11 in Ts11), and on chromosomes 8 and 19 (decreased proximity score with chromosome 11 in Ts11). One‐sample one‐sided Wilcoxon signed‐rank tests. H0: the median is greater than 0 (for chromosomes 2 and 12); the median is less than 0 (for chromosomes 8 and 19). From left to right, p value = 0.9969, 1, 8.943e−5 and 0.4255. *** p value < 0.001, NS: not significant. mESC, mouse embryonic stem cell; WT, wild‐type.

Article Snippet: When mESCs reached 70%–80% confluence on the coverslips, Alexa 647‐5‐EU was added to the culture medium at the concentration of 1 mM for 1 h. After that, cells were fixed with a mixture of methanol and acetic acid (3:1) under −20°C for 20 min. Then the samples were labelled with commercial multicolour FISH probes for specific mouse chromosomes (XMP 8/9/12, MetaSystems) and proceeded following established FISH procedures.

Techniques: Genome Wide, Expressing

Journal: Cell Proliferation

Article Title: The effect of trisomic chromosomes on spatial genome organization and global transcription in embryonic stem cells

doi: 10.1111/cpr.13639

Figure Lengend Snippet: Coupled chromosome painting and nascent RNA imaging reveal higher transcriptional activity at chromosome intermingling regions. (A) Representative simultaneous chromosome painting and nascent RNA imaging results of WT mESCs. (B) The distribution of nucleus volume of WT and Ts8 mESCs. Two‐sided Wilcoxon rank‐sum test, p value = 0.95. NS: not significant. From left to right: sample size n = 514, 324 nuclei. (C) The distribution of chromosome volume of chromosomes 8, 9 and 12 in WT and Ts8 mESCs. Two‐sided Wilcoxon rank‐sum tests. From left to right: p value = 1.7e−11, 0.0023, 2.3e−7; n = 350, 315, 424, 210, 354 and 105 chromosomes. ** p Value < 0.01, *** p value < 0.001. (D) The distribution of intermingling index between chromosomes pairs in WT or Ts8 mESCs. The intermingling index between the two chromosomes A and B is defined as (volume of interaction region) 2 /(chromosome A volume*chromosome B volume). Two‐sided Wilcoxon rank‐sum tests. From left to right: p value = 0.006 and 0.71; n = 19, 12 and 42 chromosome pairs. ** p value < 0.01, NS: not significant. (E) The distribution of the number of nascent RNAs overlapping with chromosome 8, 9 or 12 in WT and Ts8 mESCs. Two‐sided Wilcoxon rank‐sum tests. From left to right: p value = 1.6e−5, 0.00075, 0.23, 1.7e−14, 3.7e−11 and 0.94; n = 351, 431, 358, 310, 205 and 100 chromosomes. *** p Value < 0.001, NS: not significant. (F) The distribution of transcriptional intensity of individual chromosomes (8, 9 and 12) or chromosome intermingling regions in WT and Ts8 mESCs. “Chr8–9” (chr8–12) indicates the intermingling region of chromosome 8 and chromosome 9 (chromosome 8 and chromosome 12). Transcriptional intensity is defined as the number of nascent RNAs overlapping a chromosome/region, divided by the volume of the chromosome/region. From left to right: n = 325, 399, 1120, 352, 344, 2105, 277, 208, 375, 165, 107 and 741 chromosomes/regions. Two‐sided Wilcoxon rank‐sum tests, all the p values < 2.22e−16. *** p Value < 0.001. mESC, mouse embryonic stem cell; WT, wild‐type.

Article Snippet: When mESCs reached 70%–80% confluence on the coverslips, Alexa 647‐5‐EU was added to the culture medium at the concentration of 1 mM for 1 h. After that, cells were fixed with a mixture of methanol and acetic acid (3:1) under −20°C for 20 min. Then the samples were labelled with commercial multicolour FISH probes for specific mouse chromosomes (XMP 8/9/12, MetaSystems) and proceeded following established FISH procedures.

Techniques: Imaging, Activity Assay

Journal: Cell Proliferation

Article Title: The effect of trisomic chromosomes on spatial genome organization and global transcription in embryonic stem cells

doi: 10.1111/cpr.13639

Figure Lengend Snippet: Transcriptional upregulation of trisomic chromosomes. (A) The distribution of expression fold changes between trisomic and WT mESCs of the genes on each chromosome. Genes whose FPKMs less than 1 in both comparing samples are not included. (B) The distribution of differentially expressed genes (DEGs) on different chromosomes. (C). Gene ontology (GO) enrichment analysis of shared up‐ and downregulated genes in trisomic relative to WT mESCs. (D) KEGG pathway enrichment analysis of shared differentially expressed genes in trisomic relative to WT mESCs. KEGG, Kyoto Encyclopedia of Genes and Genomes; mESC, mouse embryonic stem cell; WT, wild‐type.

Article Snippet: When mESCs reached 70%–80% confluence on the coverslips, Alexa 647‐5‐EU was added to the culture medium at the concentration of 1 mM for 1 h. After that, cells were fixed with a mixture of methanol and acetic acid (3:1) under −20°C for 20 min. Then the samples were labelled with commercial multicolour FISH probes for specific mouse chromosomes (XMP 8/9/12, MetaSystems) and proceeded following established FISH procedures.

Techniques: Expressing

Journal: Cell Proliferation

Article Title: The effect of trisomic chromosomes on spatial genome organization and global transcription in embryonic stem cells

doi: 10.1111/cpr.13639

Figure Lengend Snippet: Chromosome territories, inter‐chromosomal interactions and gene transcription in WT and trisomic mESCs. Hi‐C, RNA‐seq and imaging data showed reduced chromosome volume of both trisomic and disomic chromosomes. Radial location of chromosomes and inter‐chromosomal interaction preferences were retained in trisomic mESCs, and chromosome intermingling regions were associated with active transcription in both WT and trisomic mESCs. mESC, mouse embryonic stem cell; WT, wild‐type.

Article Snippet: When mESCs reached 70%–80% confluence on the coverslips, Alexa 647‐5‐EU was added to the culture medium at the concentration of 1 mM for 1 h. After that, cells were fixed with a mixture of methanol and acetic acid (3:1) under −20°C for 20 min. Then the samples were labelled with commercial multicolour FISH probes for specific mouse chromosomes (XMP 8/9/12, MetaSystems) and proceeded following established FISH procedures.

Techniques: Hi-C, RNA Sequencing Assay, Imaging

Journal: Development (Cambridge, England)

Article Title: IRX3/5 regulate mitotic chromatid segregation and limb bud shape

doi: 10.1242/dev.180042

Figure Lengend Snippet: IRX3 associates with SMC1 and CUX1 in vivo. (A) Lysate from 30 WT E10.5 hindlimb buds was immunoprecipitated (IP) with anti-IRX3 and immunoblotted against SMC1, SMC3 or CUX1. Three separate co-IP experiments were performed using multiple limb buds on each occasion. Representative full-length western blots (WB) are shown, and arrows indicate expected band size. (B) IRX3 protein localised in anterior-proximal (AP), but not posterior-distal (PD), nuclei in mesoderm of 29 som. limb bud. (C) STED microscopy revealed that IRX3 puncta contacted those of SMC1 (anterior versus posterior, P=0.008) and CUX1 (anterior versus posterior, P=0.005) in anterior mesoderm of the hindlimb field at 29-32 som. (25-30 cells, n=3 embryos, unpaired, two-tailed t-test, error bars indicate s.e.m.). (D,E) PLA in vivo demonstrated physical association of IRX3 with SMC1 (8/82, 9.7%; D) and CUX1 (8/64, 12.5%; E) in anterior half mesoderm at 29-30 som. (n=3 embryos per condition). Scale bars: 400 µm (B, left); 20 µm (B, right); 5 µm (C-E).

Article Snippet: Antibodies company and catalogue number IRX3 [Novus Biologicals, 1D7, H0079191-M05, mouse, 1:250 – used for immunofluorescence (IF)]; IRX3 (Santa Cruz Biotechnology, G-6, 166877, mouse, 1:100 – used for co-IP in vivo ); SMC1 (Abcam, ab21583, rabbit, 1:250 IF, 1:1000 – used for immunoblotting (IB); SMC3 (Novus Biological, NB100-207 IF, in vitro IB; Abcam, ab9263, 1:1000 in vivo IB); NIPBL (Santa Cruz Biotechnology, C-9, sc-374625, 1:1000); CUX1 (Abcam, ab140042, mouse, 1:250 IF, in vitro IB; Abcam, ab230844, rabbit, 1:2500 in vivo IB); IgG (NA931V, GE Healthcare, 1:100) or donkey anti-rabbit IgG (NA934V, GE Healthcare, 1:100) co-IP in vitro , IgG (Santa Cruz Biotechnology, 2343, 1:100) co-IP in vivo ; Caspase-3 (BD Biosciences, 559565, 1:250); F-Actin (Alexa Fluor 633 Phalloidin, Thermo Fisher Scientific, A22284, 1:1000).

Techniques: In Vivo, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Microscopy, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: IRX3/5 regulate mitotic chromatid segregation and limb bud shape

doi: 10.1242/dev.180042

Figure Lengend Snippet: IRX3/5 maintain cohesin subunits and CUX1 in vivo. (A,B) Immunofluorescence intensities of SMC1, NIPBL, SMC3 and CUX1 were diminished in the absence of Irx3/5 in anterior (A), but not posterior (B), limb bud mesoderm. (C) Quantification of A and B (29-32 som.; n=3 embryos per condition; *P=0.008 for anterior SMC1, *P=0.003 for anterior NIPBL, *P=0.004 for anterior SMC3, *P=0.004 for anterior CUX1; unpaired, two-tailed t-test, error bars indicate s.e.m.). (D,E) Transcription of Smc1a, Smc1b and Cux1 were unchanged by real time RT-PCR in anterior and posterior limb bud tissue in Irx3/5+/− and Irx3/5−/− embryos (29-30 som., n=3 embryos for each of three independent experiments). Error bars indicate s.e.m. Scale bars: 20 µm.

Article Snippet: Antibodies company and catalogue number IRX3 [Novus Biologicals, 1D7, H0079191-M05, mouse, 1:250 – used for immunofluorescence (IF)]; IRX3 (Santa Cruz Biotechnology, G-6, 166877, mouse, 1:100 – used for co-IP in vivo ); SMC1 (Abcam, ab21583, rabbit, 1:250 IF, 1:1000 – used for immunoblotting (IB); SMC3 (Novus Biological, NB100-207 IF, in vitro IB; Abcam, ab9263, 1:1000 in vivo IB); NIPBL (Santa Cruz Biotechnology, C-9, sc-374625, 1:1000); CUX1 (Abcam, ab140042, mouse, 1:250 IF, in vitro IB; Abcam, ab230844, rabbit, 1:2500 in vivo IB); IgG (NA931V, GE Healthcare, 1:100) or donkey anti-rabbit IgG (NA934V, GE Healthcare, 1:100) co-IP in vitro , IgG (Santa Cruz Biotechnology, 2343, 1:100) co-IP in vivo ; Caspase-3 (BD Biosciences, 559565, 1:250); F-Actin (Alexa Fluor 633 Phalloidin, Thermo Fisher Scientific, A22284, 1:1000).

Techniques: In Vivo, Immunofluorescence, Two Tailed Test, Quantitative RT-PCR