Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: Validation of Firre knockout in MEFs. a qRT-PCR analysis of Firre expression in wild-type and knockout MEFs. Error bars: s.e.m. b Plot showing transcripts per million (TPM) values for wild-type and Firre KO MEF RNA-seq. Error bars: s.d. c CTCF ChIP-seq signal tracks showing the complete loss of CTCF binding at the Firre locus in Firre KO MEFs (mm9, chrX:47.8–49 Mb). d , e Hi-C reads per million (RPM) values for the Firre locus in ( e ) wild-type and ( f ) Firre KO MEFs

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: Biomarker Discovery, Knock-Out, Quantitative RT-PCR, Expressing, RNA Sequencing, ChIP-sequencing, Binding Assay, Hi-C

Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: Firre KO does not result in disruption of TAD boundaries. a-d Hi-C heatmaps showing ± 5 Mb of the Firre gene locus (mm9, chr.X: 45–51 Mb) in female wild-type and Firre KO MEFs, ( b ) male wild-type and Firre KO MEFs, ( c ) allele-specific haploid chromosomes for female Cast (wild type) and C57BL6 ( Firre KO), and ( d ) male C57BL6 ( Firre KO). The TAD boundaries and the insulation plot for each Hi-C dataset is depicted below. e Dot plots showing the boundary strength of the Firre -centered and the neighboring TAD boundaries in wildtype (gray) and Firre KO (red) samples. The TAD insulation scores of all TAD boundaries on chromosome X is shown as boxplots on the right panel. f Hi-C heatmaps from wild-type (grown on 2i) and Firre KO mouse embryonic stem cells (mESCs) (grown on feeders + 2i) showing ± 5 Mb of the Firre locus. g , h Boxplot showing the inter-TAD interaction frequency between the TAD domains neighboring the Firre locus in wild-type (gray) and Firre KO (red) cells ( g ) in female MEFs and ( h ) in mESCs. Error bars: s.d. (* p -value: Wilcoxon rank-sum test)

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: Disruption, Hi-C, Insulation

Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: TAD boundaries are preserved upon ectopic Firre cDNA insertion and its induced expression at target sites. a Cartoon depicting the approach for the generation of the transgenic MEFs with endogeous Firre KO and ectopic Firre cDNA insertions. b qRT-PCR showing the induced expression of the Firre cDNA in wild type, DOX − , and DOX + conditions. Error bars: s.e.m. (* p -value: t -test). c Chromosome ideograms showing the Firre cDNA insertion sites on four different chromosomes. d CTCF ChIP-seq signal from DOX − transgenic MEFs for each of the exons of Firre cDNA at randomly inserted loci. As the transgenic MEFs harbor an endogenous Firre deletion, the intronic regions did not harbor any ChIP-seq signal. e-h Hi-C heatmaps showing the TAD organization, TAD boundary position, and the insulation plots for male Firre KO, DOX − , and DOX + samples ± ~ 5 Mb of Firre cDNA insertion sites on ( e ) chr 8, ( f ) chr 10, ( g ) chr 11, and ( h ) chr 15

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR, ChIP-sequencing, Hi-C, Insulation

Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: Firre KO results in a loss of Firre -DXZ4, and changes in super-loop interactions in female MEFs. a Hi-C heatmaps at 40 kb resolution in male and female wildtype and Firre KO MEFs showing the interaction frequency between Firre and DXZ4 ± 1 Mb. b Forty-two windows of 40 kb bins were slid across the entire heatmap (2 Mb × 2 Mb in size) to compare the interaction frequency at each position of female and male WT versus KO conditions to the Firre -DXZ4 interaction by t -tests. The p -value distribution in the female or male wild-type samples indicate a drastically enriched significance of Firre -DXZ4 interactions when compared with either the female Firre KO or male samples (* p < 0.05, one-way ANOVA). c Hi-C heatmap at 100 kb resolution showing the zoomed-in interactions between the mouse Firre , DXZ4, x75, and ICCE regions ± 100 kb in female and male wild-type and Firre KO MEFs. d Boxplots showing the interactions among the super-loop regions in wild-type (gray) and Firre KO (red) female (top) and male (bottom) MEF samples. The sample sizes of the boxplots are n = 12 for Firre -DXZ4, DXZ4-x75, and DXZ4-ICCE interactions, and n = 9 for all other combinations. p -value: t -test. Error bars: s.d

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: Hi-C

Journal: Nature Communications

Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

doi: 10.1038/s41467-018-03614-0

Figure Lengend Snippet: CRISPR live-cell imaging and 3C validates the changes in super-loop interactions. a Four-color CRISPR live-cell imaging (CLING) from female wild-type and Firre KO MEFs. Firre (red), DXZ4 (green), and x75 (white) loci were simultaneously visualized with Hoechst staining (blue). Pseudo-coloring was used for visual simplicity. Scale bar: 5 μm. b Quantification of the colocalization percentages between Firre -DXZ4, DXZ4-x75, and Firre -x75 between wild-type (black) and Firre KO (red) MEFs. (* p -value: χ 2 -test, n > 80 nuclei). Error bars: s.e.m. c Chromosome conformation capture (3C) analysis showing the interaction frequency ratios of Firre -DXZ4, DXZ4-x75, and Firre -x75 in female Firre KO vs. wild-type MEFs (* p -value: t -test, n = 3). The black arc indicates 3C enrichment in the wild-type samples, whereas the red arc represents enrichment in the Firre KO samples

Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

Techniques: CRISPR, Live Cell Imaging, Staining

Journal: Molecular cell

Article Title: The Elongation Factor Spt6 Maintains ESC Pluripotency by Controlling Super-Enhancers and Counteracting Polycomb Proteins

doi: 10.1016/j.molcel.2017.09.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CF-1 MEF feeder, irradiated , MTI-GlobalStem , GSC-6001G.

Techniques: Recombinant, Transfection, Protease Inhibitor, SYBR Green Assay, Reporter Assay, Irradiation, Negative Control, Plasmid Preparation, Mutagenesis, Software

Journal: Molecular cell

Article Title: The Elongation Factor Spt6 Maintains ESC Pluripotency by Controlling Super-Enhancers and Counteracting Polycomb Proteins

doi: 10.1016/j.molcel.2017.09.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell lines E14 ESCs were grown initially on CF-1 MEF feeder (GlobalStem), passaged feeder-free on gelatin-coated dishes in ES medium (DMEM [GIBCO], 15% HyClone FBS [GE Healthcare], GlutaMAX [GIBCO], MEM nonessential amino acids [GIBCO], sodium pyruvate [GIBCO], leukaemia inhibitory factor [LIF] [Millipore; ESGRO], 0.1 mM β-mercaptoethanol, and antibiotics) or subjected to differentiation by withdrawing LIF for 3 days.

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Reverse Transcription, SYBR Green Assay, Reporter Assay, Irradiation, Negative Control, Plasmid Preparation, Mutagenesis, Software