Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: The binding of TRF2 outside of telomeric regions (non-telomeric) is dependent on telomere length. When telomeres shorten, the presence of TRF2 at telomeres diminishes, resulting in increased TRF2 binding at non-telomeric sites. This shift in TRF2 distribution triggers epigenetic modifications at promoters showing telomere-dependent gene regulation. Described in .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Binding Assay

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) Telomer length (TL) by flow-cytometry analysis of telomeric signal (FITC labelled PNA probe) showing increase in TL in HT1080-LT cells relative to HT1080 cells; plotted along x-axis in log scale. ( B ) TERT and TERC expression as expected was enhanced in HT1080-LT relative to HT1080 cells (normalised to GAPDH). [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) Immuno-histochemical (IHC) staining of previously reported and well-characterised cells with short/long telomeres (fibrosarcoma HT1080 or the long-telomere version HT1080-LT cells in cell-isogenic background) hybridised with telomere-specific FITC-labelled PNA probe, counterstained with DAPI (left panel); telomeric signal was enhanced in HT1080-LT cells (quantified in right panel). [N=15] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) Volcano plot of expressed genes in RNA-seq in HT1080 or HT1080-LT cells; x-axis has log-2 fold change for genes and the y-axis represents adjusted p values in log-10 scale. Key inflammation-related genes that have been marked in figure – most differentially expressed genes like IL1R1 , IL1B and IL10 were found to have higher expression in HT1080 cells compared to HT1080-LT cells. TERT which (as expected) is significantly lower in HT1080 cells compared to HT1080-LT cells have been also marked. Upregulated and downregulated genes listed in . ( E ) Gene Ontology (KEGG) for upregulated genes in HT1080 cells over HT1080-LT cells. ( F ) Gene Ontology (KEGG) for down-regulated genes in HT1080 cells over HT1080-LT cells. ( G ) Occupancy of TRF2 by ChIP-qPCR checked in HT1080 cells for cytokine-related genes. Enrichment was plotted relative to mock IgG (post normalisation to 1% input) [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( H ) TRF2 occupancy on the IL1R1 promoter in HT1080, MDAMB231, HEK293T or MRC5 cells by chromatin-immunoprecipitation (ChIP) followed by qPCR. Primers spanning +200 to –1000 bp of TSS used for TRF2 enrichment on the gene promoter; 3’UTR and –20 Kb upstream of TSS used as negative control for TRF2 binding; fold-change of occupancy was calculated over mock IgG after normalizing signal to 1% input. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( I ) IL1R1 protein levels in HT1080 and HT1080-LT cells by western blot; GAPDH was used as loading control. ( J ) mRNA expression of hTERT and TERC (normalised to 18 S) in xenograft tumours in NOD-SCID mice injected with HT1080 or HT1080-LT cells. 18 S has been used as a normalizing control. [N=5] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( K ) hTERT mRNA expression in hTERT-inducible HT1080 cells on treatment with different concentrations of doxycycline [N=2] (left panel) and at different days following induction [N=3] (right panel). GAPDH was used for normalisation. Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( L ) Relative telomere length by qRT-PCR in MDAMB231 or MDAMD231-LT (long telomere) cells in three independent experiments. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 1—figure supplement 2—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 1—figure supplement 2—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 1—figure supplement 2—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Flow Cytometry, Expressing, Immunohistochemistry, RNA Sequencing, ChIP-qPCR, Chromatin Immunoprecipitation, Negative Control, Binding Assay, Western Blot, Control, Injection, Quantitative RT-PCR

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) Occupancy of TRF2 was checked by ChIP-qPCR on the IL1R1 promoter spanning +200 to –1000 bp of TSS was reduced ex vivo HT1080 cells with long telomeres (HL1080-LT) relative to ones with short telomeres (HT1080) cells (N=3 in each case); IL1R1-3’UTR or a region 20 kb upstream were used as negative controls for TRF2 binding. Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( B ) mRNA expression of IL1R1 in ex vivo HT1080 or HT1080-LT cells; GAPDH was used for normalisation [N=3]. Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) IL1R1 protein expression was checked by immuno-flow cytometry in HT1080 or HT1080-LT cells in three independent replicates; Mean IL1R1 expression has been plotted along the X-axis in log scale (right panel) [N=3]. Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) IL1R1 levels by immunofluorescence microscopy in HT1080 or HT1080-LT cells; cells were stained with DAPI for marking the cell nucleus; IL1R1 levels from 25 individual cells shown in graph (right panel). [N=25] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) Telomere length of the xenograft tumours made in NOD SCID mice using HT1080 cells with long telomeres (HL1080-LT) and short telomeres (HT1080) was checked by flow-cytometry and was higher in HT1080-LT xenograft tumours. Following this, IL1R1 expression was checked by flow cytometry for these samples. Mean Telomeric signal and Mean IL1R1 expression has been plotted respectively (panels below) [N=5] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( F ) Occupancy of TRF2 by ChIP-qPCR at the IL1R1 promoter spanning +200 to –1000 bp of TSS was reduced in xenograft tumours made from HT1080 cells with long telomeres (HL1080-LT) relative to ones with short telomeres (HT1080) cells (N=3 in each case; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls for TRF2 binding). [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( G ) mRNA expression of IL1R1 in xenograft tumours (HT1080 or HT1080-LT cells); 18 S was used for normalisation. [N=5] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( H ) Telomere length by flow-cytometry of telomeric signal in HT1080 cells (hTERT-inducible stable line) following 0, 1, 6, 12, or 20 days of hTERT induction; HT1080-LT shown as positive control for enhanced telomere length; mean telomeric signal (FITC) plotted bar graph (left). [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( I ) TRF2 occupancy ChIP-qPCR on the IL1R1 promoter spanning +200 to –1000 bp of TSS in HT1080 cells following 0, 1, 6, or 12 days of hTERT induction in ex vivo culture; primers and normalisation as in ; IL1R1-3’UTR or a region 20 kb upstream used as negative controls. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( J ) IL1R1 mRNA expression (normalised to GAPDH ) in HT1080 cells following 0, 1, 6, or 12 days of hTERT induction in ex vivo culture. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( K ) TRF2 occupancy by ChIP-qPCR on the IL1R1 promoter in MDAMB23 or MDAMD231-LT cells using primers and normalisation described earlier. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( L ) Expression for IL1R1 in MDAMB231 cells or MDAMD231-LT cells; GAPDH was used for normalisation. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values.

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: ChIP-qPCR, Ex Vivo, Binding Assay, Expressing, Flow Cytometry, Immunofluorescence, Microscopy, Staining, Positive Control

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) IL1R1 expression by qRT PCR following expression of TRF2-flag or TRF2 silencing (48 hr of transient transfection); GAPDH was used for normalisation. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( B–C ) TRF2 ChIP-qPCR spanning +200 to –1000 bp of IL1R1 TSS for TRF2 occupancy ( B ) and promoter activity (luciferase reporter including –1500 bp TSS); ( C ) following expression of flag-tag TRF2 full-length, or the mutants TRF2-delB or TRF2-delM; anti-flag antibody used for ChIP; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) TRF2 and IL1R1 levels by western blots following TRF2 induction with 2 or 4 µg/ml doxycycline (Dox) in HT1080 cells (stably transformed for Dox-inducible TRF2); GAPDH as loading. ( E ) TRF2 and IL1R1 expression in control or TRF2-induced HT1080 cells by flow-cytometry; TRF2/IL1R1 in x-axis in log scale for 20,000 cells. ( F ) IL1R1 levels in control or TRF2-induced HT1080 cells. CD44 used for cell-surface marker and nuclei were stained with DAPI; 25 cells in each condition were scored and plotted in the summary graph. [N=25] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( G ) TRF2 (left panel), IL1R1 (right panel) levels from xenograft tumours in NOD-SCID mice (control or doxycycline-induced TRF2 in HT1080 cells; N=6 mice in each group) by immuno-flow cytometry; mean fluorescence signal from individual tumours in control or TRF2-induced tumours plotted in adjacent graphs. [N=6] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 2—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 2—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 2—source data 3. Original image files for western blot or .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Expressing, Quantitative RT-PCR, Transfection, ChIP-qPCR, Activity Assay, Luciferase, FLAG-tag, Western Blot, Stable Transfection, Transformation Assay, Control, Flow Cytometry, Marker, Staining, Fluorescence

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) IL1R1 protein levels on TRF2-flag induction (left) or TRF2 silencing (right) in HT1080 cells following 48 hr of transfection. ( B ) IL1R1 mRNA expression in MDAMB231 cells following expression of TRF2 cDNA or TRF2 silencing for 48 hr; GAPDH used as normalizing control (left). [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). IL1R1 protein levels in MDAMB231 cells following TRF2 induction for 48 hr by western blot (centre) and immuno-flow cytometry (right). ( C ) IL1R1 mRNA expression in HEK293T and MRC5 cells following transfection of TRF2-cDNA for 48 hr; GAPDH was used as normalizing control. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 2—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 2—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 2—figure supplement 1—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Transfection, Expressing, Control, Western Blot, Flow Cytometry

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) Schematic depicting G4 motifs ( A, B ) and their respective positions on the IL1R1 promoter; sequence scheme depicting generic G4 motifs; ( B ) Circular dichroism profile (220–310 nm wavelength) of oligonucleotides (5 µM) with sequence of the G4 motifs A or B (or respective mutants with base substitutions) in solution. ( C ) ChIP-qPCR following BG4 ChIP at the IL1R1 promoter (fold enrichment over mock) overlaid with TRF2 occupancy (fold enrichment over IgG) in HT1080 cells. [N=3] Statistical significance was calculated using Šídák’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) IL1R1 promoter activity in HT1080 cells from luciferase-reporter without or with G4-deforming substitutions against G4 motif A (IL1R1G4-MUT-A) or G4 motif B (IL1R1-G4 MUT-B) in presence or absence of TRF2 induction; G4 motifs A and B as shown in schematic; specific base substitutions have been indicated in blue font. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) IL1R1 promoter activity, mRNA and protein levels in presence/absence of the G4 binding ligand 360 A with or without TRF2 induction. For Left and centre panels- [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( F ) IL1R1 promoter-firefly luciferase reporter cassette, with or without substitutions deforming the G4 motif B, was artificially inserted at the CCR5 locus using CRISPR-cas9 gene editing in HEK293T cells (scheme in left panel). Promoter activity from (right panel). [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( G ) TRF2 occupancy by ChIP-qPCR (right panel) at the artificially inserted IL1R1 promoter without or with G4-deforming substitutions (IL1R1G4 MUT-B);ChIP-qPCR primers designed specific to the inserted promoter using the homology arms(see Materials and methods). [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 3—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 3—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 3—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Sequencing, Circular Dichroism, ChIP-qPCR, Activity Assay, Luciferase, Binding Assay, CRISPR, Western Blot

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) TRF2 ChIP-seq peak with sequence of the G4 motifs ( A, B ) and their respective positions on the IL1R1 promoter. ( B ) Occupancy of TRF2 by ChIP-qPCR at the IL1R1 promoter at the region of 0–200 bp of TSS in presence of ligand 360 A in three independent experiments in HT1080 cells. The dosage of 360 A used has been previous standardised [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C, D ) Base substitutions for the G4 motif B in the IL1R1 promoter (as shown in ) were introduced using CRSIPR in HEK293T cells. TRF2 ChIP-qPCR spanning the IL1R1 promoter (left) and IL1R1 mRNA expression (right) in HEK293T cells with or without the G4-disrupting substitution in two replicates. [N=2]. Error bars correspond to SEM from independent experiments. Figure 3—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values.

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: ChIP-sequencing, Sequencing, ChIP-qPCR, Expressing

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) Enrichment of histone marks H3K27ac, H3K4me3, H3K27me3, H3K9me3 (normalised to total H3) by ChIP-qPCR on the IL1R1 promoter in HT1080 cells in TRF2-up (induced) condition or un-induced corresponding control cells. [N=3] Statistical significance was calculated using Šídák’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( B ) Transcription factors/histone remodelers on the IL1R1 proximal promoter (–750 bp) across seven cell lines curated from ENCODE was plotted using UCSC genome browser hg19 human genome assembly. P300 enrichment marked in red box. ( C ) Immunoprecipitation using TRF2 antibody probed for p300, acp300/CBP, TRF2 or TRF1 (positive control) in input, TRF2 IP fraction or mock IP fraction in HT1080 cells. ( D ) Histone acetyl transferase (HAT) assay using purified histone H3/H4 and full-length p300 as substrate in presence/absence of recombinant TRF2; BSA (bovine serum albumin) was used as a non-specific mock protein. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 4—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 4—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 4—figure supplement 1—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: ChIP-qPCR, Control, Immunoprecipitation, Positive Control, HAT Assay, Purification, Recombinant, Western Blot

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) H3K27ac occupancy at the IL1R1 promoter spanning +200 to –1000 bp of TSS by ChIP-qPCR in HT1080 (left panel) and MDAMB231 cells (right panel) in control (uninduced), TRF2-induced (up) or TRF2-down conditions; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( B ) p300 occupancy on the IL1R1 promoter in HT1080 (left panel) and MDAMB231 cells (right panel) in control (uninduced), TRF2-induced or TRF2-down conditions; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) CBP (left panel) and ac-p300/CBP (right panel) occupancy on the IL1R1 promoter in HT1080 cells in control (uninduced) or TRF2-induced conditions; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Šídák’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) p300, CBP, acp300/CBP and H3K27Ac occupancy on the IL1R1 promoter in HT1080 and HT1080-LT cells; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Šídák’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). All protein ChIP, other than histones, were normalised to 1% input and fold-change have been calculated over respective IgG. Histone ChIP were normalised to 1% Input and fold change over total H3 have been calculated for individual samples (Further details in the Materials and methods section). Error bars correspond to SEM from independent experiments. Figure 4—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values.

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: ChIP-qPCR, Control

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) Schematic showing positions (amino acid residues) where acetylation and deacetylation of TRF2 have been reported. ( B ) IL1R1 mRNA expression post 48 hr of transient over-expression of various TRF2 acetylation mutants. GAPDH was used for normalisation. [N=3] Statistical significance was calculated using Dunnett’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) IL1R1 protein level following post 48 hr of transient over-expression of various TRF2 acetylation mutants; GAPDH used as loading control. ( D ) Immunofluorescence for IL1R1 in HT1080 cells without (control) or with induction of flag-tag TRF2 WT or mutant TRF2-293R; quantification from 29 cells in each case shown in graph. [N=29] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) Occupancy of flag-tag TRF2-WT or TRF2-293R on the IL1R1 promoter by ChIP-qPCR in HT1080 cells (left panel); IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. Occupancy by ChIP-qPCR of p300 (middle panel) and acp300/CBP (right panel) without (control) or with induction of TRF2-WT and TRF2-293R. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( F ) TRF2-wild type (WT) and TRF2-2293R mutant proteins fused with dCAS9 expressed and targeted to the IL1R1 promoter using IL1R1 -specific gRNA in HT1080 and MDAMB231 cells. Following this, expression of IL1R1 or other TRF2 target genes (non-specific with respect to the IL1R1-gRNA) in HT1080 (left) or MDMB231 cells (right). [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 5—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 5—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 5—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Expressing, Over Expression, Control, Immunofluorescence, FLAG-tag, Mutagenesis, ChIP-qPCR, Western Blot

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) TRF2-WT Flag or TRF2 K293R-Flag was stably over expressed in HT1080 cells and immunoprecipitation was performed using Anti-Flag antibody or Mock IgG. Enrichment for p300 in IP faction was tested for TRF2-WT (left) and TRF2 K293R(right). ( B ) Previously reported TRF2 target genes were checked following transient over-expression of TRF2 WT and TRF2 K293R in HT1080 cells. While K293R did not affect TRF2 mediated repression, it affected TRF2 activated genes PDGFR-B and IL1R1 but not WRNIP1. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 5—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 5—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 5—figure supplement 1—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Stable Transfection, Immunoprecipitation, Over Expression, Western Blot

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A, B ) NFKappaB activation in presence of IL1B (10 ng/ml) in HT1080 ( A ) or MDAMB231 ( B ) cells with or without TRF2 induction; activation signalling was confirmed through NFKappaB-Ser536 phosphorylation (normalised to total NFKappaB); ratio of Ser566-p/total NFKappaB plotted for respective blots from three independent replicates (right panels). [N=2]. ( C ) Expression of NFKappaB targets IL6, IL8 or TNF in presence/absence IL1A, IL1B, or TNF-a (10 ng/ml) for 24 hr in control (scrambled siRNA) or TRF2-low (TRF2 siRNA) conditions in HT1080 cells. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) Expression of IL6, IL8, or TNF in control or TRF2-induced conditions on treatment with either IL1A or IL1B (10 ng/ml) for 24 hr in absence (left panel) or presence (right panel) of the IL1-receptor-antagonist IL1RA (20 ng/ml) in HT1080 cells. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) NFkappaB/phosphor-Ser536-NFKappaB levels in xenograft tumours developed in NOD-SCID mice (control or doxycycline-induced TRF2 in HT1080 cells; N=6 mice in each group) by immuno-flow cytometry; mean fluorescence signal from individual tumours in control or TRF2-induced tumours plotted in adjacent graph; activation shown as ratio of pSer536-p-NFkappaB over total NFkappaB; significance was calculated using Wilcoxon’s non-parametric test. [N=6] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 6—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 6—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 6—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Activation Assay, Phospho-proteomics, Expressing, Control, Flow Cytometry, Fluorescence, Western Blot

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) TRF2-wild type (WT) and TRF2-2293R mutant proteins fused with dCAS9 expressed and targeted to the IL1R1 promoter using IL1R1-specific gRNA in HT1080 (left) or MDAMB231 cells (right). Following this, NFKB and p-NFKB (Ser536-phosphorylation) levels were assessed by flow-cytometry in ten thousand HT1080 or MDAM231 cells; mean fluorescence signal for IL1R1 and ratio of p-NFKB/total NFKB plotted in graphs on right panels. [N=1]. ( B ) IL1R1 knockout (KO) HT1080 cells using CRISPR were made and were transduced (lentiviral) to a doxycycline-inducible-TRF2 stable line (top left). TRF2, NFKappa-B and Ser536p-NFKappa-B in IL1R1 knockout or control HT1080 cells with or without TRF2 induction by doxycycline; GAPDH used as loading control (top right). Induction of TRF2 in HT1080 WT and IL1R1 KO cells to check for NFkappaB downstream target gene expression (bottom). Activation of NFKB target genes (~two- to fivefold) post-TRF2 over-expression was lower in IL1R1KO cells compared to WT cells (~10tenfold) [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) Phospho-NF-Kappa-B-(Ser-536p) and NFkappaB in HT1080 or HT1080-LT cells with or without IL1B stimulation (top panel); ratio of p-NFKB to total NFKB from above western blots plotted from two independent experiments (bottom panel). The NFkappa B (p65/RELA) is detected just below the 70 Kda molecular weight marker. Error bars correspond to SEM from independent experiments. Figure 6—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 6—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 6—figure supplement 1—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Mutagenesis, Phospho-proteomics, Flow Cytometry, Fluorescence, Knock-Out, CRISPR, Control, Targeted Gene Expression, Activation Assay, Over Expression, Western Blot, Molecular Weight, Marker

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: Scheme depicting relatively low infiltration of TAM in tumours with relatively long telomeres vis-à-vis tumours with short telomeres. Reduced non-telomeric TRF2 binding at the IL1R1 promoter in tumours with long telomeres, and consequent low IL1R1 activation, attenuated p65-mediated IL1-beta and macrophage infiltration. Created with BioRender.com .

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Binding Assay, Activation Assay

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Derivative Assay, Recombinant, Flow Cytometry, N-ChIP, Control, Plasmid Preparation, shRNA

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Comprehensive analysis of differentially expressed profiles of Alzheimer’s disease associated circular RNAs in an Alzheimer’s disease mouse model

doi: 10.18632/aging.101387

Figure Lengend Snippet: The hierarchical cluster, scatter plot and volcano plot of differential expression of circRNAs in 10-month-old SAMP8 versus age-matched SAMR1 and 10-month-old SAMP8 versus 5-month-old SAMP8. ( A ) Hierarchical cluster of differentially expressed circRNAs. “green” indicates low intensity, “black” indicates medium intensity and “red” indicates strong intensity. ( B ) Scatter plot of circRNA signal values. The values of X and Y axes represents the normalized signal values of the samples (log2 scaled) and the averaged normalized signal values of samples (log2 scaled) respectively. The green lines are fold change lines. The CircRNAs above the top green line and below the bottom green line demonstrates more than 1.5-fold change of circRNAs between the two compared samples. ( C ) Volcano plot of differential expression of circRNAs. The vertical lines correspond to 1.5-fold up and down, respectively. The horizontal line represents a P -value of 0.05, and the red point in the plot represents the differentially expressed circRNAs with statistical significance.

Article Snippet: Three hippocampal tissues of SAMP8 and SAMR1 were used for microarray assay to determine differentially expressed circRNAs using the circRNAs chip (Arraystar mouse circRNAs chip, AraryStar) containing 14,236 probes specific for mouse circular RNAs splicing sites.

Techniques: Quantitative Proteomics

Journal: Aging (Albany NY)

Article Title: Comprehensive analysis of differentially expressed profiles of Alzheimer’s disease associated circular RNAs in an Alzheimer’s disease mouse model

doi: 10.18632/aging.101387

Figure Lengend Snippet: The expression levels of candidate circRNAs for validation by real-time qPCR in 15 10-month-old SAMR1 and SAMP8 hippocampal tissues. Statistically differences were calculated by t -test using SPSS 13.0 software. * P <0.05, ** P <0.01 versus SAMP8 group.

Article Snippet: Three hippocampal tissues of SAMP8 and SAMR1 were used for microarray assay to determine differentially expressed circRNAs using the circRNAs chip (Arraystar mouse circRNAs chip, AraryStar) containing 14,236 probes specific for mouse circular RNAs splicing sites.

Techniques: Expressing, Biomarker Discovery, Software

Journal: Aging (Albany NY)

Article Title: Comprehensive analysis of differentially expressed profiles of Alzheimer’s disease associated circular RNAs in an Alzheimer’s disease mouse model

doi: 10.18632/aging.101387

Figure Lengend Snippet: The expression levels of candidate circRNAs for validation by real-time qPCR in 15 10-month-old SAMP8 and 5-month-old SAMP8 hippocampal tissues. Statistically differences were calculated by t -test using SPSS 13.0 software. ** P <0.01 versus 5-month-old SAMP8 group.

Article Snippet: Three hippocampal tissues of SAMP8 and SAMR1 were used for microarray assay to determine differentially expressed circRNAs using the circRNAs chip (Arraystar mouse circRNAs chip, AraryStar) containing 14,236 probes specific for mouse circular RNAs splicing sites.

Techniques: Expressing, Biomarker Discovery, Software

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: Immunohistochemistry images of testis samples are shown from male mouse foetuses. a , Z-DNA antibody staining (green) is diminishing in germ cells, as identified by PGC7 staining (red) at 13.5, 15.5 and 18.5 dpc. Scale bars, 5 µm. b , c , Loss of Z-DNA is specific to the germ cells in the foetal testis. Immunohistochemistry is shown for foetal testis sections at 13.5, 15.5 and 18.5 dpc timepoints, using Z-DNA antibody (green), germ cell marker (PGC7) and DAPI counterstain. Merged images are displayed on the right. Prospermatogonia ( b ) exhibit loss of Z-DNA. Somatic cells ( c ) maintain Z-DNA. Scale bars, 1 µm. d , ZBTB43 protein is detected in germ cells inside the testicular cords of the wild-type but not in the Zbtb43 −/− mutant testis at 15.5 dpc. Note the typical weak and diffuse DAPI staining of germ cell nuclei. The surrounding Sertoli cells, which exhibit stronger DAPI staining, are negative for ZBTB43 staining. Scale bars, 5 µm. e , Double staining of 15.5 dpc germ cells with ZBTB43 and germ cell marker OCT4 antibodies is shown in testis samples at 15.5 dpc. Scale bars, 2 µm. f , Double staining of testis samples using the ZBTB43 and germ cell marker DDX4 antibodies is shown at the foetal days as marked. Scale bars, 2 µm. The results shown represent three independent immunostaining experiments using four biologically independent testis samples per experiment ( a – c ), five independent experiments using three biologically independent testis samples per experiment ( d ), one immunostaining experiment using two biologically independent testis samples ( e ) and two independent immunostaining experiments using two biologically independent testis samples per experiment ( f ).

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Immunohistochemistry, Staining, Marker, Mutagenesis, Double Staining, Immunostaining

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a ) The fetal testis and mesonephros are shown in transmitted light to the left. Testicular cords are visualized by transgenic EGFP expression to the right. EGFP expression in the TgOG2 mouse line allows germ cell isolation by FACS. MGC: male germ cell (EGFP positive); MSC: male somatic cell (EGFP negative). FACS gating strategy is shown. R6: EGFP+, R5: EGFP-. ( b ) RNA-seq results are displayed in purified male germ cells (MGC), female germ cells (FGC) and somatic cells of male and female gonads (MSC and FSC) at 15.5 dpc ( GSE46953 ). Transcript levels of Zbtb43 are plotted among other Zbtb family members. ( c ) Zbtb43 expression is depicted among some of the most highly expressed transcripts (apart from ribosomal proteins, elongation complex members or chaperons) in purified MGC, FGC, MSC, and FSC at 15.5 dpc. ( d ) Zbtb43 expression is shown for mouse organs outside of the gonads. GEO Profiles: GDS661/116970_at from #BioProject PRJNA66167.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Transgenic Assay, Expressing, Cell Isolation, RNA Sequencing, Purification

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a ) Knockout strategy. Mice carrying the Zbtb43 tm1b(KOMP)Mbp allele. A LacZ cassette replaced exon 4 of the Zbtb43 gene. ( b ) PCR genotyping from tail DNA to distinguish Zbtb43 +/+ , Zbtb43 +/- and Zbtb43 -/- pups at weaning. Data shown represent three independent experiments. ( c ) Western blot in adult kidney samples using the anti-ZBTB43 antibody and GAPDH loading control. ( d ) Western blot in adult kidney and brain samples using the anti-LacZ antibody. Data shown represent two independent experiments in c and d.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Knock-Out, Western Blot, Control

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , Structure of the ZBTB43 protein. Location of the BTB domain and the three ZF domains are indicated. The full-length protein (ZBTB43-FL), its ZF domain (ZBTB43-ZF) and its BTB domain (ZBTB43-BTB) were purified, as shown in the protein gels to the right. Protein purification and protein gel testing was done twice for ZBTB-FL and ZBTB-ZF and once for ZBTB43-BTB. b , Outline of the affinity sequencing experiment. MBP tag was used for the capture. c , ZBTB43 protein has affinity to methylated and unmethylated genomic DNA. Heatmap analysis of MBP-ZBTB43-FL binding is shown to genomic DNA, either DNMT-TKO ES cell DNA or DNA fully methylated by SssI bacterial CpG methyltransferase centred at the TKO DNA peaks. Background level binding by MBP is shown on the right. d , Consensus binding sequences of the affinity peaks in TKO determined by RSAT (top), significance 4.5e −13 , BaMM (middle), dataset performance 0.096, motif performance 0.84, and MEME (bottom), significance 4.7e −2775 . e , Heatmap shows the match between ZBTB43 affinity binding and predicted Z-DNA sites . f , IGV browser images of selected specific MBP-ZBTB43 peaks are shown in TKO DNA samples at four genomic regions. Control samples show the background of MBP capture. The tracks for transcripts and predicted Z-DNA are displayed at the bottom. The affinity-sequencing results shown represent two independent biological replicates in c , e and f . g , EMSA confirm the binding of ZBTB43-FL or ZBTB43-ZF to the regions detected by affinity sequencing. The FAM-labelled probes (CACG) 8 , Rps6kl1 , and Ago2 , marked as a, b, and c, respectively, were competed out of the complexes by 100-fold excess of specific (Self) but not by the mutant (Mut) cold competitor. ( h) EMSA confirms the binding of ZBTB43-ZF to the consensus PPR sequences. The FAM-labelled probes (CA) and (CACG) 8 , marked as d and a, respectively, resulted in specific shift; they were competed out of the complexes by 100-fold excess of specific (Self) but not by the mutant (Mut) cold competitor. The BTB domain of ZBTB43 (ZBTB43-BTB) lacked binding activity. Data shown represent three independent experiments in panels g and h.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Purification, Protein Purification, Sequencing, Capture-C, Methylation, Binding Assay, Control, Mutagenesis, Activity Assay

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a-b ) Heatmap analysis is shown of MBP-ZBTB43 (FL) binding intensities to genomic DNA, either Dnmt1/Dnmt3a/Dnmt3b triple knockout (TKO) ES cell DNA or DNA fully methylated by SssI bacterial CpG methyltransferase (SssI) centered at the TKO DNA peaks ( a ) and at the SssI peaks ( b ). Background level binding by MBP is included to the right. ( c ) Venn diagram shows the relationship between ZBTB43 affinity peaks mapped in SssI and TKO DNA. ( d ) Venn diagram shows the relationship between ZBTB43 affinity peaks mapped in SssI DNA, TKO DNA, and predicted Z-DNA sites or enriched Z-DNA sites in activated B-cells . ( e-f ) Heatmaps show the match between ZBTB43 affinity binding peaks and Z-DNA sites. The affinity intensities are centered at predicted Z-DNA sites ( e ) and enriched Z-DNA sites mapped in activated B-cells ( f ). ( g-h ) Venn diagrams displays the relationship between predicted Z-DNA locations and ZBTB43 affinity binding sites in SssI ( g ) or TKO DNA ( h ). ( i-j ) Venn diagrams displays the relationship between in vivo mapped Z-DNA sites in activated B-cells and ZBTB43 affinity binding sites in SssI DNA ( i ) or TKO DNA ( j ). Affinity-sequencing results shown represent two biologically independent samples.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Binding Assay, Triple Knockout, Methylation, In Vivo, Sequencing

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: Venn diagrams depict the location of ZBTB43 affinity peaks mapped in fully methylated genomic DNA (SssI) and unmethylated genomic DNA (TKO) relative to the location of transcripts in the genome. In comparison, the distribution of predicted Z-DNA and enriched Z-DNA mapped in vivo in activated B-cells are also depicted to the right. ( a ) All genomic locations are plotted. ( b ) Distal intergenic regions are excluded. Affinity-sequencing results represent two independent biological replicates.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Methylation, Comparison, In Vivo, Sequencing

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: IGV browser images of selected specific MBP-ZBTB43 peaks are shown in unmethylated genomic DNA (TKO) at four genomic regions. Control samples show the background of MBP capture. Independent biological replicate samples are displayed. The tracks for transcripts and predicted Z-DNA are displayed at the bottom. The predicted Z-DNA sequence is enlarged to illustrate the underlying PPR sequences. The ZBTB43 peaks are often found in an intron of very long transcripts. ( a ) Rps6kl1 ( b ) Ago2 ( c ) Arid2 ( d ) Eml1 .

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Control, Sequencing

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , ZBTB43 binding sequence at Rps6kl1 , in forward and reverse orientation (Z1 and Z2), was inserted into the vector pUCON, and the resulting plasmids pUPZ1 and pUPZ2 were tested in LacZ mutation assays. b , Mutation frequencies in bacterial NEB Stable cells. Average values of three replicate samples are shown with standard deviation. Statistically significant differences ( P < 0.05) resulting from n = 3 independent experiments are marked. These were obtained using two-tailed Student’s t -tests (unequal variance). c , The LacZ gene is frequently mutated at the Z-DNA insert, as depicted by an agarose plate with DH5α bacteria that contain the parent plasmid (blue colonies) or its mutants (white colonies). d , Restriction digestion is shown from randomly picked clones recovered from DH5α bacteria. DNA sequencing revealed small mutations that did not change the size of the 910-bp-long PPR-containing DNA fragment (blue arrow). Molecular size marker is shown in the left lane. Wild-type plasmid (WT) is depicted in the right lane. e , Mutation frequencies in mammalian COS-7 cells. Average values of three replicate samples are shown with standard deviation. Statistically significant differences ( P < 0.05) resulting from n = 3 independent experiments are marked. These were obtained using two-tailed Student’s t -tests (unequal variance). f , Restriction digestion is shown from randomly picked biologically independent clones recovered from COS-7 cells and grown in NEB Stable or DH5α bacteria. The loss of the 910-bp-long PPR-containing fragment (blue arrow) reveals large deletions. Molecular size marker is shown in the left lane. Wild-type plasmid (WT) is depicted in the right lane. Restriction digestion results in d and f are shown from one of three independent mutagenesis experiments. g , Mutation detection in the pUPZ1 and pUPZ2 plasmids mutagenized in COS-7 cells. Structural elements of the vector plasmid are depicted at the top. Position of the inserted Z1 and reciprocal, Z2 sequences is shown in turquoise. Sequencing results are shown from biologically independent clones recovered in NEB Stable or DH5α bacteria, as indicated. Deletions are marked by blue horizontal bars. Micro-homologies that flank these deletions are shown by the DNA sequence. Sequencing results are shown from one of three independent mutagenesis experiments.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Binding Assay, Sequencing, Plasmid Preparation, Mutagenesis, Standard Deviation, Two Tailed Test, Bacteria, Clone Assay, DNA Sequencing, Marker

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , EMSA. Hexamine CoCl 3 was added to the DNA probes at increasing concentrations. The induced Z-DNA was detected as a gel shift using the anti-Z-DNA antibody Z22. b , Z-DNA formation (blue lines) is detected by CD spectroscopy at the (CACG) 8 consensus sequence in response to different concentrations of CoCl 3 . The B-DNA specific spectrum is displayed in red. c , 2D gel electrophoresis detects a kink (blue arrow) at certain plasmid topoisomers, a sign of Z-DNA formation. d , Generation of circular Z-DNA probe. When two single-stranded circles (CC) are annealed, part of the circle, which contains PPRs is forced into left-handed DNA . Circular B-DNA is prepared by annealing a circular and a linear strand followed by ligating the nick (CL). Linear B-DNA is prepared by annealing two strands of linear DNA (LL) . e , f , Z-DNA structure is confirmed in the CC probe by its insensitivity to restriction enzymes. The CC, CL and LL probes of PPR sequences, as marked above, were subjected to restriction digestion. The CC form (turquoise asterisk), was refractory. The CL form (black asterisk) is linearized, and the LL form is restricted to two fragments (red asterisks). Experiments where the (CACG) 6 sequence ( e ), and the Rps6kl1 -Y peak sequence ( f ) are digested using Hha I and Bsiw I are displayed. g , Z-DNA is formed in the CC probe at the ZBTB43 consensus sequence. Increasing amount of the Z-DNA antibody Z22 quantitatively shifts the CC probe of (CACG) 6 and Rps6kl1 -Z sequences. h , ZBTB43 binds Z-DNA. EMSA results show that ZBTB43-FL shifts the CC probe containing the (CACG) 6 and Rps6kl1 -Z sequences. Data shown represent three independent experiments in a – c and e – h . i , ZBTB43 binds both Z-DNA and B-DNA but prefers Z-DNA. Competition binding experiment is shown where the CC and CL probes were mixed at equal molar ratios and the aliquots were allowed to interact with increasing amounts of ZBTB43-FL before separating the free probes and complexes in EMSA gels. The remaining free CC and CL probes in each reaction were quantified in the gel images. Data are presented as mean ± standard error of the mean (s.e.m.) from n = 3 independent experiments.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Gel Shift, Circular Dichroism, Sequencing, Two-Dimensional Gel Electrophoresis, Electrophoresis, Plasmid Preparation, Binding Assay

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a – c show that ZBTB43 has the capacity to remove Z-DNA in vitro. a , Restriction digestion using Tai I of the Rps6kl1 -Z affinity peak in the CC probe (blue asterisk) and the LL probe confirms Z-DNA and B-DNA, respectively. b , ZBTB43 renders CC sensitive to digestion. The CC probe was reacted with TOPO1, or ZBTB43, or both, followed by phenol extraction and precipitation. The resulting DNA was run on a gel before or after Tai I digestion. TOPO1 and/or ZBTB43 resulted in a band that migrated slower than the CC form (black asterisk). Tai I produced a linear 121 bp fragment (red asterisk). c , ZBTB43 has no effect on B-DNA. The LL probe (121 bp) was reacted with TOPO1, and/or ZBTB43, and the recovered LL DNA was still sensitive to Tai I digestion into two fragments of 37 and 84 bp. d , ZBTB43 enhances the effect of TOPO1 in reducing supercoiling-induced tension. TOPO1 was reacted with plasmid DNA containing the Rps6kl1 affinity peak sequence in the presence or absence of ethidium bromide (EB6 or EB0, respectively) and increasing amounts of ZBTB43-FL. Control reactions were run without TOPO1. Supercoiled plasmid (PL) purified from bacteria and a molecular weight marker (M) are included. e – h show that ZBTB43 reverses the action of ADAR1 Z-α domain on DNA topology in vitro. e , To induce Z-DNA, the Z-α domain was added to the (CA) 16 linear DNA probe in increasing molar excess. B-DNA specific spectrum, peaking at 280 nm (orange dot) is eliminated by 20× excess of Z-α. New Z-DNA peaks (turquoise and blue dots) at 260 nm are visible at 40× and 80× excess of Z-α. f , ZBTB43 has no effect on B-DNA topology. g , Forty-fold molar excess of ZBTB43 reverses the Z-to-B shift caused by 20-fold molar excess of Z-α (280 nm peak regained, green dot). h , Forty-fold molar excess of ZBTB43 reverses the Z-to-B shift caused by 40-fold molar excess of Z-α (ZBTB43 reverts the peak from 260 nm). Data shown represent two ( a – c ) or three ( d ) independent experiments. Experiments in e – h have been performed once, and each CD spectrum is presented as an average of three scans.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: In Vitro, Extraction, Produced, Plasmid Preparation, Sequencing, Control, Purification, Bacteria, Molecular Weight, Marker

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , Global Z-DNA is eliminated by ZBTB43 in Zbtb43 +/+ , but not in Zbtb43 −/− foetal testis sections at 15.5 dpc. Images of testicular cords were obtained by immunohistochemistry and confocal microscopy using the ZBTB43 (red), and Z-DNA (green) antibodies, counterstained with DAPI. Scale bars, 10 µm. b , Images of testicular cords as above are shown using higher magnification and background reduction. Scale bar, 5 µm. c , Images of germ cells as above are shown using higher magnification. Scale bars, 2 µm. d , Quantification of the fluorescence intensities of DAPI, Z-DNA and ZBTB43 immunostaining are depicted in 15.5 dpc Zbtb43 +/+ and Zbtb43 −/− prospermatogonia. Spermatogonia ( n = 10 + 9) were quantified from two independent foetuses for each genotype. The intensity was measured by Fiji, and the quantification was done by Prism. Data are presented as mean ± s.e.m. The differences between genotypes were statistically significant for the Z-DNA and ZBTB43 intensities by multiple unpaired two-tailed t -tests. e , ZBTB43 protects from DSBs. Immunohistochemistry of the 15.5 dpc Zbtb43 +/+ and Zbtb43 −/− foetal testis sections is shown using the γH2AX (red) and DDX4 (green) antibodies, counterstained with DAPI. Scale bars, 5 µm. f , Enlarged details of the testicular cords are displayed. Scale bars, 2 µm. The results shown represent three ( a – c ) or two ( e , f ) independent experiments done using four biologically independent testis samples per experiment.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Immunohistochemistry, Confocal Microscopy, Fluorescence, Immunostaining, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a-b ) Immunohistochemistry is shown of the Zbtb43 +/+ wild-type ( a ), and Zbtb43 -/- mutant ( b ) fetal testis sections at 13.5, 15.5 and 18.5 dpc using the ZBTB43 (red), and Z-DNA (green) antibodies, counterstained with DAPI. Scale bar: 10 µm. The ZBTB43-Z-DNA double staining was done three times at all stages. ( c-e ) Quantification of signal intensities of DAPI, Z-DNA and ZBTB43 in Zbtb43 +/+ wild-type, and Zbtb43 -/- mutant fetal male germ cells shown relative to the corresponding somatic cells at 13.5 dpc (n=10 cells from one out of two independent experiments) and at 15.5 and 18.5 dpc (n=19 cells from one out of two independent experiments). Data are presented as mean values +/- SEM. Statistically significant changes as calculated by multiple 2-sided T-tests are marked with P -value.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Immunohistochemistry, Mutagenesis, Double Staining

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , Genome browser images of ZBTB43 ChIP–seq peaks (light green) obtained in purified 15.5 dpc prospermatogonia are displayed at 12 genomic locations. IgG lanes are shown as controls. The ChIP peaks align with the in vitro affinity sequencing peaks found in fully methylated genomic DNA (SssI) and in fully unmethylated genomic DNA (TKO) (dark green). MBP affinity-seq lanes are shown as controls. The scale of reads was normalized between experimental samples and their respective background control samples using the ‘group-autoscale’ function of IGV as marked on the right. b , c , Heatmap showing the ZBTB43 ChIP–seq peaks detected against IgG background in 100,000 ( b ) or 500,000 ( c ) purified prospermatogonia. The read intensities in three libraries, ZBTB43 antibody, IgG antibody and input DNA (as marked above), are plotted centred at the peak and using +1 kb and −1 kb flanking regions. d , e , Venn diagrams depicting the location of ZBTB43 ChIP–seq peaks mapped in 15.5 dpc prospermatogonia relative to the location of transcripts in the genome. In d , all genomic locations are plotted. In e , distal intergenic regions are excluded. f , The ZBTB43 ChIP–seq peaks detected in vivo are recognized by purified ZBTB43 protein in vitro. Heatmaps display the read intensities of ZBTB43-FL and the control MBP in affinity binding with unmethylated DNA (TKO) or methylated DNA (SssI). The plotted regions were centred at ChIP peaks called in 500,000 or 100,000 prospermatogonia against IgG, as indicated at the bottom. g , The ZBTB43 ChIP–seq peaks detected in prospermatogonia align with affinity-seq peaks and with predicted Z-DNA. Heat maps display the ChIP–seq log 2 IP/IgG read intensities detected in 500 K or 100 K prospermatogonia (as marked at the top) along subset of genomic regions centred at ZBTB43-FL affinity-seq peaks in methylated DNA (SssI), unmethylated DNA (TKO) and at the subset of predicted Z-DNA sites (marked at the bottom) where overlap is found with ChIP–seq peaks. The ChIP–seq and affinity-sequencing results shown represent two independent biological replicates in a , f and g .

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: ChIP-sequencing, Purification, In Vitro, Sequencing, Methylation, Control, In Vivo, Binding Assay

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: a , Z-DNA structure inhibits DNMT3A catalytic activity in vitro. The CC, CL and LL substrates were prepared from the Rps6kl1 affinity binding sequence and were subjected to methylation by DNMT3A at 30× or 60× molar excess. The level of DNA methylation at each CpG is plotted after multiplexed bisulfite sequencing in two biologically independent replicates. b , DNA methylation is aberrant in sperm of Zbtb43 −/− mice at the sites of ZBTB43 binding. IGV browser images display selected in vivo hypomethylated regions detected using MIRA–seq assays in Zbtb43 −/− versus wild-type spermatozoa (navy). The ZBTB43 affinity binding peaks (green), and ChIP–seq peaks mapped in 15.5 dpc prospermatogonia (light green) are also shown together with the MBP and IgG backgrounds. Independent replicate samples are displayed. c , DMRs originate at the time of de novo methylation in prospermatogonia. Bisulfite sequencing results show methylated and unmethylated CpGs (black and white circle, respectively) along individual chromosomes at specific DMR sequences and at one control region in adult spermatozoa (top) and 18.5 dpc prospermatogonia (bottom). The genotypes are marked on the left. d , Heatmap displaying the MIRA–seq intensities in navy at the hypomethylated and hypermethylated DMRs identified between Zbtb43 −/− and Zbtb43 +/+ spermatozoa. Heatmaps to the right in green show the affinity binding of MBP-ZBTB43-FL in unmethylated and methylated genomic DNA (TKO and SssI) centred at the sites of sperm DMRs. The sequencing results shown represent two independent biological replicates in b and d . e , ZBTB43 ChIP–seq peaks in 15.5 dpc prospermatogonia overlap with hypo-DMRs detected in Zbtb43 −/− sperm DNA. Venn diagrams. f , Model. Top: ZBTB43 binds Z-DNA in normal prospermatogonia. By removing Z-DNA, ZBTB43 creates an accessible substrate for DNMT3A and facilitates DNA methylation establishment at PPR-rich DNA regions. Bottom: Z-DNA is not removed in the Zbtb43 −/− prospermatogonia in the absence of ZBTB43, DNA methylation is not established at PPRs, and hypomethylated DMRs are found in mutant sperm. One prominent ZBTB43 region can induce large re-arrangements in mammalian mutation assays, and ZBTB43 protects from DSBs by directly binding to PPRs and removing mutagenic Z-DNA structures in the foetal germ cells.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Activity Assay, In Vitro, Binding Assay, Sequencing, Methylation, DNA Methylation Assay, Methylation Sequencing, In Vivo, ChIP-sequencing, Control, Mutagenesis

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a ) Venn diagram displays the relationship between ZBTB43 affinity peaks in fully methylated (SssI) genomic DNA and DMRs detected in Zbtb43 -/- sperm. ( b ) Venn diagram displays the relationship between ZBTB43 affinity peaks in fully unmethylated (TKO) genomic DNA and DMRs detected in Zbtb43 -/- sperm. ( c ) Venn diagram shows the relationship between predicted Z-DNA sites in the genome and DMRs detected in Zbtb43 -/- sperm. ( d ) Venn diagram shows the relationship between enriched Z-DNA sites in activated B-cells in the genome and DMRs detected in Zbtb43 -/- sperm.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Methylation

Journal: Nature Cell Biology

Article Title: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome

doi: 10.1038/s41556-022-00941-9

Figure Lengend Snippet: ( a ) Zbtb43 -/- pups out of the test cross are underrepresented at weaning. Venn diagram shows the distribution of genotypes at weaning from 55 pups out of 6 breeding pairs and 13 litters of the original Zbtb43 +/- heterozygous stock (HET x HET). The genotype distribution of live weanlings is also plotted from a test cross where the parents had been back-crossed 8-times in the JF1/Ms mouse strain (HET.JF1.N8 x HET.JF1.N8). These results were obtained from four breeding pairs, 19 litters and 70 pups. ( b ) Zbtb43 -/- pups are overrepresented among dead pups out of the HET x HET cross. Venn diagram shows the distribution of dead pups by genotype. We identified 6 Zbtb43 -/- dead pups out of 11 total dead pups from 2 litters of 1 breeding pair of the HET x HET initial cross by PCR genotyping. After backcrossing to C57BL/6N once, we identified 10 Zbtb43 -/- dead pups out 23 dead pups from 20 litters of 4 pairs of HET.B6 X Het B6. ( c ) Zbtb43 -/- dead pups (n=16) are overrepresented among all dead pups out of the HOMO x HET cross. Venn diagram shows the distribution of dead pups by genotype. ( d ) Partially penetrant lethality in the Zbtb43 mutant mouse line depends on parental genotype. The portion of newborn pups that died is depicted by bar graphs. The lethality phenotype is shown according to different parental crosses as indicated below each bar. The postnatal day of death (P0, P1, P2 and after P2) is shown by colored columns as coded to the right. Zbtb43 -/- (HOMO), and Zbtb43 +/- (HET) mice were obtained from the original JAX stock. Another set of mice were obtained after crossing to C57Bl/6N (B6) one time (HOMO B6, and HET B6). The mother is written first in each cross. The number of breeding pairs, litters, and pups born are provided under the chart. ( e ) The partial perinatal lethality phenotype of Zbtb43 -/- pups persists through generations (N1, N2, N3 and N4) in the HOMO x HOMO crosses. Total number of animals and litters in each generation are marked under the plot. ( f-g ) Zbtb43 -/- pups from Zbtb43 -/- parents exhibit reduced growth compared to Zbtb43 +/+ pups out of Zbtb43 +/+ parents. Weight of male ( f ) and female ( g ) pups is plotted at weeks 2, 3, 4, 5, 5, 6, and 8 after birth, and numbers of pups included in the measurements are given under the dot plots. Data are presented as mean values +/- SEM. Statistical analysis between genotypes at each age was done using multiple two-sided T-Tests. ( h-i ) Zbtb43 -/- pups exhibit reduced growth after weaning compared to their littermates. Growth of male ( h ) and female ( i ) pups out of HET x HET cross is depicted. Weight of WT, HET and HOMO pups is shown as coded by colors to the right and numbers of pups are given under the dot plots. Data are presented as mean values +/- SEM. Statistically significant difference is marked with P -value as determined using multiple two-sided T-Tests between pairs of genotypes at each age. ( j ) Parental genotype of Zbtb43 +/+ pups does not affect their growth. Growth curve of male and female WT pups is shown from 3 weeks to 8 weeks. Solid lines indicate the average weight of WT pups out of WT parents. Dashed lines indicate the average weight of WT pups out of the HET x HET cross. Data are presented as mean values +/- SEM. No statistically significant difference was found between crosses using multiple 2-sided T-Tests. ( k ) Parental genotype of male and female Zbtb43 -/- pups affects their growth after weaning. Growth curve of male and female HOMO pups is shown from 3 weeks to 8 weeks. Solid lines indicate the average weight of HOMO pups out of HOMO x HOMO cross. Dashed lines indicate the average weight of HOMO pups out of the HET x HET cross. Data are presented as mean values +/- SEM. Statistically significant differences between crosses at each age as obtained from multiple two-sided T-Tests are marked.

Article Snippet: The remaining 9/10 of the sample was diluted tenfold with ChIP dilution buffer containing 4 μl ml −1 50× PIC (Roche, cat. no. 11697498001), and 1 μg of the anti-ZBTB43 antibody (Aviva Systems Biology, cat. no. ARP39048-P050), or the control rabbit-IgG (Abcam, cat. no. ab171870), was added per 100,000 cells-worth chromatin and incubated overnight at 4 °C.

Techniques: Mutagenesis