Fig. 4: G4s and R-loops formation at G-rich VNTRs, along with DNA damage, contributes to gene dysregulation. | Nature Communications

Fig. 4: G4s and R-loops formation at G-rich VNTRs, along with DNA damage, contributes to gene dysregulation.

From: ATRX loss couples genome instability at a G-rich repeat to dysregulation of human alpha-globin expression

Fig. 4: G4s and R-loops formation at G-rich VNTRs, along with DNA damage, contributes to gene dysregulation.

a Expression of HBM and HBA to β-like (HBB + HBG) globin in day 8 ATRX degron and ATRX degron-VNTR KO cells following 48 h treatment with G4 stabiliser CX-5461. NT: untreated; dTAG-13: 100 nM from day 0 to day 8; CX-5461: 0.1 μM from day 6 to day 8. Statistical analysis within each cell line used one-way ANOVA followed by Dunnett test on log₂-transformed values (n = 3 biological replicates, data shown as mean ± SEM). For HBM to β-like ratio, *p = 0.0267 (NT vs dTAG-13); **p = 0.0025 (NT vs CX-5461). b S9.6 dot-blot assay measuring global R-loops in dTAG-13-neg-treated ATRX+ and dTAG-13-treated ATRX- cells. RNase H served as a negative control. Quantification is shown as mean ± SEM from three independent experiments. *p = 0.0454 for 200 ng, two-way ANOVA Šídák’s test. c DRIP-qPCR analysis showing increased R-loop levels around the VNTR region in ATRX-negative cells, but not at a distal region on chromosome 16 (n = 3 biological replicates, data shown as mean ± SEM; *p = 0.0422, two-way ANOVA Šídák’s test). d Expression of HBM to β-like globin following RNase H overexpression in ATRX-negative cells. (n = 3 biological replicates, data shown as mean ± SEM; **p = 0.0084, one-way ANOVA Dunnett test). e Schematic of the ψζ VNTR locus and the sgRNA target sites used in the CRISPR-Cas9-induced DNA damage assay in VNTR KO cells. f Expression of HBM and HBA to β-like globin in day 8 ATRX degron-VNTR KO cells after 48 h CRISPR assay (n = 3 biological replicates, data shown as mean ± SEM). AAVS1 vs sgRNA used one-way ANOVA Dunnett test, and ARTX+ vs ATRX- used a two-tailed unpaired Welch’s t-test. For HBM to β-like ratio, **p = 0.0067, 0.0013, 0.0013 for AAVS1 vs sgRNA-1, sgRNA-2, sgRNA-3 in VNTR KO-ATRX+ cells; **p = 0.0058, 0.0053 for AAVS1 vs sgRNA-2, sgRNA-3 in VNTR KO-ATRX- cells. *p = 0.0332 for ATRX+ vs ATRX- cells. For HBA to β-like ratio, **p = 0.0057, 0.0022, 0.0026 for AAVS1 vs sgRNA-1, sgRNA-2, sgRNA-3 respectively in VNTR KO-ATRX+ cells. Source data are provided as a Source Data file.

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