202 irf3  (ATCC)

Journal: bioRxiv

Article Title: ATRX Deficiency Drives Aberrant Type I Interferon Signalling Through cGAS-Dependent Transcriptional Dysregulation

doi: 10.1101/2025.11.19.685761

Figure Lengend Snippet: (A) cGAMP measured by ELISA in unstimulated control fibroblasts, ATRX patient fibroblasts (Y1758C), and fibroblasts from patients with biallelic mutations in TREX1 , DNASE2 or RNASEH2A [circles represent technical repeats: 19 repeats in controls, 16 repeats in ATRX-Y1758C patients, and three or four repeats in patients with mutations in TREX1 , DNASE2 or RNASEH2A . Kruskal-Wallis test, uncorrected Dunn’s test, mean +/-SEM]. (B) cGAMP levels measured by ELISA in unstimulated BJ-hTERT fibroblasts WT for CGAS and either WT, KI (Y1758C) or KO for ATRX , and in CGAS KO BJ-hTERT and WT fibroblasts treated with herring-testis DNA (HT-DNA), [each circle represents the mean of a clone, clones were tested two to four times; Kruskal-Wallis test, uncorrected Dunn’s test, mean +/-SEM]. (C) Immunoblot of phosphorylated STING (S366), phosphorylated and total forms of NF-κB, phosphorylated IRF3 (S396), TBK1 (S172) and STAT1 (Y701) in individual clones of BJ-hTERT fibroblasts WT or KI for ATRX . The last lane relates to WT BJ-hTERT fibroblasts treated with the STING agonist diABZI, mimicking cGAMP activity. (D) BJ-hTERT fibroblasts WT, KI or KO for ATRX were separated into cytosolic and nuclear fractions, followed by sequential elution of nuclear pellets with the indicated concentrations of NaCl. Quantification (right) of cGAS levels in the different fractions of cells WT, KI or KO for ATRX . Vinculin and H2B are cytosolic and chromatin markers respectively [three independent clones, mean +/-SEM]. (E) cGAMP levels measured in BJ-hTERT fibroblasts KO for CGAS transfected with lipofectamine only (-), WT or catalytically inactive (D319A mutant) cGAS constructs and stimulated with HT-DNA for four hours. (F) Interferon (IFN) score (median of the relative expression of the following ISGs: IFI27, IFI44L, OASL, IFIT2, IFI6, ALDHA1 ) in BJ-hTERT fibroblasts KO for CGAS , and WT, KI or KO for ATRX were transfected with lipofectamine only (-), WT or catalytic inactive (D319A mutant) cGAS constructs, [each circle represents the mean of a clone, clones were tested four times, average+/-SEM, Kruskal-Wallis test, uncorrected Dunn’s test]. (G) Gene set enrichment analysis (GSEA) comparing the expression levels of 444 canonical ISGs determined by RNA-seq in BJ-hTERT fibroblasts lacking STING1 , and KI (top panel) or KO (bottom panel) for ATRX , relative to WT. NES = Normalized Enrichment Score; FDR = False Discovery Rate. (H) Bar plots depicting NES of hallmark gene sets that were significantly different (FDR <; 0.05) in BJ-hTERT STING1 KO fibroblasts, comparing cells KI or KO for ATRX to cells WT for ATRX .

Article Snippet: BJ-hTERT WT and null for cGAS were obtained from the Chen laboratory (University of Texas, Dallas) ; THP-1 and IRF3 KO THP-1 cells were from ATCC (TIB-202) and InvivoGen respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Clone Assay, Western Blot, Activity Assay, Transfection, Mutagenesis, Construct, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: ATRX Deficiency Drives Aberrant Type I Interferon Signalling Through cGAS-Dependent Transcriptional Dysregulation

doi: 10.1101/2025.11.19.685761

Figure Lengend Snippet: (A-B) Dot plots of genes exhibiting significant (adjusted p value (q) <; 0.1, x-axis) changes (‘FC’ signifies fold change) in both transcription and in DAXX (A) or H3.3 (B) levels in ATRX KI compared to WT cells. Quadrant colour coding is as follows: blue = genes enriched in DAXX or H3.3 and downregulated in expression; orange = genes enriched in DAXX or H3.3 and upregulated in expression; brown = genes depleted of DAXX or H3.3 and downregulated in expression; green = genes depleted of DAXX or H3.3 and upregulated in expression; ISGs are denoted separately by a black asterisk, while other genes are represented by empty circles. (C) Representative genome browser tracks showing levels of H3.3, DAXX at the ISG STAT1 . (D-E) Relative expression of IFNB1 (D) and interferon (IFN) score (median of the relative expression of the following ISGs: IFI27, IFI6, STAT1, IFIT2, IRF7, IRF9, IFIH1, MX1 ) (E) measured by qPCR in BJ-hTERT fibroblasts WT or KI for ATRX , and WT or heterozygous KO for DAXX [three to four independent clones repeated three times, Kruskal-Wallis test, two-stage linear step-up procedure of Benjamin, Krieger and Yekutieli, mean +/-SEM, q values are indicated on graphs]. (F-G) Comparison of DAXX (F) and H3.3 (G) occupancy at gene bodies between BJ-hTERT fibroblasts WT and KI for ATRX on a CGAS KO background; grey dots indicate genes exhibiting no difference in DAXX or H3.3 occupancy; blue dots indicate genes with significantly altered (adjusted p value <; 0.1) H3.3 occupancy in ATRX KI cells as compared to WT cells. (H) DAXX occupancy assessed by CUT&Tag at +/-2 kb of transcription starting sites (TSS) of a curated list of 444 canonical ISGs in BJ-hTERT fibroblasts WT, KI or KO for ATRX (from left to right), and WT or KO for CGAS (Top and bottom). (I) In homeostasis (top), ATRX and DAXX bind near the promoters of ISGs to regulate their expression. In ATRX loss of function (bottom), DAXX is depleted, and H3.3 enriched, at the promoters of ISGs. cGAS, which remains normally bound to nucleosomes, mediates these alterations in nucleosomal patterning and subsequent changes in gene expression.

Article Snippet: BJ-hTERT WT and null for cGAS were obtained from the Chen laboratory (University of Texas, Dallas) ; THP-1 and IRF3 KO THP-1 cells were from ATCC (TIB-202) and InvivoGen respectively.

Techniques: Expressing, Clone Assay, Comparison, Gene Expression