Figure 7.
Endogenous cGAS Associates with Centromeres
(A) Representative immunofluorescence images of a metaphase spread of cycling U2OS cells showing endogenous cGAS enrichment at the inner centromere. CENP-A marks the centromere position. Yellow arrows point to cGAS localization at an inner centromere. Scale bar, 5 μm.
(B) Top left: magnification of a centromere as in (A) showing cGAS enrichment between two CENP-A foci. Top right: schematic of the expected CENP-A and ACA localization at the inner kinetochore and at the inner kinetochore and centromere, respectively. Bottom: normalized mean of the fluorescence intensity scan lines of ACA, CENP-A, and cGAS along the centromeres. Error bars represent the SEMs of 36 centromeres in 1 cell (representative of 2 independent experiments).
(C) Heatmaps of ACA, CENP-A, and cGAS intensities for individual chromosomes as in (B) after distance normalization (n = 22 chromosomes, representative of 2 independent experiments).
(D) Baseline expression of the indicated ISGs (Ifit1, Ifit2, Oas1a) in bone marrow-derived DCs from Cgas−/− mice or WT littermates. Bars represent means and error bars are SEMs. Each dot represents an individual mouse; n = 6 mice per genotype combined from 3 experiments; 1-way ANOVA with post hoc Tukey test; ∗∗∗∗p < 0.0001.
(E) Overview of the GFP-cGAS knockin locus.
(F) Experimental scheme for ChIP-seq of GFP-cGAS mouse DCs from GFP-cGASKI/KI mice.
(G) Annotation of the significant peaks of the ChIP-seq on CgasKI/KI mice DCs over input. Elements with <10 peaks are grayed out.
(H) Annotation of the significant peaks of endogenous GFP-cGAS over input in mouse DCs (peak intersection of replicates 1 and 2) compared to significant peaks of overexpressed GFP-NLS-cGAS over input in human DCs (donor 1). Elements with <10 peaks are not included.
(I) ChIP-seq read enrichment on repeats; CgasKI/KI over input in mouse DCs. SATMIN, mouse minor satellite DNA; GSAT_MM, mouse γ-satellite repetitive sequence; IMPB_01, consensus of repeated region of mouse chromosome 6; SQR1_MM, SQR2_MM, SQR4_MM, mouse simple repetitive DNA (sqr family); ZP3AR, satellite from Muridae.
(J) Working model of cGAS expression in the cytoplasm in interphase, followed by localization to the nucleus as a result of mitosis.