cGAS deficient mice display premature aging associated with de-repression of LINE1 elements and inflammation

Abstract

Aging-associated inflammation, or 'inflammaging" is a driver of multiple age-associated diseases. Cyclic GMP-AMP Synthase (cGAS) is a cytosolic DNA sensor that functions to activate interferon response upon detecting viral DNA in the cytoplasm. cGAS contributes to inflammaging by responding to endogenous signals such as damaged DNA or LINE1 (L1) cDNA which forms in aged cells. While cGAS knockout mice are viable their aging has not been examined. Unexpectedly, we found that cGAS knockout mice exhibit accelerated aging phenotype associated with induction of inflammation. Transcription of L1 elements was increased in both cGAS knockout mice and in cGAS siRNA knockdown cells associated with high levels of cytoplasmic L1 DNA and expression of ORF1 protein. Cells from cGAS knockout mice showed increased chromatin accessibility and decreased DNA methylation on L1 transposons. Stimulated emission depletion microscopy (STED) showed that cGAS forms nuclear condensates that co-localize with H3K9me3 heterochromatin marks, and H3K9me3 pattern is disrupted in cGAS knockout cells. Taken together these results suggest a previously undescribed role for cGAS in maintaining heterochromatin on transposable elements. We propose that loss of cGAS leads to loss of chromatin organization, de-repression of transposable elements and induction of inflammation resulting in accelerated aging.

Figure 1.. cGAS knockout mice show increased frailty and inflammation.

(a) Frailty index in 18-months old WT and cGAS KO mice. cGAS KO mice display increased frailty. p < 0.001, t-test. WT n = 64 mice; cGAS KO n = 24 mice. (b) cGAS KO male mice display higher body mass at older age. p < 0.01, t-test. WT n = 23 mice; cGAS KO n =14 mice. (c) H&E staining of lung in WT and cGAS KO mice. Relative to WT, cGAS KO tissues display substantial neutrophil infiltration (indicated by arrows). (d) H&E staining of liver in WT and cGAS KO mice. Relative to WT, cGAS KO livers display substantial increase in fibrotic tissue (indicated by arrows). (e) Survival of WT and cGAS KO female mice. cGAS KO mice show a significantly reduced lifespan (WT n = 48, cGAS KO n = 58; WT median survival = 106.7 weeks, cGAS KO survival = 92.4 weeks; p = 0.0008, Mantel-Cox test). (f) Representative images of two different pairs of cGAS KO and WT male mice at 14-months-old.

Figure 2.. RNAseq reveals pro-inflammatory signature in cGAS KO mice.

(a) Volcano plot showing differentially expressed genes in lung cells from cGAS KO vs. WT mice. Relative to WT, cGAS KO samples display 315 significantly upregulated genes and 190 downregulated genes. cGAS KO n = 6 mice; WT n = 5 mice. (b) Top biological processes GO Terms for cGAS KO versus WT mouse lung cells show pro-inflammatory signature in cGAS KO samples. (c) Top cellular components GO Terms for cGAS KO versus WT mouse lung cells. (d) qRT-PCR confirms higher levels of inflammation-related gene expression in cGAS KO mice. NFkB, IL-18, and IL-6, along with the PYHIN gene AIM2, are significantly enriched in cGAS KO mice but not WT mice. qRT-PCR was conducted on 12-month-old WT and cGAS KO lung tissue. N=3 mice per group. *p < 0.05, **p < 0.01, or ****p < 0.0001, t-test.

Figure 3.. cGAS knockout mice display increased L1 expression across multiple tissues.

(a) RNAseq analysis of primary lung cells from cGAS KO and WT mice shows changes in L1Md L1 families upon cGAS KO. L1 mRNA expression in L1MdA_I, L1Mda_II, L1MdA_III, and L1MdFanc families is higher in cGAS KO mouse lung. (b) qRT-PCR confirms elevated L1MdA expression in cGAS KO mouse tissues. Organs were harvested from 12-month-old WT and cGAS KO mice. L1 expression was measured via qRT-PCR and normalized to EEF2. n=3-6 mice per group. *p < 0.05 or **p < 0.01, t-test. (c) cGAS KO primary cells display elevated levels of L1 cDNA in the cytoplasm. smiFISH probes specific for L1 ORF1p and ORF2p L1MdA sequence were applied to WT and cGAS KO primary lung cells. Signal for cytoplasmic L1 cDNA was consistently elevated in cGAS KO cells. (d) Immunostaining of WT and cGAS KO mouse primary cells for ORF1p. Cells were stained using LINE1 ORF1p (EA13) antibody.

Figure 4.. cGAS knockdown leads to aberrant L1 expression, accumulation of L1 cDNA, and DNA damage.

(a) cGAS knockdown in MEFs. WT MEFs were treated with cGAS or control shRNA. cGAS knockdown was confirmed via qRT-PCR and Western blotting. (b) L1MdA mRNA expression was measured via qRT-PCR. EEF2 was used as a reference. (c) cGAS knockdown results in L1 cDNA accumulation. WT MEFs were treated with cGAS or control shRNA before conducting DNA smiFISH for L1MdA cDNA. Cells treated with cGAS shRNA display substantial increase in L1 cytoplasmic cDNA. (d) Quantification of (c). (e) cGAS knockdown results in L1 ORF1p accumulation. Immunostaining of WT MEFs treated with control or cGAS shRNA and stained for ORF1p (EA13). (f) Quantification of (e). (g) DNA qPCR following subcellular fractionation shows increased L1MdA cDNA in cytoplasm of cells treated with cGAS shRNA. MEFs were fractionated and cytoplasmic fraction was subjected to qPCR following DNA cleanup (Thermo) and RNase treatment. (h) Western blot to confirm successful cellular fractionation. Cytoplasmic and nuclear fractions were stained for vinculin and H3. (i) cGAS knockdown cells induces DNA damage signaling. γH2AX immunostaining of WT and cGAS KO MEFs transfected with control or cGAS shRNA. (j) Quantification of (i). Experiments were performed in triplicate **p < 0.01. ***p < 0.001, ****p < 0.0001, t-test

Figure 5.. Primary cGAS knockout cells exhibit increased accessibility on pro-inflammatory genes and young L1s.

(a) Volcano plot of differentially accessible OCRs in cGAS KO versus WT mouse primary lung cells. Relative to WT, cGAS KO samples display 1392 significantly more accessible OCRs and 200 less accessible OCRs. n = 4 mice for each group. (b) Top GO terms of differentially accessible promoters in cGAS KO versus WT mouse lung. (c) Motif discovery and enrichment using MEME-ChIP for regulators of the observed inflammatory response in cGAS KO cells. OCRs significantly more open in cGAS KO samples were enriched for Spi1 binding sites. OCRs significantly more closed in cGAS KO samples were most enriched at FoxE1 and FoxD2 binding sites. (d) AIM2 (one of the primary PYHIN genes; that stimulates expression of IL-18 and IL-6 via STING) is significantly more accessible in cGAS KO lung cells (red peaks) than in the WT lung cells (blue peaks). M/F indicate mouse sex. (e) Changes in accessibility of mouse LINE1 families upon cGAS KO. Families are sorted based on the average length of their copies, which is a proxy of evolutionary age. Teal bars indicate the evolutionary young L1Md clade. Y-axis is the fold change in cGAS KO ATACseq compared to WT (0 = no change).

Figure 6.. cGAS KO cells exhibit a decrease in methylation on genomic DNA.

(a) Volcano plot of differentially methylated repetitive element families in cGAS KO versus WT primary mouse lung cells. Relative to WT, cGAS KO samples display 84 repeat families with significantly decreased DNA methylation. The majority of repeat families have lower methylation in cGAS KO lung tissue (One sample Wilcoxon signed-rank test). n = 3 mice for each genotype. (b) Change in genomic DNA methylation on various transposable element classes in cGAS KO relative to WT primary mouse lung cells. cGAS KO samples exhibit significant global reduction of DNA methylation on DNA, LINE, LTR, and SINE elements. Stars represent significance for one sample Wilcoxon signed-rank test (Null hypothesis: median logFC is zero). (c) Change in genomic DNA methylation on Mus L1Md elements in cGAS KO primary lung cells relative to WT. (d) Change in genomic DNA methylation on Mus SINE elements by family in cGAS KO primary lung cells relative to WT.

Figure 7.. cGAS mediates chromatin organization.

(a) Nuclear cGAS co-localizes with H3K9me3. STED images of primary lung cells from WT and cGAS KO mice stained for cGAS and DAPI. Experiments were performed in triplicate; n = 3 mice for each group. (b) cGAS forms LLPS condensates in the nucleus of human diploid fibroblasts. (c) 1,6 HD treatment disrupts cGAS LLPS nuclear condensates in human fibroblasts. (d) Model for nuclear cGAS function in maintaining chromatin organization and L1 repression. cGAS binding to nucleosomes promotes heterochromatin on L1 elements via phase separation. Loss of cGAS disrupts heterochromatin organization leading to L1 de-repression and induction of inflammation. Created in BioRender. Martinez, J. (2024) BioRender.com/x29g707