KAT2A and KAT2B prevent double-stranded RNA accumulation and interferon signaling to maintain intestinal stem cell renewal

Abstract

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and sequencing of immunoprecipitated double-stranded RNA were used to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions and maintaining intestinal health.

Fig. 1.. Intestinal epithelium–specific DKO of Kat2a and Kat2b depletes H3K9ac and is required for survival.

(A) Timeline of induction of genetic KOs and harvest. Vertical arrows indicate the days of tamoxifen injection. Horizontal arrows indicate the average time range for tissue collection. (B) Transcript levels of Kat2a and Kat2b from Kat2 DKO mice in vivo, normalized to housekeeping gene controls [two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test, *adj. P < 0.05, n = 12 to 13 per group]. (C) Average weights over time in percentage, relative to first day of tamoxifen injection, of DKO and control mice show that loss of Kat2 in the intestine leads to rapid weight loss and death (n = 12 to 15 per group, mixed-effects ANOVA analysis, *adj. P < 0.05). (D) Schematic of the pipeline for histone PTM MS. Created with Biorender.com. (E) Relative abundance (%) of H3K9ac and H45acK12acK16ac from histone PTM MS of histones from Kat2 DKO IECs (n = 4 to 5 per group, Welch’s t test). (F) Immunohistochemistry (IHC) of jejunal sections using the indicated antibodies in control and DKO mice (n = 3 to 4 per group). Scale bars, 100 μm. Note efficient depletion of GCN5, PCAF, and H3K9ac in DKO tissues. (G) Additional H3K9ac immunostaining; insets outline the intestinal epithelium to highlight the epithelial-specific reduction of H3K9ac immunoreactivity. Scale bars, 100 μm (center images) and 50 μm (insets). Immunostaining is representative of four biological replicates. (H) Left: Immunoblots of two representative samples for H3K9ac and histone H3 expression on purified histones. Right: Ratios of H3K9ac to histone H3 in DKOs from densitometric analysis using ImageJ (n = 5 per group, unpaired t test, *P < 0.01). All graphs show means with error bars in SEM.

Fig. 2.. Intestinal epithelium–specific Kat2a/Kat2b DKO leads to loss of stem cell renewal and skews expression of distinct intestinal lineage markers.

(A) Left: IHC on mouse jejunum for antibodies against OLFM4 and KI67 at 4 to 7 days after onset of tamoxifen treatment (n = 3 per group). Scale bars, 100 μm. Right: RT-qPCR of Olfm4 and Ki67 transcript changes in DKO. Srsf6 was used as an additional reference gene, and all transcript expression was normalized to housekeeping gene control (n = 3 per group, unpaired t test, *P < 0.01). ns, not significant. (B) Left: Crypts were collected from mice 4 to 5 days after the onset of tamoxifen treatment and then seeded in Matrigel for organoid culture. Ex vivo primary epithelium-derived intestinal organoids grown for 6 days (n = 3 per group). Scale bars, 500 μm. Right: Survival graph of intestinal organoids (n = 3 per group, unpaired t test, *P < 0.01). (C) Experimental design for RNA-seq. Arrows indicate the days of tamoxifen injection, with tissue collection occurring on day 3. (D) Heatmap of significantly up-regulated or down-regulated RNA transcripts in Kat2a/Kat2b DKO mice (n = 4 per group, average FPKM > 1, DESeq2 adj. P < 0.05, fold change > 2 or < −2). (E) FPKM values of Olfm4, Mki67, and select intestinal cell lineage markers from RNA-seq (n = 4 per group, *DESeq2 adj. P < 0.05). GSEA enrichment profiles from RNA-seq that are (F) up-regulated or (G) down-regulated (n = 4 per group). FDR, false discovery rate; NES, normalized enrichment score. (H) PAS staining and IHC for lysozyme of intestinal tissue (n = 3 per group). Scale bars, 10 μm (middle) or 100 μm (top and bottom). All graphs show means with error bars in SEM.

Fig. 3.. Kat2a/Kat2b DKO induces epithelial-intrinsic IFN signaling, which can be suppressed with pharmacological inhibitors of cytoplasmic double-stranded nucleic acid–sensing pathways.

(A) Top up-regulated (red) and top down-regulated (blue) GO terms in DKO samples (483 and 362 genes, respectively, from RNA-seq Fig. 2D) using DAVID GO. (B) Expression profile of immune-related genes populated from RNA-seq and GO analysis (from five GO terms: GO:0051607, GO:0009615, GO:0002376, GO:0045087, and GO:0045071). (C) GSEA profiles demonstrate enrichment of IFN-β and IFN-λ targets from GSE142166 and down-regulation of mitochondrial complex V in Kat2a/Kat2b DKO. (D) Timeline of events for ex vivo epithelium-derived intestinal organoid experiments, where mice are harvested on day 0 for three-dimensional organoid culture containing antimicrobials [Primocin (0.1 mg/ml) and penicillin-streptomycin (100 U/ml)]. Induction of DKO with 16-hour tamoxifen (1 μM) treatment in ex vivo culture begins on day 4. Inhibitor treatment also begins on day 4 and ends on day 8 at the time of organoid collection. (E) Schematic of IFN signaling pathway activation via double-stranded nucleic acids. Targets of select inhibitors are shown in red. MAVS, mitochondrial antiviral-signaling protein. (F) RT-qPCR of ISGs from epithelium-derived intestinal organoids with DKO induced in culture, which were treated with pharmacological inhibitors targeting TBK1 (GSK8612; 10 μM), JAK (ruxolitinib; 2 μM), and STING (H-151; 2 μM) for 4 days. Inhibitors were added during and after induction of DKO in culture, with media and inhibitors refreshed daily (n = 3 per group, two-way ANOVA with Tukey post hoc analysis, *adj. P < 0.05 for the indicated gene versus 0.2% DMSO vehicle-treated DKO, normalized to Hprt). Mean shown with error bars in SEM.

Fig. 4.. Determination of H3K9ac status at ISGs induced in the Kat2 DKO.

(A) Mean H3K9ac coverage of all murine genes in the jejunal epithelium is shown at a metagene [TSS to transcription end site (TES) ± 2 kb (left) and only the TSS ± 2 kb (right)]. Positive and negative sense strands were reoriented into the same direction for analysis. Data visuals were generated with deepTools (v3.5.0) using ChIP-seq of H3K9ac from wild-type mice in GSE86996. (B) Left: K-means clustering of H3K9ac signal within 2 kb of the TSS forms five distinct patterns in the murine jejunal epithelium. Positive and negative sense strands were reoriented into the same direction for analysis. Heatmap visual was generated with deepTools 3.5.0. Right: Pie charts indicating the percentage of up-regulated ISGs from Kat2 DKO RNA-seq belonging to each cluster, highlighting that the presence of H3K9ac in wild-type mice does not coincide with ISG expression upon Kat2 DKO. bp, base pairs. (C) Bar charts indicating the percentage of ISGs, all DEGs up-regulated, and all DEGs down-regulated from Kat2 DKO RNA-seq in each H3K9ac ChIP-seq cluster from (B). (D) Scatter plots showing the log fold change of Kat2 DKO FPKM values versus control FPKM values from RNA-seq versus the H3K9ac counts from ChIP-seq (GSE86996) for DEGs (top) or all genes (bottom) from Kat2 DKO RNA-seq. There does not appear to be a strong correlation between H3K9ac levels and changes in RNA levels in the Kat2 DKO.

Fig. 5.. Mitochondrial proteins are differentially acetylated, and mitochondrial enzyme activity is compromised in Kat2 DKO.

(A) Schematic of the pipeline for acetylomics MS. Created with Biorender.com. (B) GO term analysis of the proteome (top) and nonhistone proteins with down-regulated acetyl-lysine residues (bottom) from Kat2 DKO IECs (n = 4 controls and 6 DKOs). (C) Volcano plot of acetyl-lysine residues from acetylomics of Kat2 DKO IECs. Orange points indicate lysine residues on mitochondrial proteins (n = 4 controls and 6 DKOs). Stains for activity of mitochondrial enzymes (D) NADH and (E) COX in jejunal intestine sections (n = 2 to 4 per group). Scale bars, 500 μm. (F) COX activity in the IECs from control and Kat2 DKO mice (n = 3 mice, Welch’s t test) using spectrophotometry. ***P < 0.001.

Fig. 6.. Mitochondrial dsRNA is a likely source of ISG induction in Kat2a/Kat2b DKO.

(A) Schematic of the workflow for dsRIP-seq. Created with Biorender.com. IP, immunoprecipitation. (B) Volcano plot of dsRIP-seq for dsRNA (K1 antibody) pull-down (n = 2 per group, DESeq2 adj. P < 0.01, likelihood ratio test, average FPKM > 1) shows enrichment in dsRNA from mitochondrial transcripts that are specific to anti-dsRNA antibodies in the DKO versus controls and normalized for baseline levels of RNA in input samples. Orange points indicate mitochondrial genes. (C) Top up-regulated (red) and top down-regulated (blue) GO terms for DKO from dsRIP-seq using dsRNA (K1) antibody and DAVID GO. (D) FPKM values of select dsRNA species up-regulated in DKO. Minimum values, maximum values, and mean of the values are shown. (E) IGV tracks for input and dsRNA (K1) pull-down samples from dsRIP-seq along the mitochondrial genome. Sense reads are on the same scale (0 to 197,239) in the top half of the track and antisense reads on the same scale (0 to 23,604) in the bottom half for each sample. (F) Number of unique mitochondrial (top) or nuclear (bottom) sense or antisense reads from each dsRIP-seq sample, normalized to the total number of all dsRIP-seq reads (including both sense and antisense transcripts).