GATA6-AS1 Regulates Intestinal Epithelial Mitochondrial Functions, and its Reduced Expression is Linked to Intestinal Inflammation and Less Favourable Disease Course in Ulcerative Colitis

Abstract

Background and aims: Widespread dysregulation of long non-coding RNAs [lncRNAs] including a reduction in GATA6-AS1 was noted in inflammatory bowel disease [IBD]. We previously reported a prominent inhibition of epithelial mitochondrial functions in ulcerative colitis [UC]. However, the connection between reduction of GATA6-AS1 expression and attenuated epithelial mitochondrial functions was not defined.

Methods: Mucosal transcriptomics was used to conform GATA6-AS1 reduction in several treatment-naïve independent human cohorts [n=673]. RNA pull-down followed by mass spectrometry was used to determine the GATA6-AS1 interactome. Metabolomics and mitochondrial respiration following GATA6-AS1 silencing in Caco-2 cells were used to elaborate on GATA6-AS1 functions.

Results: GATA6-AS1 showed predominant expression in gut epithelia using single cell datasets. GATA6-AS1 levels were reduced in Crohn's disease [CD] ileum and UC rectum in independent cohorts. Reduced GATA6-AS1 lncRNA was further linked to a more severe UC form, and to a less favourable UC course. The GATA6-AS1 interactome showed robust enrichment for mitochondrial proteins, and included TGM2, an autoantigen in coeliac disease that is induced in UC, CD and coeliac disease, in contrast to GATA6-AS1 reduction in these cohorts. GATA6-AS1 silencing resulted in induction of TGM2, and this was coupled with a reduction in mitochondrial membrane potential and mitochondrial respiration, as well as in a reduction of metabolites linked to aerobic respiration relevant to mucosal inflammation. TGM2 knockdown in GATA6-AS1-deficient cells rescued mitochondrial respiration.

Conclusions: GATA6-AS1 levels are reduced in UC, CD and coeliac disease, and in more severe UC forms. We highlight GATA6-AS1 as a target regulating epithelial mitochondrial functions, potentially through controlling TGM2 levels.

Keywords: GATA6-AS1 long non-coding RNA; inflammatory bowel disease; mitochondria.

Figure 1.

GATA6-AS1 is reduced in UC, CD and coeliac disease, in isolated epithelia in CD and UC, and is further reduced in UC cases with less favourable outcome. [A–C] GATA6-AS1 mRNA expression in CD ileum [A; RISK, SOURCE, isolated epithelia^27] and UC colon [B; PROTECT, RISK, isolated epithelia]. Lines indicate median and upper and lower quartile. [C] GATA6-AS1 is further reduced in more severe UC cases based on clinical [PUCAI], combined clinical–endoscopic [Total Mayo score] and endoscopic disease severity [Endoscopic Mayo score] in the PROTECT cohort. [D] GATA6-AS1 mRNA reduction is linked to less favourable disease outcome [W4R or W52SFR] in the PROTECT cohort. GATA6-AS1 levels in controls are given as a reference. [E] Functional annotation enrichments using ToppGene/ToppCluster and Cytoscape of the GATA6-AS1 co-expression network in PROTECT using Euclidean distance [full list and FDR values are given in Supplementary Dataset S1].

Figure 2.

GATA6-AS1 expression in the gut is confined to epithelia. [A–C] Cellular expression of GATA6-AS1 in single-cell dataset of human colon [A] and ileum [B and C]. The size and colour of the dots are proportional to the percentage of cells expressing the gene and the normalized expression level, respectively [A]. [B, C] Uniform manifold approximation and projection [UMAP] plot with the indicated cell types [B, left] and a map coloured by GATA6-AS1 expression [B, right] with further focus on epithelial cell types and expression of the mature epithelial and stem cell markers SI and LGR5 respectively [C], overall showing GATA6-AS1 specificity to epithelia. [D] GATA6-AS1 expression in patient-derived ileum and colon differentiated organoid culture, with and without 40 ng/ml IFNγ plus 20 ng/ml TNFα treatment. [E] GATA6-AS1 expression with and without 100 ng/ml LPS plus 40 ng/ml IFNγ and 100 µM DFO in HT29 [left] and 40 ng/ml IFNγ plus 20 ng/ml TNFα in Caco-2 cells. [F] Fluorescence in situ hybridization [FISH] showing GATA6-AS1 [red, arrows] distribution between the nucleus and cytoplasm in Caco-2 cells, compared to MALAT1 [red], which show predominant nuclear expression. Nuclei are stained with Hoechst [blue]; magnification ×63 oil; scale bar 10 µm. Results of a t-test or Spearman correlation with coefficients [r] are shown. All two-sided: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3.

GATA6-AS1 regulates epithelial mitochondrial genes and function. [A] Schematic representation of GATA6-AS1 interactome pull-down experiments by two independent pools of tiling antisense biotinylated oligos. Binding of GATA6-AS1 RNA was confirmed by PCR. [B, C] The GATA6-AS1 interactome in differentiated Caco-2 cells identified 285 proteins using mass spectrometry [t-test: delta LFQ ≥ 0.1 and FDR ≤ 0.05; Supplementary Dataset S2]. [B] Heatmap of GATA6-AS1 top curated interacting targets identified by mass spectrometry; green and red colours indicate low and high binding respectively; Ctl columns are binding to non-specific control oligos. [C] Functional enrichments of the 285 protein targets indicated enrichment of mitochondrial functions [ToppGene/ToppCluster^58]. [D] GATA6-AS1 silencing [sh1, sh2] was compared to scrambled non-specific shRNA [Ctl] using qRT-PCR [normalized to GAPDH and to controls]. [E] JC1 staining and FACS analysis were used to define the mitochondrial membrane potential [MMP]; schematic representation of MMP calculated as the ratio of red/green fluorescence. The analysis was performed in the absence or presence of 50 µM CCCP [carbonyl cyanide 3 chlorophenylhydrazone], which reduces MMP. Representative FACS images are presented, and the summarized experiments [n = 3] are shown as a bar-graph. [F] Representative western blots using specific antibodies in GATA6-AS1 knockdown cells indicated reduction of DECR1, MT-CO₂ and GATA6, with induction of TGM2 and EPHX proteins. GAPDH was used as a loading control [three to four independent experiments are shown in Supplementary Figure S3]. [G, H] Mitochondrial ROS levels and apoptosis were measured using MitoSOX [G] and annexin V [H] in GATA6-AS1 knockdown cells at baseline or after treatment with 500 µM H₂O₂ for 3 h. All two-sided paired t-tests: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Schemes were created with biorender.com.

Figure 4.

GATA6-AS1 regulates cellular metabolism. [A–C] Untargeted metabolomics was used to capture cellular metabolites and metabolites secreted to the media in GATA6-AS1 knockdown and control cells, untreated or treated with 100 µM DFO + 100 ng/ml LPS + 40 ng/ml IFNγ. [A] Schematic representation of the experiment and PCoA plot of the extracted metabolites based on Canberra distance, indicating differences by both GATA6-AS1 expression and by treatment. [B] Heatmap representing significantly different metabolites in cells [left heatmap] and their media [right heatmap, rank-mean test and FDR < 0.1] between GATA6-AS1 knockdown [sh1 and sh2] and control cells, with visualization of the metabolites in both treated and untreated cells [full comparisons in Supplementary Dataset S3]. Blue and red arrows indicate metabolites that were lower or higher in cells and/or media respectively. [C] Heatmap of the –log₁₀[FDR] enriched pathways obtained from metabolites increased [left two columns] or decreased in the media and cells [right two columns]. The pathways in the upper panel are increased and those in the lower panel are decreased in GATA6-AS1 silencing. [D] Peak area of specific faecal metabolites that differed in GATA6-AS1 knockdown vs controls and between CD and controls. Each case submitted two faecal samples at least 2 months apart and those are shown separately for each control and CD subject. Mann–Whitney test with *p < 0.05 and **p < 0.01.

Figure 5.

GATA6-AS1 silencing induces TGM2 expression and inhibits mitochondrial respiration while TGM2 co-silencing recovered mitochondrial respiration. [A] Schematic representation of the mitochondrial electron transport chain [I, II, III, IV] and ATP synthase [complex V], with the modulators included in the Seahorse XF Cell Mito Stress Test. [B, C] Mito Stress Test measurement of oxygen consumption rate [OCR] in basal conditions and after sequential injections of oligomycin, FCCP and rotenone/antimycin A [B] in GATA6-AS1 knockdown and control Caco-2 cells without [left] and with [right] 100 µM DFO + 100 ng/ml LPS + 40 ng/ml IFNγ; changes in basal respiration, ATP production and maximal respiration were calculated [C]. [D] OCR [as in Figure 4B and C] in GATA6-AS1 knockdown and control cells transiently transfected with TGM2 or control siRNA to test whether reduction of TGM2 can rescue the GATA6-AS1 knockdown effect of cellular respiration. Changes in basal respiration, ATP production and maximal respiration. [E] Extracellular acidification rate [ECAR] was measured [see also Supplementary Figure S4]; non-glycolytic acidification was calculated and found to differ between GATA6-AS1 knockdown and control cells, and to be rescued by reduction of TGM2. Individual values are shown in the graph with their mean. All two-sided paired t-tests: *p < 0.05, **p < 0.01, ***p < 0.001. [F] Concluding scheme; GATA6-AS1 is reduced in gut epithelia of UC, CD and coeliac cases, and mitochondrial function is impaired in epithelia of UC and CD patients.^,, To model our observations in patients mechanistically, we showed that GATA6-AS1 binds a complex that includes TGM2 [1]. GATA6-AS1 silencing resulted in TGM2 induction, similarly to the induction seen in patients [1]. Reduction of GATA6-AS1 and TGM2 induction caused attenuation of mitochondrial membrane potential [2], higher production of mitochondrial ROS [3], reduction of TCA metabolites [4] and reduced cellular respiration [5]. Schemes were created with biorender.com.