STAT1 epigenetically regulates LCP2 and TNFAIP2 by recruiting EP300 to contribute to the pathogenesis of inflammatory bowel disease

Abstract

Background: The aetiology of inflammatory bowel disease (IBD) is related to genetics and epigenetics. Epigenetic regulation of the pathogenesis of IBD has not been well defined. Here, we investigated the role of H3K27ac events in the pathogenesis of IBD. Based on previous ChIP-seq and RNA-seq assays, we studied signal transducer and activator of transcription 1 (STAT1) as a transcription factor (TF) and investigated whether the STAT1-EP300-H3K27ac axis contributes to the development of IBD. We performed ChIP-PCR to investigate the interaction between STAT1 and H3K27ac, and co-IP assays were performed to investigate the crosstalk between STAT1 and EP300.

Results: Lymphocyte cytosolic protein 2 (LCP2) and TNF-α-inducible protein 2 (TNFAIP2) are target genes of STAT1. p-STAT1 binds to the enhancer loci of the two genes where H3K27ac is enriched, and EP300 subsequently binds to regulate their expression. In mice with dextran sulfate sodium (DSS)-induced acute colitis, an EP300 inhibitor significantly inhibited colitis.

Conclusions: p-STAT1 and EP300 promote TNFAIP2 and LCP2 expression through an increase in H3K27ac enrichment on their enhancers and contribute to the pathogenesis of chronic inflammation.

Keywords: EP300; Enhancer; H3K27ac; IBD; STAT1.

Fig. 1

Increased levels of STAT1/p-STAT1 in a mouse model of DSS-induced chronic colitis and in patients with IBD are related to H3K27ac modification. a Overlap of the predicted TFs with the differentially expressed genes by RNA-seq in DSS-induced mice chronic colitis. b STAT1, HNF4A, and IRF2 mRNA expression in DSS-induced mice chronic colitis (error bars: mean ± SEM; control group n = 7, DSS group n = 9). c STAT1/p-STAT1 protein expression in DSS-induced mice chronic colitis. d-e STAT1/p-STAT1 mRNA and protein expression in IBD patients (error bars: mean ± SEM; NC group n = 8, UC group n = 8, CD group n = 10). *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significance

Fig. 2

Identification of LCP2 and TNFAIP2 as STAT1 target genes. a Overlap of the target of STAT1 on the website of GTRD and the upregulated genes in the RNA-seq whose enhancers had increased H3K27ac enrichment after DSS treatment. b TNFAIP2, LCP2, CREBBP, HNF4A, LRG1, HSD17, USP18 mRNA expression in NCM460 cell inflammation model (error bars: mean ± SD). c-d LCP2 and TNFAIP2 mRNA and protein expression in DSS-induced mice chronic colitis (error bars: mean ± SEM; control group n = 7, DSS group n = 9). e–f LCP2 and TNFAIP2 mRNA and protein expression in IBD patients (error bars: mean ± SEM; NC group n = 8, UC group n = 8, CD group n = 10). g The correlation between STAT1 and LCP2, TNFAIP2 in UC and CD patients in GEO database. h–i STAT1 mRNA and protein expression in NCM460 after transfected with siSTAT1-1, 2, 3 or the negative control (error bars: mean ± SD). j–k LCP2 and TNFAIP2 mRNA and protein expression in NCM460 after transfected with siSTAT1-1, 2 or the negative control with stimulation by IFN-γ (error bars: mean ± SD). l–m LCP2 and TNFAIP2 mRNA and protein expression in NCM460 after transfected with siSTAT1-1, 2 or the negative control with stimulation by TNF-α and IFN-γ (error bars: mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

STAT1 regulates its target genes through H3K27ac at their enhancers in mice with DSS-induced chronic colitis. a UCSC browser view showing the ChIP-seq density of H3K27ac signal in both control tissue and DSS-induced tissue located in LCP2 and TNFAIP2 gene locus. b ChIP-PCR was performed to validate H3K27ac modification at the LCP2 and TNFAIP2 enhancer in DSS-induced mice chronic colitis. c ChIP-PCR was performed to validate the occupancy of p-STAT1 on the LCP2 and TNFAIP2 enhancer where H3K27ac enriched in DSS-induced mice chronic colitis. Error bars: mean ± SEM; n = 6 per group. *p < 0.05, **p < 0.01

Fig. 4

p-STAT1 may recruit EP300 to promote target gene expression in NCM460 cells. a EP300 mRNA expression in DSS-induced mice chronic colitis (error bars: mean ± SEM; control group n = 7, DSS group n = 9). b EP300 mRNA expression in IBD patients (error bars: mean ± SEM; NC group n = 8, UC group n = 8, CD group n = 10). c H3K27ac protein expression in NCM460 after adding EP300 inhibitor C646 in different concentration (0, 10, 20, 30, 40, 50 μM) for 24 h. d–e LCP2 and TNFAIP2 mRNA expression in NCM460 after adding C646 or DMSO without or with stimulation of by TNF-α (error bars: mean ± SD). f–g EP300 mRNA and protein expression in NCM460 after transfected with siEP300-1, 2, 3 or the negative control (error bars: mean ± SD). h–i LCP2 and TNFAIP2 mRNA expression in NCM460 after transfected with siEP300 or the negative control with or without stimulation by TNF-α (error bars: mean ± SD). j LCP2 and TNFAIP2 mRNA expression in NCM460 after transfected with siEP300 or the negative control with stimulation by TNF-α + IFN-γ (error bars: mean ± SD). k LCP2 and TNFAIP2 mRNA expression in NCM460 after transfected with siSTAT1 + siEP300 or the negative control with stimulation by TNF-α + IFN-γ (error bars: mean ± SD). l co-IP experiment was performed to validate the combination between p-STAT1 and EP300. *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significance

Fig. 5

Inhibition of EP300 relieves DSS-induced colitis in mice. a Methods for DSS-induced acute colitis in C57BL/6 mice and C646 administration. b Body weight and c disease activity index of mice that received regular drinking water alone (water group n = 10) or 3.0% DSS-containing water (DSS group n = 10), or 3.0% DSS combined with C646 injection (DSS + C646 group n = 15). For statistical comparisons, asterisk indicates DSS vs. DSS + C646. d Colon length and e representative hematoxylin and eosin (H&E) staining of distal colon sections at day 11 after DSS induction (water group and DSS group n = 10, DSS + C646 group n = 15). f Colonic inflammatory cytokine analysis from colonic tissue at day 11 after DSS induction (n = 10 per group). g LCP2 and TNFAIP2 mRNA expression in colonic tissue at day 11 after DSS induction (n = 10 per group). Error bars: mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns means no significance

Fig. 6

Model explaining how p-STAT1 interacts with EP300 to promote H3K27ac enrichment on the enhancers of LCP2 and TNFAIP2 and contributes to the development of chronic inflammation. When STAT1 is activated by IFN-γ, STAT1 migrates into the nucleus and binds to the enhancers of LCP2 and TNFAIP2. p-STAT1 interacts with EP300 to promote H3K27ac enrichment on the enhancers and promote the transcriptions of LCP2 and TNFAIP2 and then contributes to the development of chronic inflammation