Cytokine-induced chromatin modifications of the type I collagen alpha 2 gene during intestinal endothelial-to-mesenchymal transition - PubMed (original) (raw)

Cytokine-induced chromatin modifications of the type I collagen alpha 2 gene during intestinal endothelial-to-mesenchymal transition

Tammy Sadler et al. Inflamm Bowel Dis. 2013 Jun.

Abstract

Background: Fibrosis of the intestine is currently an irreversible complication of inflammatory bowel disease; yet, little is understood of the underlying pathogenesis and antifibrotic strategies remain elusive. To develop effective therapies, knowledge of the mechanism of transcription and excessive deposition of type I collagen, a hallmark of fibrosis, is needed. We have shown previously that endothelial-to-mesenchymal transition (EndoMT) contributes to the pool of intestinal fibrotic cells and that a cytokine cocktail (interleukin 1-β, tumor necrosis factor α, and transforming growth factor β) induces collagen I alpha 2 (COL1A2) mRNA and protein.

Methods: Chromatin immunoprecipitation assays on pure cultures of human intestinal mucosal endothelial cells undergoing EndoMT were performed with antibodies to specific histone modifications and RNA polymerase II. Reverse transcriptase-PCR was used to quantify the levels of Col1A2 and endothelial-specific von Willebrand factor (vWF) mRNA.

Results: We showed that cytokines induce selective chromatin modifications (histone 4 hyperacetylation, and hypermethylation of histone 3) and phosphorylated RNA polymerase II at the COL1A2 promoter. Hypoacetylated and hypomethylated histone 3 was detected on the repressed vWF gene. Prolonged exposure to cytokines (16 days) retained hyperacetylation of select lysines in H4 on the COL1A2 promoter. Removal of cytokines after 16 days and continued culture for 10 days showed persistent hyperacetylation at lysine 16 in histone H4.

Conclusions: This is the first study to show that COL1A2 gene expression is associated with cytokine-induced, temporally ordered, and persistent chromatin modifications and suggests that these are important determinants of gene expression in EndoMT and intestinal fibrosis.

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Figures

Fig. 1. Induction and persistence of COL1A2 gene expression during EndoMT

HIMEC were seeded at 50 % confluency and were treated with a cytokine cocktail comprising: TGF-β1 (5 ng/ml), TNF-α (5 ng/ml), and IL-1β (0.1 ng/ml) or with cytokine-free growth medium alone for 0–20 days (A). HIMEC were monitored daily to identify endothelial-fibroblast transitions. The cytokines were replenished and the growth medium replaced every 3–4 days. At the end of the cytokine incubation period the cytokines were removed, the cells washed and the medium replaced (A). The cultures were continued for a further 10 days (“10 day wash off”). At the end of this period cells were harvested, RNA extracted and RT-PCR performed to determine the levels of COL1A2 mRNA relative to GAPDH as a reference gene after incubation with the cytokine cocktail (gray bars) and after cytokine removal/”wash off” (black bars) (B). Data represent mean +/− S.E.M. n= 4–5 experiments. * p<0.05.

Fig. 2. Cytokine-dependent enrichment of histone modifications on the COL1A2 and vWF gene promoters during EndoMT

Chromatin was prepared from HIMEC cells that had undergone EndoMT after 5 days incubation with the cytokine cocktail as described. ChIP assays were performed with antibodies to 3 modifications linked to gene activation: (Panels A, C, E) H4 and H3 acetylation (H4Ac and H3Ac) and H3K4 trimethylation (H3K4me3) and 3 modifications characteristic of repressed genes (Panels B, D, F): H3K9 dimethylation (H3K9me2), H3K27 trimethylation (H3K27me3) and H4K20 trimethylation (H3K20me3). qPCR detected the COL1A2 proximal promoter (A, & B), the COL1A2 upstream enhancer (C & D) and vWF promoter endothelial specific region (E & F) respectively. Mean values +/− SEM of % of total precipitated chromatin relative to input chromatin expressed as % input for each modification on each gene is shown. RNA was prepared from the same cells and RT-PCR performed to determine the level of COL1A2 and vWF mRNA after 5 days (Panel G). Control (open bars) and cytokine-treated (gray bars) cells were compared. Data represent mean +/− S.E.M of n=6–11 experiments. * p<0.05.

Fig. 2. Cytokine-dependent enrichment of histone modifications on the COL1A2 and vWF gene promoters during EndoMT

Fig. 3. Cytokine-induced recruitment of S2 phosphorylated RNA polymerase II to the COL1A2 gene promoter

HIMEC were treated with TGF-β1 (5 ng/ml), TNF-α (5 ng/ml), and IL-1β (0.1 ng/ml) for 5 days as already described and cells were crosslinked with 2mM freshly prepared Disuccinimidyl glutarate (DSG) for 30 mins at room temperature followed by 0.5% v/v Formaldehyde (Sigma) for 5 min. To terminate the crosslinking reaction, glycine (0.125M) was added for a further 5 min. The crosslinked chromatin was sonicated and immunoprecipitated with antibodies to the S2, S5 and unphosphorylated forms of RNA polymerase II (polIIS2ph, polIIS5ph and PolII respectively). The proximal promoter region of the COL1A2 promoter was analyzed. Mean values +/− S.E.M of % of total precipitated chromatin relative to input chromatin expressed as % input for each form of the RNA polymerase is shown. Control (open bars) and cytokine-treated (gray bars) cells were compared. Data represent mean +/− S.E.M of n=3 experiments. ** p<0.01.

Fig. 4. Cytokines increase the acetylation of Histone H4 at specific lysine residues on the COL1A2 gene

Chromatin was prepared from HIMEC cells that had undergone EndoMT after 1 day (Panel A & B) and 5 days (Panel C & D) incubation with the cytokine cocktail as described. ChIP assays were performed with antibodies to individual acetylated lysine residues in histone H4: H4K5Ac, H4K8Ac, H4K12Ac and H4K16Ac (Panels A – D). qPCR of the ChIP samples detected the COL1A2 promoter (A & C) and the COL1A2 upstream enhancer (B & D). Mean values +/− S.E.M of % of total precipitated chromatin relative to input chromatin expressed as % input for each modification on each gene is shown. RNA was prepared from the same cells incubated with the cytokine cocktail for 1 day and RT-PCR performed to determine the level of COL1A2 mRNA (Panel E). Control (open bars) and cytokine-treated (gray bars) cells were compared. Data represent mean +/− S.E.M of n=8–11 experiments. * p<0.05.

Fig. 5. Prolonged COL1A2 gene expression in EndoMT is associated with persistent histone H4 acetylation

Chromatin was prepared from HIMEC cells after 16 days incubation with growth medium (open bars), the cytokine cocktail (gray bars) as described and after removal of cytokines and a further 10 day incubation (wash off, black bars). ChIP assays were performed with antibodies to individual acetylated lysine residues in histone H4: H4K16Ac (Panels A & B), H4K12Ac (Panels C & D), H4K8Ac (Panels E & F), H4K5Ac (Panels G & H). qPCR of the ChIP samples was performed to detect the proximal promoter (Panels A,C,E,G) and upstream enhancer region (Panels, B,D,F,H) of the COL1A2 gene promoter. Mean values +/− S.E.M of % of total precipitated chromatin relative to input chromatin expressed as % input for each modification on each gene is shown (Panels A–H). Data represent mean +/− S.E.M of n=3 experiments. * p<0.05.

Fig. 6. Prolonged COL1A2 gene expression in EndoMT is associated with persistent acetylation of H4K16

Chromatin was prepared from HIMEC cells after 16 days incubation with growth medium (open bars), the cytokine cocktail (gray bars) as described and after removal of cytokines and a further 10 day incubation (wash off, black bars). ChIP assays were performed with antibodies to H3K9Me2 (Panels A & B) and H3K4Me3 (Panels C & D). qPCR of the ChIP samples was performed to detect the proximal promoter (Panels A & C) and upstream enhancer (Panels B & D) of the COL1A2 gene promoter (A & C) and upstream enhancer (B & D). Mean values +/− S.E.M of % of total precipitated chromatin relative to input chromatin expressed as % input for each modification on each gene is shown (Panels A–D). Fold increase in Col1A2 mRNA (Panel E) over background is shown after incubation with the cytokine cocktail (open bars), and after cells treated with cytokine cocktail for 16 days followed by an additional 10 days of culture after the removal of cytokines (washoff, black bars) were compared. Data represent mean +/− S.E.M of n=3 experiments. * p<0.05.

Fig.7. Schematic summary of cytokine-induced, time dependent changes in histone acetylation and methylation at the COL1A2 promoter

Induction of COL1A2 gene expression after 5 days of cytokine (IL-1β, TNFα and TGFβ,) treatment is associated with acetylation (Ac) of H4 lysines 8, 12 and 16 and dimethylated H3K9 (Me) at the regions encompassing the proximal promoter and upstream enhancer regions (Panel A). H4 acetylation at the 3 aforementioned sites is retained during prolonged expression of COL1A2 following 16 day cytokine treatment whereas dimethylated H3K9 is lost and acetylation at lysine 5 of histone H4 is enhanced over this time period (Panel B). Cytokine treatment for 16 days followed by removal of the cytokines for a further 10 days resulted in cytokine dependent retention of H4K16 acetylation at the upstream enhancer region of the COL1A2 promoter (Panel C).

Fig.8. Schematic model of cytokine-induced modification of chromatin and regulation of COL1A2 gene transcription

IL-1β, TNFα and TGFβ induce H4 acetylation, H3K9 methylation and RNA polymerase II (PolII) binding on induction of COL1A2 gene expression. The increase in histone acetylation and methylation are likely to be markers of increased activity of specific chromatin modifying enzymes e.g. increased activity of specific histone acetylases (HATs) or decreased activity of histone deacetylases (HDAC). Hyperacetylated and hypermethylated histones are also potential binding sites for the recruitment of transcription factors (TF) and other coregulator proteins. These contribute to the formation of an active transcription complex responsible for the induction and prolonged expression of COL1A2.

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Cytokine-induced chromatin modifications of the type I collagen alpha 2 gene during intestinal endothelial-to-mesenchymal transition - PubMed (original) (raw)