DNA Methylation Analysis in the Intestinal Epithelium—Effect of Cell Separation on Gene Expression and Methylation Profile

Jenke, Andreas; Postberg, Jan; Raine, Tim; Nayak, Komal; Molitor, Malte; Wirth, Stéfan; Kaser, Arthur; Parkes, Miles; Heuschkel, Robert; Orth, Valerie

doi:10.1371/journal.pone.0055636

Cited by 24 publications

(20 citation statements)

References 16 publications

(16 reference statements)

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“…Statistical algorithms that can provide estimated cell proportions and allow adjustment for cellular heterogeneity are now widely performed throughout the EWAS literature17. There are comparatively fewer epigenetic studies with separated cell data, particularly disease-relevant cells and this is a significant strength of the present study43. Detailed characterization of matched genetic, DNA methylation and expression data in separated leukocytes has highlighted several cell-specific findings.…”

Section: Discussionmentioning

confidence: 95%

Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease

Ventham¹,

Kennedy²,

Adams³

et al. 2016

Nat Commun

199

158

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Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression.

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Section: Discussionmentioning

confidence: 95%

Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease

Ventham¹,

Kennedy²,

Adams³

et al. 2016

Nat Commun

199

158

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“…Colonic mucosal tissues from patients with IBD and patients without mucosal inflammation or injury (control subjects) were collected, and colonic epithelial cells purified by cell sorting using immunomagnetic beads as described previously. 33,34 SIRT2 mRNA ( Figure 7A) and protein levels ( Figure 7B) were significantly reduced in the intestinal epithelium of IBD patients as compared with control (ie, non-IBD) samples, indicating a possible role of SIRT2 in regulating human intestinal epithelial homeostasis.…”

Section: Expression Of Sirt2 Is Decreased In Inflamed Colitis From Pamentioning

confidence: 96%

“…Human colonic epithelial cell isolation was performed as described. 33 Briefly, human colon epithelial samples were delivered from the endoscopy room in ice-cold PBS. Whole tissue biopsies, placed in collagenase IV with Dulbecco's modified Eagle medium/10% fetal bovine serum in a 1:10 dilution, chopped with scissors and agitated gently with a 1-mL pipette tip, remained in this room-temperature solution for 20-30 minutes.…”

Section: Human Biopsy Samples and Human Colonic Epithelial Cell Isolamentioning

confidence: 99%

SIRT2 Contributes to the Regulation of Intestinal Cell Proliferation and Differentiation

Zhou

Rychahou

et al. 2020

Cellular and Molecular Gastroenterology and Hepatology

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Using complementary approaches (intestinal epithelial cell lines, intestinal organoids and SIRT2 knockout mice), we demonstrated that SIRT2, decreased in intestinal tissues from patients with inflammatory bowel diseases, contributes to the maintenance of intestinal epithelium homeostasis through regulation of Wnt-b-catenin signaling. BACKGROUND AND AIMS: Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD). We investigated the role of Sirtuin 2 (SIRT2), a NAD-dependent protein deacetylase, in intestinal epithelial cell (IEC) proliferation and differentiation and the mechanism by which SIRT2 contributes to maintenance of intestinal cell homeostasis. METHODS: IECs were collected from SIRT2-deficient mice and patients with IBD. Expression of SIRT2, differentiation markers (mucin2, intestinal alkaline phosphatase, villin, Na,K-ATPase, and lysozyme) and Wnt target genes (EPHB2, AXIN2, and cyclin D1) was determined by western blot, real-time RT-PCR, or immunohistochemical (IHC) staining. IECs were treated with TNF or transfected with siRNA targeting SIRT2. Proliferation was determined by villus height and crypt depth, and Ki67 and cyclin D1 IHC staining. For studies using organoids, intestinal crypts were isolated. RESULTS: Increased SIRT2 expression was localized to the more differentiated region of the intestine. In contrast, SIRT2 deficiency impaired proliferation and differentiation and altered stemness in the small intestinal epithelium ex vivo and in vivo. SIRT2-deficient mice showed decreased intestinal enterocyte and goblet cell differentiation but increased the Paneth cell lineage and increased proliferation of IECs. Moreover, we found that SIRT2 inhibits Wnt/b-catenin signaling, which critically regulates IEC proliferation and differentiation. Consistent with a distinct role for SIRT2 in maintenance of gut homeostasis, intestinal mucosa from IBD patients exhibited decreased SIRT2 expression. CONCLUSION: We demonstrate that SIRT2, which is decreased in intestinal tissues from IBD patients, regulates Wnt-b-catenin signaling and is important for maintenance of IEC proliferation and differentiation.

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“…DNA methylation/hydroxymethylation was assessed via methylated DNA immunoprecipitation (MeDIP/hMeDIP) as described previously [ 40 ]. Briefly, genomic DNA was isolated by phenol:choroform:isoamylalcohol extraction, sheared by ultrasonic treatment prior to pull-down of 5meC or 5hmeC containing fragments using antibodies selective for either methylated or hydroxymethylated DNA (Diagenode, Liège, Belgium).…”

Section: Methodsmentioning

confidence: 99%

Restitution of gene expression and histone acetylation signatures altered by hepatitis B virus through antiviral microRNA-like molecules in nontransformed murine hepatocytes

et al. 2014

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BackgroundVirus-host interactions result in altered gene expression profiles in host cell nuclei and enable virus particle production, thus obligatorily involving changes in their epigenomes. Neither such epigenome changes nor their response to antiviral treatment have been extensively studied to date, although viral infections are known to contribute to the long-term development of severe secondary diseases, for example, hepatocellular carcinoma. This may be causally linked to virus-induced persistent plastic chromatin deformations.ResultsWe studied whether impaired hepatitis B virus (HBV) replication can lead to the restitution of epigenome signatures hypothesizing that hepatocytes alternatively could adopt a ‘memory’ state of the infection; that is, the chromatin could persist in a HBV-induced configuration potentially inheritable between dividing hepatocytes. We therefore determined epigenomic signatures and gene expression changes altered by HBV and the effects of suppressed HBV replication in nontransformed hepatocytes of newborn mice. Further we investigated differential histone acetyltransferase and histone deacetylase activities in HBV-negative and HBVpositive hepatocytes, as well as the effects of HBV suppression on gene expression and the chromatin landscape. We show that the expression of several genes and the chromatin landscape become altered upon HBV infection, including global hypoacetylation of H2A.Z and H3K9. Reporter assays monitoring the activities of histone acetyltransferases or histone deacetylases, respectively, suggest that hypoacetylation most probably depends on elevated sirtuin deacetylase activity, but not on class I/II histone deacetylases. Using Micrococcus nuclease to study the chromatin accessibility in met murine-D3 and hepatitis B virus met murine hepatocytes, we demonstrate that the observed differences in H2A.Z/H3K9 acetylation lead to global chromatin structure changes. At all selected sites examined by chromatin immunoprecipitation and quantitative real-time PCR, these effects can be partly restituted via the nucleoside analog reverse transcriptase inhibitor 3TC or using anti-HBV microRNA-like molecules.ConclusionsIncreased sirtuin activity might lead to global histone hypoacetylation signatures, which could contribute to the HBV-induced pathomechanism in nontransformed hepatocytes. Using several techniques to suppress HBV replication, we observed restituted gene expression and chromatin signature patterns reminiscent of noninfected hepatocytes. Importantly, ectopic expression of antiviral short-hairpin RNA, but not microRNA-like molecules, provoked intolerable off-target effects on the gene expression level.Electronic supplementary materialThe online version of this article (doi:10.1186/1868-7083-6-26) contains supplementary material, which is available to authorized users.

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DNA Methylation Analysis in the Intestinal Epithelium—Effect of Cell Separation on Gene Expression and Methylation Profile

Cited by 24 publications

References 16 publications

Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease

Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease

SIRT2 Contributes to the Regulation of Intestinal Cell Proliferation and Differentiation

Restitution of gene expression and histone acetylation signatures altered by hepatitis B virus through antiviral microRNA-like molecules in nontransformed murine hepatocytes

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