Epigenetic regulation of IBD-associated fibrosis: potential for

novel anti-fibrotic therapies

 Tammy Sadler1,2, Angela Ting3, Yaomin Xu4, Xiuli Liu5, Eleni

Stylianou1,2

 1Department of Pathobiology, Lerner Research Institute, and 2Department of Gastroenterology and Hepatology, Digestive

Disease Institute, 3Genomic Medicine Institute, 4Department of Quantitative Health Sciences, 5Department of Anatomic Pathology.

Cleveland Clinic Foundation, Cleveland, USA

Crohn’s DiseaseInflammation of small bowel & colon:

transmural

Environmental factorsGenetic susceptibility

Abnormal interaction of commensal gut flora, epithelial barrier and innate immunity

Inflammatory Bowel Disease – etiology?

FIBROSIS

Increased matrix depostion e.g.Type I collagen (COL1A1, COL1A2)

EPIGENETICS?

Endothelial cells

Mucosal innate immune cells

Mesenchymal cells/fibroblasts/myofibrobla

sts

EpitheliumApoptotic &

necrotic epithelial cells

GUT LUMEN

Gut flora

SUBMUCOSA

FIBROSIS

Intestinal inflammation and fibrosis in IBD

Epithelial barrier dysfunction

Cytokines, chemokines e.g TNFa, IL-8,

TGFb, IL-1b, IL-17

Endogenoussignals

Cytokines

TYPE I COLLAGEN

Epigenetics

• Heritable changes in phenotype that are independent of changes to the DNA sequence

• 2 examples: Histone modifications and DNA methylation

• Epigenetic mechanisms determine whether a gene is silenced or activated and allows the cell to adapt to environmental cues.

• Epigenetics bridges the gap between genotype and environment

K9me

H3

H4

K27meCorepressors

K4me

H3

H4Kac

RNA Pol II

Coactivators

LPSIL-1b

TNF-a

K4me

H3

H4

Kac

Repressed, histone/DNA methylated promoter:condensed chromatin

(heterochromatin)

Histone-modified, remodelled, decondensed promoter

(euchromatin)

Accessible, transcriptionally active promoter

DNA methylation

Scarpa & Stylianou Inflamm Bowel Dis 2012

Epigenetic regulation of gene transcription

Clinical Significance

•Epigenetic modifications required for Type I collagen gene expression and a fibrotic DNA methylome or epigenotype for CD could have diagnostic and prognostic utility.

•New mechanistic insights into clinically relevant mechanisms of disease pathogenesis could be provided.

•The conceptual framework for identification of therapeutic targets and selective and efficacious anti-fibrotic "epi-pharmaceuticals” that could prevent or treat fibrosis.

COL1A2

H4

H3

Ac16

H4

H3

12 16

AcAc8Ac Ac

TGFbTGFb

H4

H3

Ac16

H4

H3COL1A2

AcCytokines removed after 16

days + 10 day washoff

-1599bp -25bp

IL1bIL1b

TNFaTNFa

H4

H3

Ac AcAc8 12 16

H4

H3

Ac Ac812 16

Me9

COL1A2

AcIL1bIL1b

TNFaTNFa

TGFbTGFb

5 days

16 days

Specific histone modifications are associated with induction of COL1A2 gene expression in intestinal EndoMT

MeMe9

Me

AcAc812

Ac5

Ac5Ac

A

B

C

Sadler et al Inflamm Bowel Dis 2013 in press

Fibrosis-specific epigenetic signatures occur in the fibrotic intestine in IBD.

 This hypothesis will be tested by 2 specific aims: Aim 1. Determine the fibrosis-specific histone modification signature in human fibrotic intestine in IBD in vivo. Aim 2. Identify the fibrosis-specific DNA methylome that defines human intestinal fibrogenesis in IBD in vivo.

Hypothesis and Aims

Research plan• Year 1

• Collect age and gender matched normal control and CD fibrotic tissue specimens

• Optimize conditions for ChIP of tissue

• Establish conditions for isolation of fresh human fibroblasts

• Demonstrate feasibility of performing epigenetic analysis in fresh HIF

• Year 2

• ChIP assays of tissue and fresh HIF to define type I collagen specific histone modification signature

• Employ MBD-isolated genome sequencing (MiGS) to profile changes in DNA methylation in human fibroblasts from fibrotic intestine.

• Integrate methylome with fibrotic transcriptome

Tissue source Diagnosis Number of specimens

Colon Diverticular disease 7

Polyps 3

UC inflamed 8

CD fibrotic 10

CD inflamed 5

Ileum Polyps 1

Ileocolic anastamosis 1

CD fibrotic 8

CD inflamed 4

Intestinal tissue specimens procured during year 1