1. Cleveland Clinic Foundation, Cleveland, USA
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

2. Inflammatory Bowel Disease – etiology?
Environmental factors
Genetic susceptibility
Abnormal interaction of commensal gut flora, epithelial barrier and innate immunity
Crohn’s Disease
Inflammation of small bowel & colon: transmural
EPIGENETICS?
Increased matrix depostion e.g.Type I collagen (COL1A1, COL1A2)
FIBROSIS

3. Intestinal inflammation and fibrosis in IBD
Gut flora
Apoptotic &
necrotic
epithelial cells
GUT LUMEN
Epithelium
Endogenous
signals
Mucosal
innate immune cells
Cytokines, chemokines
e.g TNFa, IL-8,
TGFb, IL-1b, IL-17
Epithelial barrier dysfunction
Cytokines
SUBMUCOSA
Endothelial cells
TYPE I COLLAGEN
Mesenchymal cells/fibroblasts/myofibroblasts
FIBROSIS

4. 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

5. Epigenetic regulation of gene transcription
IL-1b
LPS
TNF-a
K9me H3 H4
K27me
Corepressors
Repressed, histone/DNA methylated promoter:
condensed chromatin (heterochromatin)
Histone-modified, remodelled, decondensed promoter
(euchromatin)
Accessible, transcriptionally
active promoter
K4me H3 H4
Kac
DNA methylation
K4me H3 H4
Kac
RNA Pol II
Coactivators
Scarpa & Stylianou Inflamm Bowel Dis 2012

6. 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.

7. removed after 16 days + 10 day washoff
COL1A2 H4 H3 Ac 16 12 8
TGFb
Cytokines
removed after 16 days day washoff
-1599bp
-25bp
IL1b
TNFa Me 9
5 days
16 days
Specific histone modifications are associated with induction of COL1A2 gene expression in intestinal EndoMT 5 A B C
Sadler et al Inflamm Bowel Dis 2013 in press

8. Hypothesis and Aims
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.

9. 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

10. Intestinal tissue specimens procured during year 1
Tissue source
Diagnosis
Number of specimens
Colon
Diverticular disease 7
Polyps 3
UC inflamed 8
CD fibrotic 10
CD inflamed 5
Ileum 1
Ileocolic anastamosis 4