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

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

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

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

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

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.

removed after 16 days + 10 day washoff COL1A2 H4 H3 Ac 16 12 8 TGFb Cytokines removed after 16 days + 10 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

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.

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

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