# Poster Session II - A219 CHARACTERIZATION OF METHYLATION MODIFICATIONS OF HUMAN COLONOID MONOLAYERS ESTABLISHED AS IN VITRO CHRONIC DAMAGE MODEL

## TL;DR

This study characterizes methylation changes in human colonoid monolayers after repeated injury, linking these epigenetic modifications to chronic damage and diseases like IBD and colorectal cancer.

## Contribution

The study introduces an in vitro model to investigate chronic epithelial injury and identifies specific methylation modifications associated with disease-like states.

## Key findings

- Repeated injury leads to loss of barrier integrity and significant genotypic and phenotypic changes in colonoid monolayers.
- Increased DNA methylation and histone assembly correlate with chronic damage and disease-like states.
- Upregulated genes in damaged monolayers are linked to oxidative phosphorylation, a process relevant to cancer progression.

## Abstract

Our lab established an in vitro damage model using human colonoids grown as 2D Intestinal Epithelial Cells (IECs) monolayers in Air-Liquid Interface (ALI) culture. Upon repeated injury by submergence, these colonoid monolayers loose their epithelial barrier integrity and regrowth potential. Changes in mRNA expression and global proteomic profiling of this human model of injury were found to be very similar to those found in Inflammatory Bowel Disease (IBD) and pre-colon cancer like state

Preliminary proteomics studies on these monolayers has been suggestive of significant changes in expression of key proteins after progressive rounds of injury. These are quite like that seen in IBD. These could be strongly associated with epigenetic modifications, such as, DNA methylation which could potentially impact the level of gene expression in these epithelial monolayers.

This study aims to assess the methylation modifications of these injured IECs monolayers which would help in identification and validation of methylation at specific sites.

To assess the epigenetic modifications, these IEC monolayers from 4 human colonoids [3 from ascending colon and 1 from transverse colon], 2 males and 2 females, were analyzed pre-ALI, during growth and differentiation in ALI before injury, and after 1st, 3rd and 5th round of injury and recovery.

Global methylation, at each of the conditions was assessed using the Illumina Infinium Methylation EPIC BeadChip, an array-based technology, to assess the bisulphite-converted cellular DNA for methylation at CpG sites. Genomic DNA was extracted and bisulfite conversion was done using Zymo EZ DNA methylation kit. Methylation at specific sites has been calculated using the fluorescence intensity of methylated and unmethylated alleles. DNAm age estimator software, HOMER, was used to calculate the epigenetic age.

Gene ontology process enrichment analysis has been used to identify changes in gene expression pathways at above mentioned stages.

Progressive rounds of submergence injury lead to loss of barrier integrity, strongly correlated with significant genotypic and phenotypic changes. Increased DNA methylation and histone assembly found in our studies are reflective of chronic damage. Epigenetic characterization has led to identification of key specific targets which play a role in modification of pathways leading to chronic epithelial injury.

Correlative evidence from proteomics profiling and epigenetic characterization of this in vitro chronic damage model provides for an enhanced understanding of the initiation and progression of IBD and IBD associated colorectal cancer.

Significantly upregulated genes in FliC stimulated damaged monolayers compared to FliC stimulated monolayers after first round of damage. These genes are associated with oxidative phosphorylation, a key mechanism for providing energy to differentiated cell types but also when cells become cancerous, especially in context of colorectal cancer.

CCC