Analyzing The Epigenomic Effects of Butyrate on Human Colon Cancer Cells

Cribas, Emily

1
S. ;

Fremin, Brayon

2
J. ;

Bhatt, Ami

2
S.

1Pennsylvania

State University, University Park, PA

2Department of Genetics, Stanford School of Medicine, Stanford, CA

Abstract
Commensal bacteria within the human colon produce a metabolite known as butyrate through fiber fermentation. Studies show butyrate induces expression of a differentiation marker in a subset of human colon
cancer cells. This suggests that butyrate induces differentiation of cells, which has been explored as a method of cancer therapy. It is hypothesized that butyrate induces differentiation through its role in histone
deacetylase (HDAC) inhibition. HDAC inhibition induces decondensation of chromatin and an increase in chromatin accessibility. The whole-epigenomic effects of butyrate on different subsets of colon cancer cells
remain unknown. We hypothesize that butyrate results in differential chromatin decondensation in various colon cancer cell lines. Furthermore, we hypothesize that the extent and pattern of decondensation
will be different between those cell lines that differentiate in response to butyrate treatment vs. those that do not. In this study, we perform ATAC-Seq, an assay for tranposase-accessible (open) chromatin on different
subsets, to assess areas where the chromatin has been decondensed in response to butyrate. The results from these experiments provide a genome-wide perspective on what genes are exclusively affected by butyrate
HDACi. Further studies linking these affected genes with induced differentiation are needed to elucidate the role of butyrate, or a high-fiber diet, in serving as a cancer differentiation therapy.

Introduction

ATAC-Seq Protocol

Butyrate Production

Bacteria in our colon (gut)

ferment resistant starch
and fiber
This process produces
metabolites: butyrate,
propionate, and acetate
Butyrate is the main
energy source of epithelial
colon cancer cells

Nature Chemical Biology

Butyrate as an HDAC Inhibitor

H i s t o n e d e a c e t y l a s e s
(HDACs): enzymes that
remove (-) acetyl groups
from histones wrapped
around the DNA

Condenses chromatin
Decreases gene expression
Butyrate inhibits HDACs1

Gene Hits

Sequencing Protocol and Methods

1. Isolate nuclei

Transposase
Adaptor 1
Adaptor 2
Nucleosome
DNA

2. Transpose and purify

Annotating Genes of Interest

3. PCR

Nuclei
Amplifiable fragments

ATAC-Seq Analysis Methods

Fastqc 0.11.24

Bowtie 1.1.15

MACS 2.1.06

Homer 4.77

Assess quality of
reads

Align reads to
reference genome

Call peaks

Annotate all
differential peaks

Nature Drug Discovery

Sequencing Analysis

Butyrate and Differentiation

Butyrate induces ALPi (cell
differentiation marker)
expression in some colon
cancer cell lines2
Sensitive: Cells in
which butyra induced
ALPi expressio
Resistant: Cells in
which butyrate did not
induce ALPi expression

Genes of interest that were exclusively decondensed in

sensitive cell lines included:
KLF5, a gene that encodes a transcription factor that
increases ALPi expression
CDX2, a gene involved in epithelial cell maturation
as well as goblet and Paneth cells
XIAB, a gene that codes for an Xs-linked inhibitor of
apoptosis
TRIM genes were decondensed in butyrate-treated
HT29 and none were decondensed in HCT116
Most of the genes that code for HDACs are condensed in
both butyrate-treated cell lines

Conclusions and Future Work

Conclusions
Butyrate potentially indirectly inhibits HDACs
Genes that were induced by butyrate were decondensed by
butyrate, suggesting HDACi as its mechanism
Results provide genome-wide perspective on what genes
could undergo induced expression by butyrate
More genes can be affected by butyrate once enhancer and
transcription factor genes are appropriately accounted for

Alignment and Peak Calling

Reference Genome

Peak

Sequenced reads

Future Work

Optimize peak calling parameters

Transcription factor footprinting
Correlate open areas with gene expression levels
Measure protein expression of overexpressed genes

Acknowledgements
Bhatt Lab:
PI: Dr. Ami Bhatt
Mentor: Brayon Fremin

HT29 Peak View of KLF5 Gene

Experimental Design
Cell Preparation
RKO

HCT-116

Differential Peak Calling and Annotation

HCT116 Butyrate

HT29 Butyrate

HT-29

1656
Butyrate

Stanford Summer Research Program:

Director: Terrance Mayes
Manager: Samar Fahmy
Assistant: Clayton Brown

247

829

4 Conditions at 2.5 mM (48 hrs):

Propionate Acetate MS-275(HDACi) Control

References
1.
2.

ATAC-Seq3
Assay for Transposase-Accessible Chromatin
3-step protocol for integrative epigenomic analysis
Captures open chromatin sites at nucleotide resolution

Gene overlap between resistant and sensitive cell lines. Peaks that were exclusively found in
butyrate treatment for 2 cell lines were called and overlapped using statistical analysis tools.
These peaks were then annotated to their corresponding gene. Genes of interest include
those that were uniquely found in butyrate treated sensitive cell lines.

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4.
5.
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