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Page 4 Qu et al. J Transl Genet Genom 2023;7:3-16 https://dx.doi.org/10.20517/jtgg.2022.16

also associated with Wnt signaling. Downregulated genes were associated with antigen presentation and
processing.

Conclusion: Three days of HFD-induced epigenomic and transcriptomic changes involving metabolic and
immunologic pathways that may promote tumor growth in the genetically predisposed murine intestine without
affecting key cancer signaling pathways.

Keywords: Obesogenic diet, epigenetic changes, lipid metabolic processes, Apc Min/+

INTRODUCTION
Colorectal cancer is an obesity-associated cancer where excess body fat promotes cancer development and
[1]
worsens outcomes in patients with these tumors . Using mouse models, we and others have shown that
obesogenic diets and obesity accelerate the development of intestinal neoplasia in C57 BL6J mice with
[2,3]
mutation or deletion of the APC gene . Obesity has been postulated to accelerate cancer development
partly through epigenetic mechanisms . Murine models in which mice were fed a high-fat diet (HFD) for at
[4]
least 15-20 weeks have, in fact, shown changes in histone H3 acetylation and DNA methylation associated
with remodeling of chromatin regulatory regions that resemble cancer progression . However, we have
[5,6]
previously shown appearances of intestinal polyps inApc Min/+ mice within three days of starting on high-fat
[2]
obesogenic diets, even prior to excessive weight gain . Accordingly, we used ChIP-Seq, and RNA-Seq
approaches to investigate if a three-day HFD was sufficient to induce epigenetic or transcriptional changes
supportive of tumor growth in intestinal epithelia ofApc Min/+ and C57 BL6J mice. ChIP-Seq analysis focused
on identifying VEL and the proximal genes they may regulate. In mammalian cells, active enhancer
elements that regulate transcriptional promoters by cis-acting mechanisms are located within open
chromatin and are characterized by epigenetic alterations in histone methylation and acetylation. Enhancers
have been shown to vary under multiple conditions, including stages of cell differentiation and malignant
transformation. These regions, marked with high levels of H3K4me1 and H3K27ac, have been termed
variant enhancer loci (VELs) and are associated with transcriptional changes of crucial cancer genes in
human colon cancer lines and human colorectal cancer tumors .
[7,8]

METHODS
Mice
+/+
C57BL/6J (denoted as Apc or wild-type) and C57BL/6J-Apc Min/+ (denoted asApc Min/+ ) mice were purchased
from the Jackson Laboratory (Bar Harbor, ME) to develop breeding colonies that supplied all mice used in
the study. Mice were maintained in micro isolator cages at 25 °C on a 12 h reverse light/dark cycle at the
Case Western Reserve University (CWRU) Animal Resource Facility. Breeding was accomplished by
placing one adult mutant (Apc Min/+ ) male mouse with two WT adult female Apc mice to generate mutant
+/+
and WT littermates, which were subsequently distributed to experimental diets as previously described .
[2]
Ear punch samples were taken from mice at 14 days of age for genotyping. Offspring were weaned and
separated by sex at 21 days of age. Siblings were incubated together and fed a standard chow diet Prolab
RMH 3000 (5P00; LabDiet; Brentwood, MO) with autoclaved water ad libitum. At 30 days of age, sibling
male mice were separated by genotype, wild-type orApc Min/+ , distributed to an experimental diet (high-fat or
low-fat) with autoclaved water, and then incubated in groups of two or three mice per cage until sacrifice
after three days on experimental diet.

Experimental diets
Experimental diets consisted of a high-fat diet (HFD) and a low-fat diet (LFD), as previously described .
[2,9]
These diets differed in amounts of fats from hydrogenated coconut oil. The HFD contained 58.0% kcal/g of
fat, 25.5% kcal/g of carbohydrate, and 16.4% kcal/g of protein (D12330; Research Diets; New Brunswick,