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Published in final edited form as: Cell Metab. 2014 Apr 1;19(4):702–711. doi: 10.1016/j.cmet.2014.03.012

Obesity, rather than diet, drives epigenomics alterations in colonic epithelium resembling cancer progression

Ruifang Li 1, Sara A Grimm 2, Kaliopi Chrysovergis 1, Justin Kosak 1, Xingya Wang 1, Ying Du 2, Adam Burkholder 2, Kyathanahalli Janardhan 3, Deepak Mav 4, Ruchir Shah 4, Thomas E Eling 1, Paul A Wade 1
PMCID: PMC4050048  NIHMSID: NIHMS577800  PMID: 24703701

Summary

While obesity represents one of several risk factors for colorectal cancer in humans, the mechanistic underpinnings of this association remain unresolved. Environmental stimuli, including diet, can alter the epigenetic landscape of DNA cis-regulatory elements affecting gene expression and phenotype. Here, we explored the impact of diet and obesity on gene expression and the enhancer landscape in murine colonic epithelium. Obesity led to the accumulation of histone modifications associated with active enhancers at genomic loci downstream of signaling pathways integral to the initiation and progression of colon cancer. Meanwhile, colon-specific enhancers lost the same histone mark, poising cells for loss of differentiation. These alterations reflect a transcriptional program with many features shared with the program driving colon cancer progression. The interrogation of enhancer alterations by diet in colonic epithelium provides insights into the biology underlying high-fat diet and obesity as risk factors for colon cancer.

Introduction

Colorectal cancer (CRC) is the third most common human cancer worldwide with more than 1 million new cases every year (Ferlay et al., 2010). Above normal body weight and obesity are among the principal risk factors for development of colorectal cancer in human populations as well as in animal models (Larsson and Wolk, 2006; Moghaddam et al., 2007; Reddy et al., 1977; Wasan et al., 1997). In humans, the relationship between obesity and colorectal cancer incidence appears to be modified by gender. In men, incidence of colorectal cancer is increased in the obese; in women, this association is weaker (Bardou et al., 2013). While several hypotheses rationalize the link between obesity and colorectal cancer, it seems likely that contributing factors are complex.

To explore the effects of Western diet, weight gain, and obesity on the epigenomic landscape of colonic epithelial cells, we employed a mouse model of diet-induced obesity. C57BL/6 mice were fed a diet wherein either 10% of calories (LF) or 60% of calories (HF) come from fat. To globally map alterations of the epigenomic landscape induced by high-fat diet and obesity, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of histone H3 lysine 27 acetylation (H3K27ac). Our study revealed striking alterations in enhancer utilization, consistent with a dietary predisposition to cancer.

Results

Acetylation of H3K27 as an Indicator of Chromatin Landscape Changes

To assess the impact of diet and gender on the epigenome in colonic epithelia, male and female C57BL/6 mice raised on low-fat (10% of calories from fat) and high-fat (60% of calories from fat) diets for periods of 15 to 20 weeks. Colonic epithelial cells were harvested using a well-established protocol (Roediger and Truelove, 1979) to generate RNA and chromatinTranscript abundance was determined by conventional microarray; chromatin was evaluated by ChIP for H3K27ac . Analyses were performed on biological replicate animals (2 replicates per dietary regimen for females, 3 replicates per dietary regimen for males).

Local enrichment of H3K27ac was determined using standard peak-calling; this analysis revealed ~ 40,000 significant H3K27ac peaks in each diet group at a false discovery rate of less than 0.001. H3K27ac peaks showed a significant overlap (~75% – 91% of peaks) across biological replicates regardless of gender (Table S1A). The distribution of H3K27ac relative to annotated genes was similar among groups regardless of diet or gender, with peaks mapping to intragenic (~43%), intergenic (30%) and promoter regions (±1kb around TSS, ~24%) (Table S1B). Chromatin state models predict a correlation between H3K27ac proximal to the transcription start site and gene expression (Ernst et al., 2011). We determined the relationship between transcript abundance and H3K27ac level in a 2 kb window centered on transcription start site (Table S1C), observing robust correlation. We concluded that the H3K27ac ChIP-seq data were of sufficient quality to support detailed analysis.

High-fat Diet Induces Changes in the Enhancer Landscape of Colonic Epithelium

A statistical method (http://www.bioconductor.org/packages/release/bioc/html/DiffBind.html) that detects differential binding sites consistently changed in biological replicates comparing groups identified 876 regions that gained acetylation at H3K27 and 691 regions that lost this mark in females as a function of diet (Figure 1A); 231 regions gained H3K27ac and 1238 regions lost H3K27ac in males(Figure S1A). A subset of these differentially acetylated regions was validated by ChIP-qPCR (Figure S1C). Exemplar loci for each category were depicted in Figure 1B. Differentially enriched H3K27ac regions predominantly localized outside known promoters (>2 kb away from the nearest TSS) (Figure 1C), at regions that are moderately conserved across placental mammals (Figure S1B) and overlap ENCODE marks (H3K4me1, H3K27ac and DNAse HS) (Figure 1D), suggesting they function as enhancers. We tested this prediction using luciferase reporter assays, finding detectable enhancer activity in10 of 11 regions tested (Figure S1D).

Figure 1. High-fat Diet Changes the Enhancer Landscape in Colonic Epithelium.

Figure 1

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(A) The heatmap depicts H3K27ac levels at differentially enriched H3K27ac regions (female mice) extending 5kb from the midpoint of the regions. The images were generated using Partek Genomics Suite version 6.11.0321 based on normalized counts of H3K27ac ChIP-seq fragment using 250 bp bins. Read counts were normalized by millions of uniquely mapped, non-duplicate reads, and fragment centers were estimated to be 100 nt downstream of the 5’ mapping location. (B) UCSC genome browser views illustrating representative examples of gained H3K27ac region (top panel) and lost H3K27ac region (bottom panel) in wild type mice. (C) Gained and lost H3K27ac regions were mapped to the closest Refseq gene TSS and the distribution of distance relative to the nearest TSS is shown. (D) Percent of gained and lost H3K27ac regions overlapping with each ENCODE enhancer mark. See also Figure S1 and Table S1.

Direct overlap of differentially acetylated loci was very infrequent across gender, occurring less than 5% of the time. As individual promoters are frequently regulated by multiple enhancers (Thurman et al., 2012), we wondered whether gender differencesseen here might reflect epigenomic alterations impacting the same promoter by influencing different enhancers. We tested this hypothesis by asking whether differentially acetylated loci were colocalized in genomic space consistent with the distances between promoter and enhancer defined by current models (Dixon et al., 2012; Sanyal et al., 2012). A statistically significant majority of regions either gaining acetylation in males (126 of 231, p<0.0001 Fisher’s exact test) or losing acetylation in females (376 of 691, p=0.0015 Fisher’s exact test) were localized within a genomic interval consistent with this hypothesis (Table S1D).

Diet Reprograms the Enhancer Landscape to Resemble Colon Cancer

A recent report describing the enhancer landscape of human colon cancer concluded that cis-acting regulatory DNA with decreased levels of histone marks prototypical of enhancers in tumor versus normal tissue were enriched in loci with specific functions in intestinal crypts. Conversely, loci with elevated levels of enhancer marks in the same comparison were not enriched in any tissue specific pattern (Akhtar-Zaidi et al., 2012), supporting the conclusion that the tumor enhancer landscape reflects a loss of differentiated status. Given the association of obesity with elevated risk of colorectal cancer, we hypothesized that dietary fat and/or obesity might remodel the enhancer landscape of colonic epithelium to resemble the cancer enhancer profile. To test this hypothesis, we assessed the tissue specificity of differentially acetylated (H3K27ac) regions using a function of Shannon Entropy (Schug et al., 2005), as employed previously (Akhtar-Zaidi et al., 2012), to rank these regions in order of their specificity in colon and nine other tissue types. Loci losing H3K27ac were enriched in colon specific acetylated loci, whereas regions gaining H3K27ac were non-colon specific regardless of gender (Figure 2A). These observations suggest that high-fat diet and obesity prompt colonic epithelial cells to remodel their enhancer landscape in a manner analogous to the remodeling of the enhancer signature associated with colorectal cancer.

Figure 2. Diet Induced Enhancer Alterations in Colonic Epithelium Resemble the Enhancer Signature of Colon Cancer.

Figure 2

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(A) Tissue specificity of differentially enriched H3K27ac regions. Tissue-specificity scores (Q-Statistic, Y axis) for H3K27ac signals in 10 different tissues are plotted for gained (left panels) and lost (right panels) H3K27ac regions in female mice (upper panels) and male mice (lower panels). (B) De novo discovery of motifs associated with differential H3K27ac regions. The HOMER package (version 4.1) was used to search for de novo DNA sequence motifs. The motifs were then assigned to transcription factors or transcription factor families based on similarity with known motif matrices. In addition, the fraction of H3K27ac regions containing the motifs and p-value for the overrepresentation of each motif were shown. See also Figure S2 and Table S2.

To decipher the mechanism underlying enhancer reprogramming, we used the HOMER de novo motif algorithm (Heinz et al., 2010) to discover enriched transcription factor motifs at differentially acetylated regions. The most significantly enriched motifs(Figure 2B) exhibited striking concordance across gender, suggesting mechanistic similarities in the biological response to diet. Differential H3K27ac loci were enriched for binding sites of transcription factors downstream of two pathways commonly disrupted in colon cancer (The Cancer Genome Atlas Network, 2012) – ETS (RAS signaling pathway), Foxo1 (PI3K signaling pathway) – and AP1 (JNK signaling pathway) which regulates cell proliferation and differentiation in response to a variety of growth factors and cytokines (Hess et al., 2004). To assess the significance of enrichment of the RAS signaling pathway, a constitutively active KRAS (G12V) allele was introduced into a mouse colon epithelial cell line (CMT-93, wild type for Kras), and a subset of regions which contain putative ETS binding sites were assessed by ChIP-PCR (Figure S2A). The resulting acetylation changes at these loci closely parallel those observed in the ChIP-Seq experiment, suggesting that KRAS activation, presumably acting through an ETS family transcription factor(s), could be responsible for a subset of the H3K27ac alterations.

Binding sites of transcription factors essential for colon development and differentiation were also enriched, including Cdx2, Hnf4a, and Klf4 (Cattin et al., 2009; Gao et al., 2009; Garrison et al., 2006; Ghaleb et al., 2011; Hryniuk et al., 2012; Katz et al., 2002; Verzi et al., 2010). To interrogate the significance of this finding, we compared our data to published ChIP-Seq data for Cdx2 and Hnf4a in small intestine (Verzi et al., 2011; Verzi et al., 2013). Hnf4a peaks colocalized with diet-specific differentially acetylated loci containing putative Hnf4a binding motif at high frequency in females (81% of expected) and a reasonable frequency in males (39% of expected). Cdx2 peaks in small intestine overlapped with diet-specific differentially acetylated loci in colon containing Cdx2 motif with lower frequency in males (Table S2A). These findings are consistent with the hypothesis that diet-specific differential acetylation may reflect alterations in the maintenance of normal colon homeostasis.

We then investigated the biological functions of the differentially enriched H3K27ac regions (Table S2) using Genomic Regions Enrichment of Annotations Tool (GREAT) (McLean et al., 2010). Gained H3K27ac regions in females were enriched in genes involved in regulation of protein kinase activity ( MAP kinase and JAK), EGFR and IL6-mediated signaling events, presenilin action in Notch and Wnt signaling, and pathways controlling cell adhesion and cell migration - α6β1 and α6β4 Integrin signaling (Giancotti and Ruoslahti, 1999). Strikingly, among enriched disease ontology terms, the most enriched terms were colon cancer in addition to diseases known to be correlated with high-fat diet, such as type 2 diabetes and hyperlipidemia. In addition, gained H3K27ac regions were also significantly enriched with genes up-regulated by activated KRAS and with genes up-regulated in metastatic compared with non-metastatic tumors (Table S2B). Lost H3K27ac regions in females were associated with genes integral to antigen presentation (Table S2C), suggesting that the colonic epithelial cells from mice on high-fat diet may be impaired in this function. In addition, lost H3K27ac regions also associated with genes down-regulated by activated Kras (Table S2C).

In keeping with the hypothesis that diet impacts similar genes in males and females, the GREAT analysis of differential H3K27ac regions in male mice revealed biological functions similar to those observed in females (Table S2). These included regulation of kinase activity, growth factor signaling (including EGFR, PDGF and FGFR), phosphatidylinositol signaling and IL6-mediated signaling events. In the disease ontology category, tumor related terms were also enriched. These results underscore the similarities in the biological response to diet and obesity in male and female mice.

However, human studies suggest gender-specific differences in the relationship between obesity and cancer. In our genomic regions enrichment analysis, we observed several enriched pathways and terms that were unique to male mice. In males, differential H3K27ac regions were correlated with the RAC1 and RhoA signaling pathways involved in cytoskeletal reorganization, cellular adhesion and cellular movement (Moorman et al., 1999). Stem cell-related terms were also enriched in males, including stem cell maintenance, stem cell development and stem cell differentiation (Table S2D). Thus, gender modifies the remodeling of the enhancer landscape by diet.

Diet Induces a Transcriptional Program Enriched in Features of Cancer Progression

To assess whether the observed alterations in active enhancer profile were reflected in the transcriptional program, we performed gene expression microarray analysis. Initial analysis of the data included determination of expression value and ANOVA analysis of differentially expressed genes within gender (Table S3A and S3B). Gene ontology and pathway analysis indicated similarities in the pattern of genes responsive to diet across gender (Table S3C and S3D). We focused detailed analysis on the expression data obtained from females.

Consistent with the ChIP-Seq , we observed that targets of the RAS, PI3K and JNK signaling pathways were up-regulated (Figure S2B), suggesting that differentially enriched H3K27ac regions are biologically functional and drive a specific transcriptional program in colonic epithelia cells. In line with the GREAT analysis, genes dysregulated by high-fat diet were connected to inflammation, cancer, cellular movement and tissue development (Figure S4A and Tables S3C and S4A).

Since the epigenomics remodeling by diet resembled the enhancer signature of colon cancer, we asked whether the resulting gene expression profile also recapitulated gene expression profiles in colon cancer. Among the most differentially expressed genes, approximately half were also dysregulated in mouse colon tumor samples when compared with normal colon (Figure S3A). To determine whether the gene expression changes induced by high-fat diet are relevant to human colorectal cancer, we compared differentially expressed genes in our study to colorectal cancer datasets in Oncomine. We evaluated the top 20 genes most highly up- and down-regulated genes in our study across 6 different tumor/normal datasets (Figure S3B); the majority were changed in the same direction as by diet when comparing cancer with normal colon (Figure 3A).

Figure 3. Diet Induced Gene Signatures in Colonic Epithelium Are Significantly Enriched with Gene Features in Human Colorectal Cancer.

Figure 3

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(A) The top 20 most up- (left) and down-regulated genes (right) in wild type mice were identified. Heatmaps display the expression profiles of those genes in human normal colon, rectum and colon cancer. (B) Colorectal cancer features enriched in the up-regulated genes induced by diet in wild type female mice using Oncomine concepts analysis. (C) Gene Set Enrichment Analysis using signature genes of CCS3 subtype and stem-like subtype as gene sets. Genes in our study were ranked by Signal2Noise score comparing WT HF vs WT LF. ES, enrichment score. NES, normalized enrichment score. See also Figure S3 and Table S3.

To ascertain whether the gene signatures induced by high-fat diet associate with particular features of colorectal cancer, we performed Oncomine concepts analysis (http://oncomine.org). Strikingly, the up-regulated genes significantly associated with gene signatures characterizing the progression of colorectal cancer to advanced stages (Figure 3B), with enrichment in functions pertinent to tumor invasion and migration (Table S3D). In keeping with the identification of the RAS and PI3K signaling pathways (Figure 2B) and the up regulation of their target genes (Figure S2B), the up-regulated gene signature significantly overlapped with gene signatures in KRAS mutant tumors and PTEN mutant cell lines (Figure 3B). Accordingly, we also observed significant overlap with gene signatures in cell lines sensitive to inhibitors of these pathways (Figure 3B and Table S3E). However, concepts discriminating colon cancer from normal colon were associated with only down-regulated genes at a marginal significance (Table S3E). Finally, we performed Gene Set Enrichment Analysis, asking whether genes dysregulated in colonic epithelial cells are enriched with signature genes classifying colorectal cancer (De Sousa et al., 2013; Sadanandam et al., 2013). We observed significant enrichment with signature genes of the poorprognosis CCS3 category (De Sousa et al., 2013) and the stem-like category (Sadanandam et al., 2013) in the high-fat diet-induced differentially expressed genes (Figure 3C demonstrating that the gene expression signature induced by diet is complex with features consistent with a loss of differentiated status and other features associated with colorectal cancer progression.

To validate the specific correlation of gene expression alterations by high-fat diet with colon cancer, we also performed comparison with gene expression in colon of a mouse model of inflammatory bowel diseases (IBD) at both early and late inflammation stages (Russ et al., 2013), showing no correlation with IBD gene expression (Figure S3C).

Obesity, not Diet, Drives Remodeling of the Enhancer Landscape

Our results suggest that remodeling of the enhancer landscape by diet and obesity affects critical genes and pathways important for intestinal homeostasis and may render colonic epithelium prone to cancer. It is unclear from any of the above analyses whether the altered landscape results from direct exposure of the colonic epithelia to the contents of the lumen, or whether the status of the organism (i.e. obesity) drives the process. To distinguish between these possibilities, we employed transgenic mice (females only) expressing human NAG-1/GDF15 (NSAID activated gene-1) (Baek et al., 2006; Eling et al., 2006). NAG-1 transgenic animals, when placed on an identical high-fat diet, failed to gain weight in the same manner as their wild-type counterparts (Figure 4A) and had a lower ratio of fat mass to body weight (Figure 4B). We reasoned that these animals would provide us with a suitable system to ask whether exposure to the colon contents resulting from a western diet predominate over obesity in remodeling the colonic enhancer program.

Figure 4. Obesity drives alterations in enhancer landscape in colon.

Figure 4

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(A) Body weight of mice on either low-fat diet or high-fat diet. (B) The column graph depicts the ratio of abdominal fat mass to body weight in mice on either low-fat or high-fat diet. (C) The heatmap depicts expression of genes dysregulated in wild type mice by diet adjacent to the same genes in NAG-1 transgenic mice. (D) Diet induced enhancer alterations were not observed in NAG-1 transgenic mice. The RPKM fold change was calculated for gained and lost H3K27ac regions as identified in the wild type samples by high-fat diet treatment, with a floor for the ratio denominator set at the estimated noise level (defined as the 2nd percentile in the distribution of RPKM scores for peaks called in non-self samples). The box is the 25th– 75th percentile, and the whiskers are the 5th and 95th percentiles; the black dot is the median, and the yellow dot is the mean. Grey circles are points beyond the 5th/95th percentiles. (E) The column graphs depict quantitative RT-PCR of the indicated inflammatory cytokines in colon tissue. The expression levels were normalized to β-actin. (F) Immunohistochemistry staining of macrophage marker F4/80 reveals high-fat diet induced inflammation was suppressed in NAG-1 transgenic mice. See also Figure S4 and Table S4.

We first assessed the impact of the transgene on the gene expression alterations induced by diet. A heat map was generated displaying genes exhibiting diet-induced dysregulation in wild-type animals with the same genes displayed from NAG-1 transgenic animals (Figure 4C). The transgenic mice displayed a pattern of expression of these genes that completely differed from wild-type mice (Figure S4A, S4B and Table S4), suggesting a dominant role of obesity in directing the alterations in transcriptional program. Comparison of H3K27ac ChIP-Seq data collected from wild-type and NAG-1 transgenic animals (Figure 4D) demonstrated that loci with significant gains in acetylation in wild-type animals had, on average, no diet induced change in acetylation level in the NAG-1 transgenic animals. Likewise, loci losing acetylation in wild-type animals were unchanged in the high-fat/low-fat comparison in their transgenic counterparts. These results suggested that obesity, not diet, may be the driving factor in altering the epigenomics landscape in the colons of these mice.

Obesity is closely linked to chronic inflammation, also a hallmark of cancer. Consistent with this notion, expression of inflammatory cytokines in colon tissue was elevated in a diet-specific manner in wild-type, but not transgenic animals (Figure 4E). Histopathologic examination of colon tissue revealed the presence of substantial macrophage infiltration in wild-type, but not in NAG-1 transgenic animals on high fat diet (Figure 4F). To exclude the possibility that NAG-1 transgenic mice are defective in fat absorption, we performed the steatocrit test with feces from either WT mice or NAG-1 mice on high-fat diet. NAG-1 transgenic mice did not show malabsorption of fat (Figure S4C). These data demonstrate that NAG-1 transgenic mice were resistant to both the molecular alterations and phenotypes induced by high fat diet, suggesting that they result from obesity, not diet per se.

Discussion

During intestinal tumorigenesis, the epithelial cells undergo dedifferentiation and acquire stem-cell-like properties (Schwitalla et al., 2013); therefore, colon cancer cells lose enhancer marks that typify normal crypt differentiation status while acquiring enhancer marks that are normally found in noncolon cell types (Akhtar-Zaidi et al., 2012). The differentially acetylated regions described here were enriched with binding sites of transcription factors downstream of signaling pathways commonly disturbed in colon cancer and also binding sites of transcription factors essential for colon development. These findings suggest that alterations in the H3K27 acetylation program induced by obesity may reflect both the loss of activities integral to normal colon homeostasis in addition to gain of activity downstream of signaling pathways activated in cancer. Although diet induced alterations in the landscape of H3K27 acetylation, most changes were not readily associated with alterations in expression of a linked gene. This finding implies that the enhancer alterations described here may reflect subtle changes that prime the genome to respond to a subsequent event.

In the absence of oncogenic stimuli, the occurrence of obesity-associated cancer was not increased by high-fat diet in wild-type mice maintained in a specific pathogen free (SPF) environment (Yoshimoto et al., 2013). However, following treatment with azoxymethane or via introduction of mutant APC, mice on a high-fat diet had a higher incidence of colon tumors than mice fed normal chow (Moghaddam et al., 2007; Reddy et al., 1977; Tuominen et al., 2013). In our study, obesity induced widespread remodeling of the acetylation landscape at presumptive cis-regulatory regions that likely influence the transcriptional response suggestive of cancer predisposition. The associated gene expression profile was specifically enriched in genes implicated in cancer progression. These data suggest that a premalignant lesion in the colon from obese individuals has an increased probability of evolving into a clinically detectable malignancy in part due to ‘priming’ of the enhancer landscape for cancer progression.

Experimental Procedures

Mice

Female h-NAG-1 transgenic mice and wild type C57BL/6J mice at 5 weeks of age from a colony known to be positive for Helicobacter were placed on either 10% fat diet (D12450B) or 60% fat diet (D12492, Research Diets, New Brunswick, NJ) for 20 weeks. Male C57BL/6J mice on either 10% fat diet (380056 DIO controls) or 60% fat diet (380050 DIO high fat diet) for 15 weeks were purchased from Jackson Laboratories and acclimated for one additional week. All mice were handled and treated according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).

Isolation of Colonic Epithelial Cells

Colonic epithelial cells were isolated as previously described (Roediger and Truelove, 1979). See also Supplemental Experimental Procedures.

ChIP Sequencing and Data Analysis

Standard cross-linked ChIP DNA was used to construct sequencing libraries which were processed as single end 36 mers (detailed methods in Supplemental Experimental Procedures). ChIP data were filtered for quality score, aligned and deduplicated (methodology is detailed in Supplemental Experimental Procedures). Peaks were assigned by SICER (Zang et al., 2009), regions of differentially enriched H3K27ac were identified using the diffBind (v1.6.2) R package, motifs were extracted with HOMER (Heintz et al., 2010).

Accession Numbers

The ChIP-seq data are available at NCBI Gene Expression Omnibus (GEO) website under accession id GSE46748. The microarray data are available at NCBI Gene Expression Omnibus (GEO) website under accession id GSE46843.

Supplementary Material

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Highlights.

  • High-fat alters the enhancer landscape of colonic epithelium

  • These alterations the enhancer signature in colon cancer progression

  • The enhancer landscape reflects an expression program resembling cancer progression

  • Obesity, not diet, appears to be the relevant driver of epigenomic alterations

Acknowledgments

We thank Kevin Gerrish and the NIEHS microarray facility for their assistance in microarray processing, NIEHS pathology core for technical assistance with immunohistochemistry, the NIEHS Epigenomics Core for NextGen Sequencing support and NIEHS viral vector core facility for helping with virus packaging. We express gratitude to Dr. Scott Bultman for many useful suggestions and vigorous discussion throughout the course of this work. This research was supported [in part] by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Project number ZO1ES101965 to PAW).

Footnotes

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Disclosure Declaration

The authors have no conflicting financial interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS577800-supplement-01.pdf (6.2MB, pdf)
02
NIHMS577800-supplement-02.xlsx (15.7KB, xlsx)
03
NIHMS577800-supplement-03.xlsx (37.3KB, xlsx)
04
NIHMS577800-supplement-04.xlsx (6.5MB, xlsx)
05
NIHMS577800-supplement-05.xlsx (192.6KB, xlsx)

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