Estrogen Receptor Beta Influences the Inflammatory p65 Cistrome in Colon Cancer Cells

Abstract

Inflammation is a primary component of both initiation and promotion of colorectal cancer (CRC). Cytokines secreted by macrophages, including tumor necrosis factor alpha (TNFα), activates the pro-survival transcription factor complex NFκB. The precise mechanism of NFκB in CRC is not well studied, but we recently reported the genome-wide transcriptional impact of TNFα in two CRC cell lines. Further, estrogen signaling influences inflammation in a complex manner and suppresses CRC development. CRC protective effects of estrogen have been shown to be mediated by estrogen receptor beta (ERβ, ESR2), which also impacts inflammatory signaling of the colon. However, whether ERβ impacts the chromatin interaction (cistrome) of the main NFκB subunit p65 (RELA) is not known. We used p65 chromatin immunoprecipitation followed by sequencing (ChIP-Seq) in two different CRC cell lines, HT29 and SW480, with and without expression of ERβ. We here present the p65 colon cistrome of these two CRC cell lines. We identify that RELA and AP1 motifs are predominant in both cell lines, and additionally describe both common and cell line-specific p65 binding sites and correlate these to transcriptional changes related to inflammation, migration, apoptosis and circadian rhythm. Further, we determine that ERβ opposes a major fraction of p65 chromatin binding in HT29 cells, but enhances p65 binding in SW480 cells, thereby impacting the p65 cistrome differently in the two cell lines. However, the biological functions of the regulated genes appear to have similar roles in both cell lines. To our knowledge, this is the first time the p65 CRC cistrome is compared between different cell lines and the first time an influence by ERβ on the p65 cistrome is investigated. Our work provides a mechanistic foundation for a better understanding of how estrogen influences inflammatory signaling through NFκB in CRC cells.

Keywords: ChIP; ERβ; colon cancer; colorectal cancer (CRC); estrogen receptor; p65.

Figure 1

Genomic distribution of p65 binding sites in colon cell lines. (A) Identified p65 binding sites in three replicates of colon cancer cell lines HT29 and SW480 with those detected in at least two replicates used for further analysis and highlighted (top), and their overlap between cell lines (bottom), represented using Venn diagram. (B) Heatmap representing p65 binding sites in the two cell lines. (C) Motifs highly enriched in p65 binding sites identified by HOMER using de novo motif analysis and sorted by p-value. (D) Genomic distribution of p65 binding sites in relation to gene locations. (E) Biological functions enriched in genes nearest p65 binding sites (-50kb +2kb).

Figure 2

p65 chromatin binding sites. Peaks of p65 chromatin binding mapped to gene sequences in HT29 (A) and SW480 (B) cells using UCSC genome browser. Bp indicates distance from corresponding gene’s TSS.

Figure 3

p65 transcriptional regulation in colon cell lines. (A) Number of genes with p65 chromatin binding sites and corresponding transcriptional regulation upon TNFα (10ng/ml, 2 h) treatment, per cell line. (B) The top-10 TNFα upregulated and downregulated genes with p65 binding sites in both cell lines (HT29, SW480). (C) Enrichment signal of p65 binding sites present in both cell lines, illustrated using UCSC genome browser.

Figure 4

p65 cistrome in colon vs breast. (A) Heatmap illustrating p65 binding sites in breast (MCF7) and colon cancer cell lines (HT29, SW480). (B) Venn diagram comparing the p65 binding sites in MCF7, HT29, and SW480 cells. (C) DNA motifs located in MCF7-specific p65 binding sequences (D). Pathways enriched among the gene ontology functions assigned to genes located nearest to MCF7-specific p65 binding sites.

Figure 5

ERβ diminishes p65 chromatin binding in HT29 cells. (A) Venn diagram comparing p65 binding sites in HT29 cells with and without expression of ERβ. (B) Density plot representing the distribution of p65 tag densities in three replicates each of HT29 cells with and without ERβ. (C) Volcano plot highlighting statistically significant differences of p65 binding in HT29 cells in presence and absence of ERβ, using sliding window approach. (D) Motifs highly enriched in p65-bound sequencing in HT29 cells only in absence of ERβ, regardless of ERβ expression (core genes), and only in presence of ERβ, respectively. HOMER was used to identify genomic distribution and motifs of p65 binding sites across the genome. (E) Biological functions enriched among genes located nearest to p65 binding sites in HT29 cells depending on ERβ expression. (F) Overlap of genes located nearest to the p65 binding sites and those genes where ERβ expression impacted TNFα gene response in HT29 cells. (G, H) Heatmap representing ERβ modulation of the TNFα-regulated genes, of genes located nearest to p65 binding that was (G) inhibited and (H) enhanced by ERβ in HT29 cells. Z score values were calculated from the logarithmic fold changes, which represent a value’s relationship to the mean of a group of values. A positive Z score indicates the values above the mean and negative if it is below the mean.

Figure 6

ERβ enhances P65 chromatin binding in SW480 cells. (A) Venn diagram of p65 binding sites in SW480 cells with and without expression of ERβ. (B) Density plot representing the distribution of p65 tag densities in three replicates each of SW480cells with and without ERβ. (C) Biological functions enriched among genes located nearest to p65 binding sites in SW480 cells depending on ERβ expression. (D) Motifs highly enriched in p65-bound sequencing in SW480 cells only in absence of ERβ, regardless of ERβ expression (core genes), and only in presence of ERβ, respectively. HOMER was used to identify genomic distribution and motifs of p65 binding sites across the genome. (E) Overlap of genes located nearest to the p65 binding sites and those genes where ERβ expression impacted TNFα gene response in HT29 cells. (F, G) Heatmap representing ERβ modulation of the TNFα-regulated genes, of genes located nearest to p65 binding that (F) required ERβ or (G) was not affected by ERβ. Z score values were calculated from the logarithmic fold changes, which represent a value’s relationship to the mean of a group of values. A positive Z score indicates the values above the mean and negative if it is below the mean.

Figure 7

ERβ influences P65 chromatin binding in colon cancer cells. (A) Heatmap representing p65 chromatin binding over the genome in absence or presence of ERβ, in HT29 and SW480 cells. (B) Venn diagram comparing p65 chromatin binding sites in HT29, SW480 cell lines with and without ERβ. (C) Heatmap illustrating the impact of ERβ on TNFα modulated genes with identified p65 chromatin binding sites in HT29 and SW480 cells, respectively. Z score values were calculated from the logarithmic fold changes, which represent a value’s relationship to the mean of a group of values. A positive Z score indicates the values above the mean and negative if it is below the mean. (D) Examples of p65 chromatin binding sites that was identified only in presence of ERβ in SW480 cell lines, but bound the same sites in HT29 in absence of ERβ and p65 chromatin binding sites enhanced by ERβ in both HT29 and SW480 cells illustrated using the UCSC genome browser.