BACKGROUND: Arsenic (As) exposure is a significant worldwide environmental health concern. Low dose, chronic arsenic exposure has been associated with a higher than normal risk of skin, lung, and bladder cancer, as well as cardiovascular disease and diabetes. While arsenic-induced biological changes play a role in disease pathology, little is known about the dynamic cellular changes resulting from arsenic exposure and withdrawal.

RESULTS: In these studies, we sought to understand the molecular mechanisms behind the biological changes induced by arsenic exposure. A comprehensive global approach was employed to determine genome-wide changes to chromatin structure, transcriptome patterns and splicing patterns in response to chronic low dose arsenic and its subsequent withdrawal. Our results show that cells exposed to chronic low doses of sodium arsenite have distinct temporal and coordinated chromatin, gene expression, and miRNA changes consistent with differentiation and activation of multiple biochemical pathways. Most of these temporal patterns in gene expression are reversed when arsenic is withdrawn. However, some gene expression patterns remained altered, plausibly as a result of an adaptive response by cells. Additionally, the correlation of changes to gene expression and chromatin structure solidify the role of chromatin structure in gene regulatory changes due to arsenite exposure. Lastly, we show that arsenite exposure influences gene regulation both at the initiation of transcription as well as at the level of splicing.

CONCLUSIONS: Our results show that adaptation of cells to iAs-mediated EMT is coupled to changes in chromatin structure effecting differential transcriptional and splicing patterns of genes. These studies provide new insights into the mechanism of iAs-mediated pathology, which includes epigenetic chromatin changes coupled with changes to the transcriptome and splicing patterns of key genes.

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Published by BMC Genomics, v. 16, article 212, p. 1-19.

© 2015 Riedmann et al.; licensee BioMed Central.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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s12864-015-1295-9-s1.pdf (227 kB)
Figure S1.: iAs modulates the expression of key EMT genes in a dose-dependent manner (Figures 1D and 7A). Protein levels from 3 gels were normalized to the expression of β-actin in both BEAS-2B and HeLa cells. Data are mean S.E.M. of 3 independent experiments; P < 0.01.

s12864-015-1295-9-s2.pdf (1038 kB)
Figure S2.: Low dose of sodium arsenite does not induce DNA fragmentation in A) BEAS-2B cells and B) HeLa cells. DNA from control non-exposed cells and arsenic exposed cells were purified (see Figure 2) and ran on a 3.3% Nusieve agarose gel electrophoresis. Number of days in culture is shown in figure. NT: non-treated or control cells. T: iAs treated cells. High concentration of iAs (100 μM) shows the typical DNA fragmentation pattern (as indicated by arrows).

s12864-015-1295-9-s3.pdf (1110 kB)
Figure S3.: Arsenite treatment and transformation resulted in cells with more compact chromatin. Equal amount of nuclei was used and digest of chromatin from NT, iAs-T with 0.5 μM and 1μM iAs respectively cells showed more resistance to micrococcal nuclease (MNase). Interestingly removal of iAs from 1μM iAs-T, showed an increase in chromatin accessibility to MNase. Chromatin accessibility is dose-dependent as more resistance to MNase is seen in chromatin from cells transformed with 0.5 μM iAs compared to 1 μM iAs. Additionally, increase in accessibility is observed in cells from which iAs is removed (iAs-rev).

s12864-015-1295-9-s4.pdf (527 kB)
Table S1.: Genes upregulated and downregulated by iAs exposure.

s12864-015-1295-9-s5.pdf (518 kB)
Table S2.: Heatmap showing the differential gene expression patterns of different solute carrier proteins and zinc-finger binding proteins modulated in the different experimental conditions.

s12864-015-1295-9-s6.pdf (979 kB)
Table S3.: Transcription factors whose binding sites were detected at the promoters of iAs-target genes.

s12864-015-1295-9-s7.pdf (999 kB)
Table S4.: Pathways involved by the genes targeted by iAs. Detailed analyses of the iAs-targeted genes and their association with cancer (analyzed using GSEA).

s12864-015-1295-9-s8.pdf (978 kB)
Table S5.: Genes altered by iAs and their association with various cancer modules.

s12864-015-1295-9-s9.pdf (133 kB)
Figure S4.: Genes common in both iAs-T and iAs-Rev cells. These analyses show genes that did not revert to NT conditions. Also shown are the functions of some of these genes.

s12864-015-1295-9-s10.pdf (57 kB)
Figure S5.: Heatmap of the expression pattern of genes common in iAs-T, iAs-rev and iAs-rev-reTreat cells. Red: indicates upregulation and green indicates down regulation. From dark green to light green = low fold change downregulation to more down regulation. Dark red to light red = degree of upregulation: small to high upregulation.

s12864-015-1295-9-s11.pdf (5120 kB)
Figure S6.: Protein interaction map of altered iAs-target genes. The bioinformatics STRING database (version 9.1) was used to generate a protein interaction map with known and predicted protein associations that include direct physical and indirect functional protein linkages of microarray identified iAs-target genes at protein level from Additional file 10: Figure S5). Shown also are the interactors based on evidence (left side) and confidence level (right side). i) Evidence view: different line colors represent the types of evidence for the association. Green: neighborhood; red: gene fusion; blue: co-occurrence; black/grey: co-expression; pink: experiments; teal: databases; Pea green: textmining; purple: homology ii) Confidence view: Thicker lines represent stronger associations.

s12864-015-1295-9-s12.pdf (71 kB)
Table S6.: Genes with iAs-mediated alternatively spliced events.

s12864-015-1295-9-s13.pdf (490 kB)
Table S7.: MicroRNAs altered during arsenite exposure.