Nickel exposure induces persistent gene expression changes
BEAS-2B cells were exposed to non-cytotoxic and physiologically relevant concentration of 100 μM NiCl2 for 6 weeks (nickel-exposed) [4, 19]. After exposure, the cells were washed and plated in nickel-free medium at colony forming density and allowed to grow as colonies originating from individual nickel-exposed cells. The colonies were then isolated and expanded as populations of nickel-washed-out cells for > 6 months (Fig. 1a). Our earlier studies had shown that all nickel-exposed cells displayed similar transcriptional profiles [4]. Therefore, we randomly selected a population of nickel-washed-out cells for a detailed examination. Gene expression analysis of the untreated cells (UT), nickel-exposed cells (Ni-E) and nickel-washed-out cells (Ni-W) cells was performed using RNA-Seq.
Examination of the differentially expressed genes revealed six gene groups based on their expression profiles (Fig. 1b): (i) transiently upregulated (TU) genes—nickel exposure upregulated 211 genes (in Ni-E cells), whose expression reverted to basal levels after the termination of exposure (in Ni-W cells); (ii) transiently downregulated (TD) genes—nickel exposure downregulated 114 genes (in Ni-E cells), whose expression reverted to normal levels after the termination of exposure (in Ni-W cells). In addition to the transiently differentially expressed genes, we found a number of genes (1597 genes) that remained differentially expressed even after the cells were in culture for > 6 months after the termination of nickel exposure. We termed these genes as persistently upregulated (PU) or persistently downregulated (PD). Interestingly, we identified two categories of PU genes: (iii) persistently upregulated-A (PU-A) genes—a subset of the PU genes (115 genes), which were upregulated during nickel exposure (in Ni-E cells) and the increased expression continued after the termination of exposure (in Ni-W cells). (iv) persistently upregulated-B (PU-B) genes—a subset of PU genes (963 genes), whose expression remained unaltered during nickel exposure (in Ni-E cells). However, the genes were upregulated after the termination of exposure (Ni-W cells) and remained upregulated persistently. Similarly, the persistently downregulated genes could be classified into two categories: (v) persistently downregulated-A (PD-A) genes—this set of genes were downregulated during nickel exposure (26 genes, in Ni-E cells) and continued to remain downregulated after the termination of exposure (in Ni-W cells); and (vi) persistently downregulated-B (PD-B) genes—expression levels of these genes were not altered during nickel exposure (in Ni-E cells). However, the genes were downregulated only after the termination of exposure (493 genes, in Ni-W cells).
Nickel induces post-exposure genome-wide H3K4me3 changes
Next, we aimed at understanding the mechanisms underlying nickel-induced persistent gene expression alterations. Nickel is a non-mutagen and previous studies have shown that nickel exposure causes extensive changes to the epigenome [4, 16, 20]. Heritability of epigenetic changes through mitosis is well characterized. Therefore, it is conceivable that changes to the chromatin features could be an underlying cause for the nickel-induced gene expression changes that persist through cell division after the termination of exposure. To examine the relationship between nickel-induced gene expression changes and alterations to chromatin features, we profiled the genome-wide distribution of H3K4me3, a histone modification associated with transcriptional activation, in UT, Ni-E and Ni-W cells, using ChIP-Seq. We measured the levels of H3K4me3 at the gene promoters (+ 2 kb to − 2 kb from transcription start site [TSS]) and quantified the changes in the H3K4me3 levels among the different categories of genes that we have identified (Fig. 1b) (see “Methods” for details).
Transiently upregulated (TU) genes
Nickel exposure transiently upregulated 211 genes. Examination of H3K4me3 at TU gene promoters showed no significant difference between Ni-E and UT cells (Fig. 2a–e). However, Ni-W cells showed significantly reduced H3K4me3 levels as compared to Ni-E and UT cells (Fig. 2a–e). This suggests that the TU gene upregulation in the presence of nickel is not accompanied by increase in H3K4me3 levels. However, the decrease in the expression of these genes after the termination of exposure is associated with loss of H3K4me3.
Since the increase in the expression of TU genes was not associated with increase in H3K4me3 levels, we reasoned that that the genes that are upregulated in the presence of nickel could have permissive chromatin structure prior to nickel exposure. To examine this, we quantified the absolute H3K4me3 levels of all the gene promoters in the genome (all-genes) (+ 2 kb to − 2 kb from TSS). We then compared the promoter H3K4me3 levels of TU genes and all-genes. Our analysis showed higher H3K4me3 levels at the promoters of TU genes compared to all-genes (Fig. 2f). Since H3K4me3 marks ‘open’ or accessible chromatin regions, our results suggest that the TU gene promoters exist in an accessible chromatin environment prior to nickel exposure.
Persistently upregulated (PU) genes
Nickel exposure persistently upregulated 1078 genes. Interestingly, while only 115 genes (10.7% of PU genes) were upregulated during nickel exposure (PU-A), 963 genes (89.3% of PU genes) were upregulated after the termination of exposure (PU-B). Examination of H3K4me3 at PU-A gene promoters did not reveal significant changes in H3K4me3 levels in Ni-E or Ni-W cells compared to UT cells (Fig. 3a–e). In addition, we did not find significant variations in H3K4me3 levels between Ni-E and Ni-W cells (Fig. 3a, b, d, e). This suggests that PU-A gene upregulation is not associated with increase in H3K4me3 levels. Interestingly, quantification of the H3K4me3 levels in untreated cells revealed higher enrichment of H3K4me3 at the promoters of PU-A genes compared to all-genes (Fig. 3f). This suggests that PU-A gene promoters exist in an ‘open’ chromatin configuration prior to nickel exposure. PU-B gene promoters, on the other hand, showed significant increase in H3K4me3 levels in Ni-W cells compared to both UT and Ni-E cells (Fig. 3g–k). As expected, we did not detect H3K4me3 increase at PU-B gene promoters in Ni-E cells compared to UT cells (Fig. 3g, h, i, k), since this set of genes were upregulated only after the termination of nickel exposure (in Ni-W cells).
Collectively, our results show that upregulation of gene expression in the presence of nickel (TU-A and PU-A) does not involve changes in H3K4me3 profiles. These genes possessed high levels of H3K4me3 levels prior to exposure (in UT cells), suggesting that they exist in an open chromatin environment in untreated cells. In contrast, the genes that were upregulated only after the termination of exposure (in Ni-W cells) were associated with significant increase in the levels of H3K4me3.
Transiently downregulated (TD) genes
Nickel exposure transiently downregulated 114 genes. Although we detected modest H3K4me3 loss in Ni-E and Ni-W cells compared to UT cells (Fig. 4a–d), these changes were not statistically significant (Fig. 4e), suggesting that both downregulation of gene expression in Ni-E cells and the subsequent reversal of downregulation in Ni-W cells were not associated with H3K4me3 changes.
Persistently downregulated (PD) genes
Nickel exposure persistently downregulated (PD) 519 genes. Interestingly, while merely 26 genes (5%) were downregulated during nickel exposure (PD-A), 493 genes (95%) were downregulated only after the termination of exposure (PD-B). Examination of PD-A gene promoters did not reveal changes in H3K4me3 levels in Ni-E and Ni-W cells as compared to UT cells (Fig. 5a–e). In contrast, we detected a robust loss of H3K4me3 at the PD-B gene promoters in Ni-W cells compared to both UT and Ni-E cells (Fig. 5f–j). These results suggest that while downregulation of gene expression that happens in the presence of nickel does not involve changes in H3K4me3, downregulation that occurs after the termination of exposure is associated with significant H3K4me3 loss. As anticipated, we did not detect decrease in H3K4me3 levels at PD-B gene promoters in Ni-E cells compared to UT cells (Fig. 5f, g, h, j), since these genes were downregulated only in Ni-W cells.
Nickel-induced persistent gene upregulation is associated with loss of H3K27me3
Our results show that gene expression changes that occur in the presence of nickel were not associated with H3K4me3 alterations. Therefore, we next asked whether repressive histone modifications contribute to persistent transcriptional changes during nickel exposure. To accomplish this, using ChIP-qPCR, we examined the levels of repressive histone modification, H3K27me3, at the promoters of PU-A, PU-B, PD-A and PD-B genes. Our analyses detected significant decrease in the levels of H3K27me3 at PU-A (Fig. 6a) and PU-B (Fig. 6b) gene promoters in Ni-W cells compared to UT cells. This suggested that H3K27me3 loss could underlie gene upregulation both in the presence of nickel and after the termination of exposure. Next, to understand whether the persistently downregulated genes gained repressive epigenetic marks, we examined H3K27me3 levels at the promoters of several PD-A and PD-B gene promoters. Our analysis did not reveal significant increase in the levels of H3K27me3 at the PD-A or PD-B gene promoters (Fig. 6c, d). These results suggest that persistent gene downregulation is not associated with the gain of repressive H3K27me3.
In summary, our results show that the up- and down-regulation of gene expression that occur during nickel exposure do not involve changes in the levels of the activating histone modification, H3K4me3. However, the gene expression changes that occur after the termination of exposure are associated with significant changes in H3K4me3. In contrast, the gene upregulation that occurred both in the presence of nickel and after the termination of exposure was associated with loss of repressive H3K27me3.
Post-exposure transcriptional changes occur immediately after the termination of nickel exposure
In this study, the Ni-W cells that were used to identify post-exposure persistent transcriptional changes were in culture for > 6 months after the termination of 6-week nickel exposure (Fig. 1a). Hence, the post-nickel-exposure transcriptional changes that we have identified could have either occurred immediately after termination of nickel exposure or it could be the cumulative outcome of transcriptional changes in the cells over the course of > 6 months. Therefore, to understand when the post-exposure transcriptional changes happened, we next compared the gene expression profiles of Ni-W cells (6-week nickel exposure followed by > 6 months in culture without nickel) with that of Ni-W-2W cells (6-week nickel exposure followed by 2 weeks in culture without nickel). As shown in Fig. 7, Ni-W and Ni-W-2W cells exhibit similar gene expression profiles, which are clearly different from that of the gene expression profile of Ni-E cells. These results suggest that the post-exposure persistent transcriptional changes that we have identified in Ni-W cells is established soon after the termination of nickel exposure and remained stable for > 6 months.
RT4 cells undergo transcriptional alterations after the termination of nickel exposure
Our results show that nickel could induce significant post-exposure effects in the lung epithelial BEAS-2B cells. We next asked whether non-lung cells undergo similar post-nickel-exposure transcriptional changes. To investigate this, we exposed human non-invasive bladder cancer RT4 cells to 100 μM NiCl2 for 6 weeks. After exposure, the cells were washed and plated in nickel-free medium for 2 weeks. Following this, gene expression analysis was carried out using RNA-Seq. As shown in Fig. 8a, we identified the same six differentially expressed gene groups that we identified in BEAS-2B cells (Fig. 1b). This suggests that nickel exposure induces gene expression changes after the termination of exposure (PU-B and PD-B) in non-lung RT4 cells as well. However, relatively fewer genes were differentially expressed after the termination of nickel exposure in RT4 cells as compared to BEAS-2B cells.
We next asked whether the genes that are differentially expressed after the termination of nickel exposure are conserved between BEAS-2B and RT4 cells. Surprisingly, our analyses showed that the PU-B and PD-B genes identified in BEAS-2B cells displayed similar patterns of differential expression in RT4 cells (Fig. 8b, c). This suggests that common upstream regulator(s) may regulate the genes that are differentially expressed after the termination of nickel exposure in different cell types.