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. 2014 Jul;88(7):1451-68.
doi: 10.1007/s00204-014-1279-6. Epub 2014 Jun 17.

From transient transcriptome responses to disturbed neurodevelopment: role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects

Nina V Balmer  1 , Stefanie Klima, Eugen Rempel, Violeta N Ivanova, Raivo Kolde, Matthias K Weng, Kesavan Meganathan, Margit Henry, Agapios Sachinidis, Michael R Berthold, Jan G Hengstler, Jörg Rahnenführer, Tanja Waldmann, Marcel Leist
Affiliations

From transient transcriptome responses to disturbed neurodevelopment: role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects

Nina V Balmer et al. Arch Toxicol. 2014 Jul.

Abstract

The superordinate principles governing the transcriptome response of differentiating cells exposed to drugs are still unclear. Often, it is assumed that toxicogenomics data reflect the immediate mode of action (MoA) of drugs. Alternatively, transcriptome changes could describe altered differentiation states as indirect consequence of drug exposure. We used here the developmental toxicants valproate and trichostatin A to address this question. Neurally differentiating human embryonic stem cells were treated for 6 days. Histone acetylation (primary MoA) increased quickly and returned to baseline after 48 h. Histone H3 lysine methylation at the promoter of the neurodevelopmental regulators PAX6 or OTX2 was increasingly altered over time. Methylation changes remained persistent and correlated with neurodevelopmental defects and with effects on PAX6 gene expression, also when the drug was washed out after 3-4 days. We hypothesized that drug exposures altering only acetylation would lead to reversible transcriptome changes (indicating MoA), and challenges that altered methylation would lead to irreversible developmental disturbances. Data from pulse-chase experiments corroborated this assumption. Short drug treatment triggered reversible transcriptome changes; longer exposure disrupted neurodevelopment. The disturbed differentiation was reflected by an altered transcriptome pattern, and the observed changes were similar when the drug was washed out during the last 48 h. We conclude that transcriptome data after prolonged chemical stress of differentiating cells mainly reflect the altered developmental stage of the model system and not the drug MoA. We suggest that brief exposures, followed by immediate analysis, are more suitable for information on immediate drug responses and the toxicity MoA.

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Figures

Fig. 1
Fig. 1
Gene expression and histone methylation patterns of the neuroectodermal marker genes PAX6 and OTX2. a For all experiments, human embryonic stem cells (hESC) were differentiated to neuroepithelial precursor cells (NEP). Marker genes for hESC and NEP at the time points of analysis are indicated in the differentiation scheme. b Samples were taken at the indicated days of differentiation (DoD), and transcript levels of marker genes of neural differentiation were determined by RT-qPCR. Data (gene expression relative to hESC) are mean ± SEM of 3–5 experiments. c The differentiation was performed in the presence of non-cytotoxic concentrations of TSA (10 nM) or VPA (0.6 mM). At the indicated time points, transcript levels were determined by RT-qPCR. Data (expressed relative to control differentiated for the same time in the absence of drug) are mean ± SEM of 3–5 experiments. d Differentiating cells were treated with TSA or VPA for different time periods, and protein acetylation was analyzed by Western blots (WB) with antibodies specific for acetylated histone H3 (H3Ac) or acetylated α-tubulin (tubAc). WB against total histone 3 (H3) or α-tubulin (tub) was performed for normalization. One representative blot for the 6-h time point is displayed. The graphs are based on densitometric analysis of WB from three independent experiments. The levels of acetylated protein are given relative to untreated controls differentiated for the same time period. Data are mean ± SEM of three experiments. e Differentiating cell was treated with TSA (10 nM) for 4 days (day 4), 6 days (day 6), and for 4 days followed by a 2-day washout of the drug (TSA-w, purple bars). Chromatin immunoprecipitation (ChIP) was performed with antibodies specific for H3K4me3 or H3K27me3. Samples were taken on day 4 of differentiation (day 4) or on day 6 of differentiation (day 6; TSA-w). The figure displays the ratio of the enrichment factors for H3K4me3 and H3K27me3 (individual values are found in supplemental files). A ratio >1 points to open chromatin (more H3K4me3) and a ratio <1 suggests a more silenced chromatin (more H3K27me3). Hash symbol indicates a bivalent state. Data are mean ± SEM of three experiments. *p < 0.05; **p < 0.01; ***p < 0.01 (color figure online)
Fig. 2
Fig. 2
Consequences of different drug washout periods for gene expression and histone methylation patterns. For all experiments, hESC were differentiated to NEP. a Samples for chromatin immunoprecipitation (ChIP) were prepared at the indicated days of differentiation. ChIP was performed with antibodies specific for H3K4me3 or H3K27me3 or control IgG. The enrichment factors of OTX2 and PAX6 promoter sequences are given as % input for H3K4me3 (dark blue) and H3K27me3 (light blue). Data are mean ± SEM of three independent cell preparations. b Differentiating cells were treated with TSA (10 nM) for the indicated time periods, and ChIP was performed with the same antibodies as described in a. The ratio of enrichment factors of H3K4me3 and H3K27me3 was calculated as measure of chromatin opening. Data are given relative to values of untreated control cells at the same time point (n = 3). c Scheme of experimental treatment and sampling for the following experiments. Gray bars indicate the period of drug exposure (e.g., P2d: pulsed drug treatment for 2 days) with 10 nM TSA, and white bars indicate medium without TSA. All samples were analyzed on day 6 of differentiation for each treatment scenario. d Protein levels of PAX6 or OTX2 were determined by Western blot, and relative (vs ctr.) protein levels (n = 3) were quantified. e Transcript levels of PAX6 and OTX2 were determined. They are expressed relative to untreated control on DoD6 (ctr). f ChIP was performed for H3K27me3 (purple) or H3K4me3 (black) on promoter regions of PAX6 and OTX2, and enrichment factors were calculated relative to ChIP with control IgG. Then, these data were normalized to the values obtained for control cells (ctr). For instance, on day 6 of the differentiation, H3K27me3 was 15-fold higher in cells treated for 1 day with TSA and then left in control medium (P1d), compared to cells that were differentiated under control conditions. Data of d–f are mean ± SEM of 3–5 experiments (color figure online)
Fig. 3
Fig. 3
Transcriptome analysis after different treatments with TSA and VPA. a Overview of the different exposure scenarios during differentiation. Bars indicate the duration of differentiation before samples were taken, and gray shading indicates the period of drug exposure within that time. b Differentiating cells were treated as indicated in a; mRNA was prepared and analyzed by Affymetric DNA microarrays. The numbers of probe sets (PS) regulated upwards (red) or downwards (blue) in the presence of TSA (10 nM) or VPA (600 µM) are given in the table. c Venn diagrams display absolute numbers of regulated PS (p < 0.05, FC > 1.5 or FC < 0.67) induced by TSA, VPA, or both during the indicated treatment periods. The percentage of overlap of the drug treatments is indicated in purple. d Venn diagrams display PS altered by TSA treatment (p < 0.05, FC > 1.5 or FC < 0.67) for different time periods. The numbers in the sectors are given as percentages of the number at T4d (absolute numbers are in supplements). e Overrepresented gene ontology terms (oGOs) were determined from the regulated PS indicated in D. Individual oGOs were classified by their assigned to superordinate cell biological processes, and the number of oGO for each of these (e.g., migration or neuronal pathways) is displayed. Complete data sets on regulated PS, oGO, and superordinate biological processes are given in Tables S2 and S3 (color figure online)
Fig. 4
Fig. 4
Concordance of transcript changes after continued treatment or drug washout on DoD4. a Cells were differentiated in the presence of TSA for 6 days (T6d) or for 4 days followed by a drug washout period of 2 days (medium pulse, MP) and analyzed by Affymetrix DNA microarrays. The absolute numbers of up- and down-regulated PS are indicated in Venn diagrams. The percentage of overlap is indicated in purple. b The oGOs were determined from the up-regulated PS of T6D treatment as well as from the overlap of T6D with MP (T6d ∩ MP). The oGOs were further classified into superordinate cell biological processes, and the numbers in these categories are displayed. For full information on regulated PS, oGO, and superordinate biological processes, see Tables S2 and S4. c Significantly down-regulated PS were determined for T6d ∩ MP. GOs overrepresented among the PS down-regulated are displayed as word cloud. The character size is scaled according to the p value of the corresponding GO (color figure online)
Fig. 5
Fig. 5
Comparison of the acute and chronic (long-term treatment) effects of TSA. a Cells (hESC) were differentiated for 6 days to NEP, and TSA (10 nM) was added only during the last 6 h or 24 h. Then, protein acetylation was determined by Western blot as in Fig. 1. Data are mean ± SEM of three experiments. *p < 0.05. b Venn diagrams display the number of PS altered by drug treatment (p < 0.05, FC > 1.5 or FC < 0.67) after continuous (T6d, black circle) exposure, or after late, pulsed (LP) treatment (last 6 h of the 6-day differentiation, red circles). c Venn diagrams compare PS triggered by a late drug pulse (LP, red circles) with PS regulated by continuous drug exposure, but not found under MP washout conditions (T6d without—MP, black circles). The percentage of overlap is indicated in purple. d The GOs overrepresented among up-regulated PS of LP incubations were classified into superordinate cell biological processes (full information on regulated PS, oGO, and superordinate biological processes: Tables S2 and S4) (color figure online)
Fig. 6
Fig. 6
Differences of acute effects of TSA either early or late during NEP differentiation. a Overrepresented GOs were determined from significantly up- (upper panel) and down-regulated (lower panel) PS from T6h samples. They were displayed as word clouds with character size scaling according to the p value of the corresponding GO. For full information, see Table S2 and S3. b Venn diagrams compare numbers of up-regulated PS of long-term (T6d, black circle) and early short-term (T6h, blue circle) treatment with TSA. c. GOs overrepresented among the PS of the overlap shown in b are displayed as a word cloud. d Venn diagrams compare numbers of regulated PS of short-term TSA treatments at early (T6h, blue circle) and late (LP, red circle) time points. The percentage of overlap is displayed in purple (e). The oGOs determined from significantly up-regulated (blue) or down-regulated (red) PS of the LP and T6h incubations were assigned to superordinate cell biological processes. These were then used as axes on a radar plot, with the distance from the center indicating the number of oGO on each axis. For full information on regulated PS, oGO, and superordinate biological processes, see Tables S2 and S3 (color figure online)
Fig. 7
Fig. 7
Summary. a Neuroepithelial precursors (NEP) were generated from hESC within 6 days of differentiation (DoD). Main cell type markers are indicated. b Continuous drug treatment led to an altered NEP differentiation (NEP*) as indicated by transcriptome changes (blue), reduced marker expression (PAX6, OTX2), and permanent changes in histone methylation (after 2–3 days) at the promoters of the markers. Acetylation changes were only transient. c Drug exposure for 4 days resulted in the same disturbed NEP differentiation as continued drug exposure. The number of altered PS and the alterations of histone methylation were not affected by drug washout. d Short-term drug treatment induced a strong, but fully reversible change in gene expression. Methylation of marker promoters was not altered, and marker expression was normal. The transition of transient drug-induced gene expression and histone acetylation changes to a permanently altered transcriptome and NEP differentiation correlated with the permanence of promoter histone methylations (color figure online)
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