Tranylcypromine inhibits histone H3K4 demethylation in S2 cells
Drosophila S2 cells were treated with Tranylcypromine and the amount of di-and trimethylation of H3K4 (H3K4me2/3) related to histone H3 quantified by immunoblotting. Since Tranylcypromine is soluble in both, either water or DMSO, we considered an increased bio-availability of Tranylcypromine when dissolved in DMSO. S2 cells were cultured either in standard medium as control or in the presence of 2µM Tranylcypromine dissolved either in water or DMSO. Total proteins were immunoblotted, and histone H3 and histone H3K4me2/3 quantified by fluorescent imaging (Fig. 1A). The fold changes of the relative quantities of H3K4me2/3, i.e. the ratios of H3K4me2/3 to H3, related to the mean ratio of the controls on the same blot are given in Fig. 1B. Our results revealed that Tranylcypromine treatment significantly raised the relative amount of H3K4me2/3 (Fig. 1B). [Student’s T-test, one-sided, homoscedastic, related to control: Tranylcypromine in water p*, Tranylcypromine in DMSO p**. Three biological replicas each for control and Tranylcypromine treatments. Individual measurements for control n=9, Tranylcypromine in water n=7, Tranylcypromine in DMSO n=8. Data are normally distributed.] The data, furthermore, indicated that DMSO intensifies the effect of Tranylcypromine treatment on H3K4 methylation.
Enhanced eye pigmentation in generation F1 by feeding with Tranylcypromine
We asked whether Tranylcypromine, dissolved in water, affects gene expression when used as food supplement for Drosophila. To this end, eye colour expression in male flies of consecutive generations was used as read-out. As controls, sibling flies of the parental generation were raised under the same conditions but without Tranylcypromine, and eye colour expression investigated in the same generations, under the same conditions, and in parallel to the treated animals. Increasing concentrations of Tranylcypromine (from 1.25 mg up to 4 mg per culture vessel) were fed to the parental fly generation (P/F0) and their F1 progenies, and eye colour analysed by optical measurement of pigment extracts. We found a significant increase in eye pigmentation in generation F1 (Student’s T-test: p**) (Fig. 2A). To investigate white gene expression, flies were fed with 1.25 mg Tranylcypromine in water, and RNA of male heads extracted. In generation F3, a significant decrease in relative white gene expression compared to the control was found (Student’s T-test: p**) (Fig. 2B). All data are normally distributed. The discrepancy found in generation F1 between eye colour (Fig. 2A) and expression of white (Fig. 2B) is most likely caused by the large variability in eye colour and the restricted number of probes available for qRT-PCR. To summarize, Tranylcypromine feeding increased eye colour expression in treated flies of generation F1 but significantly decreased white gene expression in generation F3. An increase of Tranylcypromine administration beyond that is not feasible due to harmful side-effects. We observed that larval and pupal development was compromised resulting in a severe decrease in hatching rate. When using 2.5 mg Tranylcypromine as additive we observed a reduction of hatched flies to 67% to those of the control (four-fold experiment).
Transgenerational effect of Tranylcypromine and DMSO feeding on eye colour expression
In the following feeding experiments, we used 2.5 mg Tranylcypromine per culture vessel but used DMSO as solvent to increase its bio-availability. Flies were fed once in the parental (P/F0) generation and their F1 progenies, and eye colour expression measured in males of the parental and consecutive generations. We found a significant shift of eye colour expression related to the control throughout generations. The strongest effects were observed in the F1 generation, which showed an approximate fourfold increase in eye colour (p ***; Student’s T-test, one-sided, homoscedastic) related to the control, and a strong decrease in F2 (p***) and F3 (p***). In the succeeding generations F4, F5 and F6 no significant differences to the control were found (Fig. 3A).
By feeding of DMSO solely, we observed similar changes in eye colour expression as in the presence of Tranylcypromine (Fig. 3B). According to Student’s T-test (one-sided, homoscedastic) we found significant differences to the eye colour of control flies for F1 (p***), F2 (p***), F3 (p***), F4 (p**), and F6 ***, but not for F0/P, F5, F7 and F8. The data indicated that DMSO itself intensified eye colour expression in F1 followed by its suppression in F2 and F3, and a slow-increase towards the original level in consecutive generations. Additionally, we observed a harmful effect of DMSO itself on viability causing strong reduction of hatching in F1.
Side-by-side presentation of all data revealed subtle differences (Fig. 4). In the presence of Tranylcypromine, eye colour expression in F1 generation is more intense, resulting in an approximate 4-fold increase compared to that of control flies, whereas DMSO itself resulted in a less than 2-fold increase. However, Student’s T-test revealed no significant difference between DMSO and Tranylcypromine in DMSO fed flies of generation F1 (p=0.0624), most likely due to the large variation and the small number of male flies that could be investigated because of the reduced hatching rate. Furthermore, no significant differences in eye colours between DMSO fed flies and flies fed with Tranylcypromine dissolved in DMSO (all fed in F0/P//F1 generations) were found in the consecutive generations F2 and F3, but they are significantly different in F4 (p*) and F6 (p**). Relating to the control, DMSO fed flies have significantly reduced eye colours in F4 (**) and F6 (***) (Fig. 3B) whereas no significant differences were found in F4 to F6 flies fed with Tranylcypromine in DMS0 (see Fig. 3A). Return of eye pigment expression to that of the control level in Tranylcypromine fed flies of generation F4 and F6, compared to the reduced eye pigment expression in DMSO fed flies, indicates that previous feeding with Tranylcypromine still has a supporting effect on eye pigment expression. Besides, flies fed with Tranylcypromine dissolved in DMSO showed enlarged eye colour variation throughout generations compared to flies fed with DMSO alone (Fig. 4). Thus, presumably the supporting effect of Tranylcypromine on eye pigment expression is counteracted by a repressive effect of DMSO. The strong increase of eye pigmentation in DMSO-treated F1 flies is probably caused by its function as a polar aprotic solvent able to easily penetrate biological membranes and promoting cellular import of other substances, e.g. of eye pigment. To conclude, Tranylcypromine and its solvent DMSO significantly affected eye colour expression at least up to generation F3, which is the first generation that has never been in contact with the additive.
Eye pigment expression corresponds to white gene expression
Optical measurement of eye pigment extracts revealed significant changes in eye colour expression. As expected, gene activation most likely is caused by the rise of H3K4 methylation provoked by Tranylcypromine-mediated LSD1-inhibition. In fact, H3K4 di-and trimethylation increased approximately 6-fold in F1 embryos when flies were fed with Tranylcypromine dissolved in DMSO in the parental generation (Student’s T-test, p**) but not when DMSO solely was fed (Fig. 5). In F2 embryos the relative amount of H3K4me2/3 is slightly but non-significantly reduced compared to that of control F2 embryos but significantly reduced compared to F1 embryos (Student’s T-test, p**). The increase in H3K4 methylation indicates activation of the white gene, which we confirmed by quantitative RT-PCR. To this end, the relative expression of the white gene to actin as housekeeping gene was determined and calculated as fold change to the relative expression in the controls (Fig. 6). Throughout generations, eye pigment expression obtained by optical measurements, and relative white gene expression are nearly congruent (see Fig. 4 and Fig. 6). White gene expression increased in F1, with a stronger effect when flies were fed with Tranylcypromine dissolved in DMSO than DMSO alone, decreased in F2 and slowly increased again in consecutive generations. In F4 and F5 we observed a stronger relative white gene expression in Tranylcypromine fed flies (fed in F0/P//F1) than in DMSO fed flies – once again reflecting eye pigmentation. All data are normally distributed. Student’s T-test revealed significant differences between the control and treated flies in the following generations: DMSO F1, T in DMSO F1, DMSO F2, T in DMSO F2, T in DMSO F4, DMSO F5 all p***, T in DMSO F3 p**, and between DMSO F5 and T in DMSO F5 p***. However, there is a sole discrepancy between eye colour and white gene expression in generation F3 owing most likely to their large variation and the limited number of probes available for qRT-PCR. qRT-PCR was performed on individual biological replicas of one single experiment, using seven to eight technical replicas each, whereas the eye colour measurements (Fig. 3, 4) resulted from 4 individual experiments with up to 12 biological replicas. Additionally, biological replicas #5 and #6, which have reduced eye colour expression, could not be investigated by qRT-PCR due to probe limitations (Supplementary Fig. 1). Comparison of eye colour and white gene expression of individual biological replicas in F3 revealed similar tendencies (Supplementary Fig. 1) indicating an overall reduced white gene expression when all replicas (#1 to #6) could be included. An overall reduction of eye pigment expression in F3 is in all probability, by taking into consideration the large number of biological and technical replicas used for eye pigment measurement compared to the limited number of probes available for qRT-PCR, and additionally the reduced white gene expression in F3 of flies fed previously with Tranylcypromine in water.