Possible upstream regulators of DNA demethylating enzymes in the central nervous system

da | Giu 28, 2020 | Biologia Molecolare

Abstract

How are DNA methylation and the environment connected in the central nervous system? To explore this question, Grassi end collaborators [1] considered whether neuronal activity with BDNF and TGFβ controls transcription of DNA modifying enzymes specifically members of the DNA-damage-inducible (Gadd) family. Finally, they studied whether Gadd45 family members were associated with psychiatric disorder-related genes. The team applied MeDIP analysis to study DNA methylation, and they used unpredictable chronic mild stress (UCMS) as established model for depression to mice.

DNA methylation controls transcription of DNA, and in the central nervous system (CNS) it is regulated and associated with experience and learning as well as memory formation [2]. Alteration of DNA methylation has been observed in different pathological conditions [3] in which neuronal activity is reduced [4]. DNA demethylases are implicated in learning and memory. Grassi et al. [1] studied DNA demethylating enzymes, specifically the growth arrest and DNA-damage-inducible (Gadd) family. Gadd45 family members, Gadd45α, Gadd45β and Gadd45ϒ encode three GADD45 proteins: GADD45α, GADD45β and GADD45ϒ. GADD45 proteins participate in DNA repair mechanism and in demethylation processes [5]. However, the upstream regulators of DNA demethylating enzymes are unknown. The authors of this research proposed BDNF and TGFβ-signaling pathways as candidates for a link between environment and chromatin. Indeed, BDNF and TGFβ are important in the physiology of the CNS. For this reason, the researchers verified whether BDNF and TGFβ control transcription of Gadd45 family members. The authors used hippocampal neurons that derived from E18.5 mouse brains and cultured for 11 days in vitro (DIV). On DIV11, they treated the cultures with 55 mM KCl at different time points from 30 min to 6 h before harvesting. Indeed, KCl led to depolarization and neuronal activity. qRT-PCR analysis showed that the transcription of Tgfβ2 and Tgfβ3 increased, following a depolarization with a 4–6 h delay and hippocampal neurons secreted more TGFβ. KCl treatment also induced Bdnf transcription. This delayed transcriptional response suggested alterations at the chromatin level. To study transcriptional changes after depolarization, Grassi et al. employed a microarray screen comparing cDNA from control cells and neurons harvested 4 h after KCl treatment. They identified that DNA demethylases Gadd45β and Gadd45ϒ transcription increased. These results indicate that the transcriptional profile might be epigenetically adapted in response to neuronal activity. At this point, the authors verified whether Gadd45 family members were regulated by BDNF- and TGFβ-pathways in response to neuronal stimulation. Therefore, they blocked BDNF-signalling with a neurotrophic tyrosine kinase receptor type 2 (TRKB)-inhibiting antibody (TrkB-Fc), TGFBR1-mediated signaling with the activin A receptor, type II-like 1 (ALK1)/TGFBR1 inhibitor SB431542. Gadd45β expression was decreased by TRKB inhibition, whereas increased by TGFBR1 inhibition. Gadd45ϒ expression was activated through KCl-, TRKB- and TGFBR1-signaling because the inhibition of both pathways at the same time suppressed them. The expression of the Gadd45 family members can be regulated by either BDNF- and/or TGFBR1-signaling pathways. MeDIP analysis did not detect differences in the global pattern of DNA methylation upon KCl treatment and non-treated. This result indicated that loss of methylation did not affect large regions but single CpGs. Gene ontology analysis detected 218 genes that showed both differentially methylation and expression. These genes were associated with transcriptional control, signal transduction and nervous system development and function. Grassi et al. identified 15 genes with increased expression after KCl treatment, 6 of which were associated with psychiatric diseases. These 6 genes were Tshz1, Foxn3, Jarid2, Per1, Arc and Map3k5. The researchers confirmed that 4h after depolarization expression of these genes increased, according to reduced DNA methylation. To verify whether the expression of Arc, Map3k5 and Per1 was related to BDNF- and TGFβ-signalling, the authors used the same inhibitors previously described. Arc and Per1 did not depend on TGFBR1- and BDN-signaling, while Map3k5 did. However, using knockdown of TGFBR2 and SMAD4, they observed that Arc and Map3k5 depended on TGFBR2- and SMAD4-signaling whereas Per1 depended only on TGFBR2.

Finally, the team studied if the altered expression of Gadd45 family members was connected with psychiatric disease-associated genes in vivo in a mouse model system of depression (UCMS). Expression of Gadd45α, Gadd45β and Gadd45ϒ was reduced in the hippocampus and only Arc was significantly less expressed in UCMS compared to control mice. Also, expression of TGFB family members was reduced. In contrast to the in vitro results, by making a knockout of Tgfbr2, the main receptor of TGFβ1, 2 and 3 ligands, the expression of Gadd45 family members decreased overall. Moreover, knockout of Gadd45β reduced Arc’s expression. Therefore, TGFβ-signalling involving GADD45β is most likely important for the transcriptional control of this gene. In conclusion, these data suggest that TGFβ-signalling controls Gadd45 family members (Fig. 1), especially Gadd45β which is involved in the expression of psychiatric disease-associated genes.

Figure 1. TGFβ-signaling controls Gadd45 family members.

According to these results, Gadd45 genes probably depend on multiple stimuli derived by BDNF- and TGFβ-pathways.

Furthermore, altered neural activity, which is a marker of the neurons in people with psychiatric diseases, is related to the reduced expression of these genes. GADD45 proteins functions are similar, but not identical, and their induction differs under diverse physiological conditions or in different cell types, so this is probably the reason because they responded differently to the different signalling. For Gadd45α, the researchers supposed that Activin and/or BMP-ligands might activate SMAD4. However, why they did not study these other ligands too? And given that TGFβ-pathway includes not only TGFβ ligands, it is possible that the other members of the Gadd45 family may also be controlled by different TGFβ ligands, such as BMP or Activin. Therefore, this hypothesis should be evaluated. Moreover, we do not understand why the tests on BDNF were left out in vivo. This work is interesting because it reveals important epigenetic components, but it contains conflicting data. For example, in vivo TGFβ-pathways activated Gadd45β, whereas in vitro it inhibited Gadd45β. Clearly, the in vivo context is much more complex. In the future, new tests should be carried out to better evaluate these pathways from a molecular point of view, in order to develop possible treatments for people suffering from neurological diseases. This research provides important results for future analyses.

References

  1. Grassi et al. 2017, Neuronal Activity, TGFβ-Signaling and Unpredictable Chronic Stress Modulate Transcription of Gadd45 Family Members and DNA Methylation in the Hippocampus. Cerebral Cortex. 27:4166–4181 doi: 10.1093/cercor/bhx095.2
  2. Miller CA., Sweatt JD. 2007. Covalent modification of DNA regulates memory formation. Neuron. 53:857–869 doi: 10.1016/j.neuron.2007.02.022.
  3. Córdova-Palomera A. et al. 2015, Genome-wide methylation study on depression: differential methylation and variable methylation in monozygotic twins. Translational psychiatry. 5(4): e557 doi: 10.1038/tp.2015.49.
  4. Chen X. et al. 2015. Ablation of type III adenylyl cyclase in mice causes reduced neuronal activity, altered sleep pattern, and depression-like phenotypes. Biological Psychiatry. 80(11): 836–848 doi: 10.1016/j.biopsych.2015.12.012.
  5. Tamura RE. Et al. 2012. GADD45 proteins: central players in tumorigenesis. Current Molecular Medicine. 12(5): 634–651.

Andrea Vassena

Master Industrial Biotechnology student

Christian Villanti

Master Industrial Biotechnology student