Blockade of histone deacetylase activity affects transcription and splicing of neuronal and glial genes
- Authors: Borodinova A.A.1, Beletskiy A.P.1, Balaban P.M.1
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Affiliations:
- Institute of Higher Nervous Activity and Neurophysiology of Russian Academy of Sciences
- Issue: Vol 18, No 4 (2023)
- Pages: 698-701
- Section: Conference proceedings
- URL: https://genescells.ru/2313-1829/article/view/623416
- DOI: https://doi.org/10.17816/gc623416
- ID: 623416
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Abstract
The study of the molecular mechanisms underlying plastic processes in the nervous system is of great interest in modern neuroscience. It is important to understand that epigenetic modifications, which are crucial for the development and cellular differentiation, can also be involved in plastic processes in the adult nervous system.
In our early work, we provided evidence that the expression of important memory-related genes, such as Prkcz and Prkci, can be regulated epigenetically [1]. In the current study, we extended the previous work to the systemic level by applying the RNA sequencing approach to evaluate global changes in the expression patterns of various genes during the induction of epigenetic rearrangements. Rat cortical neuron cultures were incubated with one of the nonselective histone deacetylase inhibitors (trichostatin A, TSA; sodium butyrate, NaB) to change the level of epigenetic regulation. Next, the total RNA was extracted and subjected to RNA-Seq libraries preparation and subsequent NGS-sequencing.
Bioinformatics analysis of transcriptomic data revealed substantial overlapping of differentially expressed genes (DEGs) in NaB-treated and TSA-treated groups, indicating that different histone deacetylase (HDAC) inhibitors induce transcriptional changes in primary neuron cultures through common regulatory pathways irrespective of chemical structure of applied inhibitor. We found that histone deacetylase blockade is accompanied by a transition from proliferative processes to cellular differentiation. Gene Ontology (GO) analysis of DEGs datasets revealed that the upregulated genes were engaged in cell differentiation and specialization, tissue and embryonic morphogenesis, and the development of various peripheral tissues and organs. On the contrary, genes that reduce the expression under induction of epigenetic rearrangements were involved in biological processes associated with cell proliferation and, most interestingly, the specialization of various brain cells (neurons, astrocytes, oligodendrocytes). It was shown that the expression of a number of glial markers typical for astrocytes and oligodendrocytes was significantly reduced after application of HDAC inhibitors, which was also confirmed by quantitative PCR using specific primer pairs on selected target genes. During the data analysis, we also found a significant decrease in the expression of various neuronal markers associated with the cytoskeleton, the organization of pre- and postsynaptic endings, synaptic transmission.
It is known that fine-tuning of various processes in the central nervous system is due to the production of different isoforms of proteins from the same gene due to the process of alternative splicing of the resulting mRNA. According to the literature, epigenetic rearrangements create a certain environment for the regulation of alternative splicing of different genes [2, 3]. It is shown that production of alternative isoforms can play an important role in various plastic processes [4, 5]. Therefore, using IsoformSwitchAnalyzeR and DEXSeq packages we analyzed the possibility of alternative splicing during the induction of epigenetic rearrangements in rat cortical neuron cultures by evaluating the abundance of various transcripts based on exon usage. We found that some glial genes and a large number of neuronal genes, especially those associated with postsynaptic organization and cell communication, were alternatively spliced after application of histone deacetylase inhibitors. Inhibition of HDAC activity in cortical neuron cultures mainly affected the choice of alternative transcription starts (ATSS) and terminators (ATTS), and to a lesser extent alternative splicing of exons. Obtained results were selectively confirmed by the quantitative PCR using specific pairs of primers for individual exons of different transcript isoforms.
Thus, within this study, it was found that histone deacetylases play an important role in the specialization of various brain cells, and the suppression of their activity affects the expression and alternative splicing of various glial and neuronal marker genes. We do not exclude that global transcriptome changes caused by alternative splicing will lead to qualitative rearrangements of the neuron network, and this is the direction of future research.
Full Text
The investigation into the molecular mechanisms that govern plastic processes in the nervous system holds significant interest to modern neuroscience. Importantly, epigenetic modifications, which play a central role in cellular differentiation and development, may also influence adult nervous system plasticity.
In our previous research, we presented evidence indicating the epigenetic regulation of significant memory-related genes, including Prkcz and Prkci [1]. In this study, we further explored the subject at the systemic level through the application of RNA sequencing to assess the widespread alterations in the expression of several genes during the induction of epigenetic rearrangements. Rat cortical neurons were treated with nonselective inhibitors of histone deacetylases, including trichostatin A (TSA) and sodium butyrate (NaB), to alter the level of epigenetic regulation. Subsequently, total RNA was extracted, and RNA-Seq libraries were prepared, followed by NGS-sequencing.
Bioinformatics analysis of transcriptomic data showed significant overlap of differentially expressed genes (DEGs) in the NaB-treated and TSA-treated groups. This suggests that different histone deacetylase (HDAC) inhibitors induce transcriptional changes in primary neuron cultures through common regulatory pathways, regardless of the chemical structure of the applied inhibitor. Histone deacetylase inhibition leads to a shift from proliferative mechanisms to cellular differentiation. GO analysis of DEG datasets revealed upregulated genes were involved in cell differentiation, tissue, embryonic morphogenesis, and development of varied peripheral tissues and organs. Contrary to expectations, genes that decrease expression under epigenetic rearrangement induction play a role in biological processes associated with cell proliferation and, notably, the differentiation of different types of brain cells, including neurons, astrocytes, and oligodendrocytes. The expression of several glial markers found in astrocytes and oligodendrocytes significantly diminished following HDAC inhibitor application. We confirmed these findings using targeted gene primer pairs and quantitative PCR methods. Furthermore, data analysis revealed a noticeable decline in neuronal markers associated with cytoskeletal organization, pre- and postsynaptic ending organization, and synaptic transmission.
Fine-tuning of various processes in the central nervous system is accomplished by the production of different isoforms of proteins from the same gene through alternative splicing of the resulting mRNA. Epigenetic rearrangements create an environment for regulating alternative splicing of different genes [2, 3]. Studies reveal that alternative isoform production plays a vital role in various plastic processes [4, 5]. Therefore, we used the IsoformSwitchAnalyzeR and DEXSeq packages to investigate the potential for alternative splicing in rat cortical neuron cultures induced with histone deacetylase inhibitors by assessing the abundance of transcripts based on exon usage. Our findings demonstrate alternative splicing in several glial genes and numerous neuronal genes, with a particular emphasis on those genes associated with postsynaptic organization and cell communication. Inhibition of HDAC activity in cortical neuron cultures primarily impacted the selection of alternative transcription starts and terminators, with a minor effect on alternative exon splicing. The observed outcomes were confirmed using specific quantitative PCR primers for distinct exons of various transcript isoforms.
In this study, histone deacetylases were found to play a critical role in the specialization of multiple brain cells, and when their activity is repressed, it affects the expression and alternative splicing of several neuronal and glial marker genes. Possibly, global transcriptome changes resulting from alternative splicing could lead to qualitative reorganization of neuronal networks, a promising avenue for future research.
About the authors
A. A. Borodinova
Institute of Higher Nervous Activity and Neurophysiology of Russian Academy of Sciences
Author for correspondence.
Email: borodinova.msu@mail.ru
Russian Federation, Moscow
A. P. Beletskiy
Institute of Higher Nervous Activity and Neurophysiology of Russian Academy of Sciences
Email: borodinova.msu@mail.ru
Russian Federation, Moscow
P. M. Balaban
Institute of Higher Nervous Activity and Neurophysiology of Russian Academy of Sciences
Email: borodinova.msu@mail.ru
Russian Federation, Moscow
References
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