Projects

Neuronal plasticity, the ability of the nervous system to adapt to internal or external stimuli, is a fundamental property that underlies brain development, learning, memory, and resilience to neurodegenerative disorders. At the molecular level, neuronal plasticity depends on the activation of neuronal activity-regulated genes (nARGs), a process tightly controlled by enhancer regions—short regulatory DNA sequences. Recent findings reveal that active enhancers produce enhancer-derived RNAs (eRNAs), which may play crucial roles in gene regulation through complex interactions with DNA, RNA, and proteins. Despite their potential significance, the function of eRNAs in neuronal activity remains poorly understood due to their transient nature and technical challenges in studying them. This project aims to develop innovative experimental approaches to unravel the role of eRNAs in nARG activation. Using rodent primary neuronal culture —a well-established model for studying neuronal plasticity—the project will systematically analyze eRNA responses to external stimuli with high temporal resolution. Comprehensive sequencing technologies will be combined to characterize the molecular features of eRNAs. These data will be complemented by epigenomic profiling to correlate enhancer activity with transcriptional dynamics. The project will also investigate potential links between eRNA features and neurodegenerative disease-associated mutations, providing insights into how brief stimuli-related gene activation may contribute to disease phenotypes. Together, these studies will create a framework for understanding the regulatory roles of eRNAs in neuronal plasticity and their broader implications for brain development and disorders.

Enhancers are short distal cis-regulatory DNA regions that drive expression of a gene. However, enhancers do not function exclusively as DNA entities. Activated enhancers are transcribed by RNA polymerase II (RNAPII), which produces enhancer-derived RNAs (eRNAs). Production of eRNA creates additional trans-regulatory mechanisms facilitated by DNA-RNA, RNA-RNA, or protein-RNA interactions. Due to eRNAs’ fast degradation rates, and lack of robust and standardized sequencing methods, reports about the molecular nature of eRNA molecules and their processing are conflicting, making mechanisms of gene regulation by eRNA controversial. Even less is known about co- and post-transcriptional processing of eRNA. This project aims to overcome the controversy and fill the knowledge gap by studying a well-defined experimental system, cultured rat cortical neurons, and activation of immediate-early gene (IEG) response, perturbing the core eRNA endonuclease and combining this with eRNA-tailored sequencing, computational and biochemical methods. The developed integrative approach will reveal molecular features of eRNA molecules and their precursors genome-wide, opening the opportunity to study eRNA biogenesis to further understand molecular mechanisms behind the eRNA-mediated gene regulation.

Formation of new synapses, and alteration of the strength and stability of existing synapses are regarded as the main cellular basis for memory and long-term behavioral adaptations. Neuronal activity-regulated gene expression plays a crucial role in synaptic development and function, and its deregulation gives rise to various nervous system disorders. Knowledge about the regulatory mechanisms of activity-dependent gene expression is important both for understanding of nervous system function and for finding new drug targets. The aim of this project is to study the molecular mechanisms of neuronal activity-regulated gene expression, including transcription, translation and posttranslational modifications, in the nervous system health and disease. The studies are focused on two genes, the neurotrophin BDNF and the basic helix-loop-helix transcription factor TCF4.

"Pitt-Hopkins syndrome is a cognitive functional disorder, caused by a de novo genetic mutation of one allele of the transcription factor 4 (TCF4) gene. It has been reported that postnatal restoration of TCF functions in Pitt-Hopkins syndrome animal model (partially) rescues the phenotype, indicating that therapeutic approaches increasing TCF4 levels or activity might also help patients. It has also been reported that inhibiting histone deacetylase activity increases TCF4 transcriptional activity and rescues memory deficiencies associated with TCF4 haploinsufficiency in a mouse model. These effects are likely conveyed by some TCF4 co-repressor, such as ETO/RUNX1T1 recruiting HDACs. However, HDAC inhibitors have a very broad effect on the cellular transcription and can cause various side-effects. Here, we hypothesize that by modulating the activity of specific TCF4 co-activators or co-repressors or their interaction with TCF4 could increase TCF4-dependent transcription, thus alleviating the symptoms of Pitt-Hopkins syndrome and have less side effects for the patients. To this end, we pursue to thoroughly identify the co-regulators participating in TCF4-dependent transcription, and to find means to modulate their activity. The specific aims are as follows: (1) Identify the co-regulatory proteins of different TCF4 protein isoforms. (2) Determine the mechanism of action and the interacting regions between TCF4 and co-regulatory proteins. (3) Develop means to modulate the transcriptional activity or binding of the TCF4 co-regulatory proteins."

The aim of this project was to investigate the regulation of stimuli-activated neuronal enhancers through enhancer-derived RNA (eRNA), addressing the challenge posed by the unstable and transient nature of eRNAs. We combined advanced next-generation sequencing techniques with molecular biology assays to gain a mechanistic understanding of eRNA function in activity-dependent gene expression. The project's critical task was establishing a robust genome-wide method to precisely define the eRNA 5’ to 3’ end sequence. We optimized the MAPcap method, which emerged as the preferred technique for transcription start site (TSS) detection due to its low sequencing depth requirement, compatibility with previously collected RNA samples, and ease of integration into research workflows. As model systems we selected primary rat cortical neurons and Neuro2A mouse neuroblastoma cells. For both systems, we tested and optimized cultivation, treatment, and subcellular fractionation protocols. We also established RT-qPCR assays to validate eRNAs and immediate-early genes. To prepare for the functional validation of eRNAs, we established a complete procedure encompassing in vitro transcription, biochemical pull-down assays, and mass spectrometry analysis. This workflow will be applied after we complete the analysis of our NGS datasets and define eRNAs of interest. In conclusion, this project has successfully established methods and generated data that advance our understanding of eRNA roles in neuronal gene regulation. The outcomes of this research pave the way for significant discoveries in neurobiology and provide a robust platform for future studies. The support from this grant has been instrumental in achieving these results, promising impactful contributions to the field.

This project took a closer look at how BDNF, an important protein usually studied in neurons, is regulated in heart cells and in special brain cells called astrocytes. First, we figured out signals that “switch on” BDNF, such as noradrenaline (similar to adrenaline), and studied DNA regions that help controlling this switch, specifically in cardiac cells. We then focused on astrocytes, a kind of brain cell that supports neurons, among other functions. We observed that when neurons and astrocytes are maintained together and neurons are activated, astrocytes respond by producing more BDNF. BDNF is an actively studied protein, given its critical roles in the central nervous system and especially in neurons. More recently, BDNF expression and function have been studied in other cell types as well, revealing a larger spectrum of roles for this neurotrophin. Furthermore, its dysregulation in several pathological conditions make it an interesting target for therapeutic interventions. The results obtained in the frame of this project are therefore interesting to the neurotrophin community, and more broadly to the neurobiology and cardiac biology fields, and provide the fundamental knowledge required to design and implement treatment strategies. Different methodologies needed to be put in place an optimized in order to achieve the goals of this project. As a result, these are now part of the group’s diverse tool kit and can be implemented to address several of our research questions, which I find to be an important outcome of the project. Finally, the successful defence of the MSc thesis of a co-supervised student is an important milestone and a key achievement associated with this grant.

The aim of the centre is to translate the rapid progress in the field of genomics and other “-omics” technologies into improved understanding of molecular and evolutionary mechanisms of disease as well as improved prevention, diagnosis and clinical care. The Centre integrates 12 research units from University of Tartu, Estonian Biocentre and Tallinn University of Technology.

The project is focused on developing molecular diagnostics tools for early detection of schizophrenia. The research program of SZ_TEST will include three interrelated lines of research: 1: Deciphering molecular mechanisms of schizophrenia. 2: Identifying molecular biomarkers for early detection of schizophrenia. 3: Developing reliable protocols for diagnostic use of newly identified biomarkers in clinical settings.