Single cell transcriptomics reveal regulators of progenitor cell fate and postmitotic maturation during brain development

In this thesis the NanoString nCounter technology was applied to validate an improved PCR-based strategy for quantitative and qualitative global single cell transcriptome analysis. RNA isolates from whole tissue were quantitatively compared with cDNA amplificates prepared from single cell equivalent dilutions of the same RNA. A significant correlation was obtained between the values measured for RNA and cDNA amplificates for transcript copy numbers from 10 to several thousand. This method was then used to investigate two distinct neurodevelopmental issues. The first neuroscientific question dealt with the generation of cortical projection neurons, whose fate is specified at the progenitor level and depends on the mode of division. With the help of the high resolution of the single cell amplification method, it was possible to provide evidence for an extra-cortical fine-tuning of cortical progenitor output via the Eph-receptor/ephrin-ligand system. The method was further applied to investigate cortical interneuron development, especially to their post-mitotic phase of tangential migration. To identify factors orchestrating the migration, we analyzed single cell transcriptomes of cells derived from distinct proliferative niches of interneurons in the basal telencephalon. Interestingly, we observed DNA methyltransferase 1 (Dnmt1) expression in a fraction of post-mitotic POA-derived cortical interneurons. Deletion of Dnmt1 in this interneurons caused defective migration, resulting in drastically reduced numbers of cortical interneurons in adults. Next generation sequencing analysis of FAC-sorted Dnmt1 wildtype and knockout mice revealed DNMT1-dependent repression of genes involved in late maturational processes like neurite outgrowth. In this context, further experiments provide evidence that DNMT1 preserves the migratory shape of postmitotic GABAergic interneurons in part through negative regulation of Pak6, which stimulates neuritogenesis at post-migratory stages

The Expression of the Cancer-Associated lncRNA Snhg15 Is Modulated by EphrinA5-Induced Signaling

The Eph receptor tyrosine kinases and their respective ephrin-ligands are an important family of membrane receptors, being involved in developmental processes such as proliferation, migration, and in the formation of brain cancer such as glioma. Intracellular signaling pathways, which are activated by Eph receptor signaling, are well characterized. In contrast, it is unknown so far whether ephrins modulate the expression of lncRNAs, which would enable the transduction of environmental stimuli into our genome through a great gene regulatory spectrum. Applying a combination of functional in vitro assays, RNA sequencing, and qPCR analysis, we found that the proliferation and migration promoting stimulation of mouse cerebellar granule cells (CB) with ephrinA5 diminishes the expression of the cancer-related lncRNA Snhg15. In a human medulloblastoma cell line (DAOY) ephrinA5 stimulation similarly reduced SNHG15 expression. Computational analysis identified triple-helix-mediated DNA-binding sites of Snhg15 in promoters of genes found up-regulated upon ephrinA5 stimulation and known to be involved in tumorigenic processes. Our findings propose a crucial role of Snhg15 downstream of ephrinA5-induced signaling in regulating gene transcription in the nucleus. These findings could be potentially relevant for the regulation of tumorigenic processes in the context of glioma

Single-cell transcriptomics reveals regulators of neuronal migration and maturation during brain development

The correct establishment of inhibitory circuits is crucial for cortical functionality and defects during the development of γ-aminobutyric acid–expressing cortical interneurons contribute to the pathophysiology of psychiatric disorders. A critical developmental step is the migration of cortical interneurons from their site of origin within the subpallium to the cerebral cortex, orchestrated by intrinsic and extrinsic signals. In addition to genetic networks, epigenetic mechanisms such as DNA methylation by DNA methyltransferases (DNMTs) are suggested to drive stage-specific gene expression underlying developmental processes. The mosaic structure of the interneuron generating domains producing a variety of interneurons for diverse destinations complicates research on regulatory instances of cortical interneuron migration. To this end, we performed single-cell transcriptome analysis revealing Dnmt1 expression in subsets of migrating interneurons. We found that DNMT1 preserves the migratory morphology in part through transcriptional control over Pak6 that promotes neurite complexity in postmigratory cells. In addition, we identified Ccdc184 , a gene of unknown function, to be highly expressed in postmitotic interneurons. Single-cell mRNA sequencing revealed a positive correlation of Ccdc184 with cell adhesion–associated genes pointing to potential implications of CCDC184 in processes relying on cell-cell adhesion–like migration or morphological differentiation of interneurons that deserves further investigations

Neuronal Lhx1 expression is regulated by DNMT1-dependent modulation of histone marks

Apart from the conventional view of repressive promoter methylation, the DNA methyltransferase 1 (DNMT1) was recently described to modulate gene expression through a variety of interactions with diverse epigenetic key players. We here investigated the DNMT1-dependent transcriptional control of the homeobox transcription factor LHX1, which we previously identified as an important regulator in cortical interneuron development. We found that LHX1 expression in embryonic interneurons originating in the embryonic pre-optic area (POA) is regulated by non-canonic DNMT1 function. Analysis of histone methylation and acetylation revealed that both epigenetic modifications seem to be implicated in the control of Lhx1 gene activity and that DNMT1 contributes to their proper establishment. This study sheds further light on the regulatory network of cortical interneuron development including the complex interplay of epigenetic mechanisms

DNMT1 modulates interneuron morphology by regulating Pak6 expression through crosstalk with histone modifications

Epigenetic mechanisms of gene regulation, including DNA methylation and histone modifications, call increasing attention in the context of development and human health. Thereby, interactions between DNA methylating enzymes and histone modifications tremendously multiply the spectrum of potential regulatory functions. Epigenetic networks are critically involved in the establishment and functionality of neuronal circuits that are composed of gamma-aminobutyric acid (GABA)-positive inhibitory interneurons and excitatory principal neurons in the cerebral cortex. We recently reported a crucial role of the DNA methyltransferase 1 (DNMT1) during the migration of immature POA-derived cortical interneurons by promoting the migratory morphology through repression of Pak6. However, the DNMT1-dependent regulation of Pak6 expression appeared to occur independently of direct DNA methylation. Here, we show that in addition to its DNA methylating activity, DNMT1 can act on gene transcription by modulating permissive H3K4 and repressive H3K27 trimethylation in developing inhibitory interneurons, similar to what was found in other cell types. In particular, the transcriptional control of Pak6, interactions of DNMT1 with the Polycomb-repressor complex 2 (PCR2) core enzyme EZH2, mediating repressive H3K27 trimethylations at regulatory regions of the Pak6 gene locus. Similar to what was observed upon Dnmt1 depletion, inhibition of EZH2 caused elevated Pak6 expression levels accompanied by increased morphological complexity, which was rescued by siRNA-mediated downregulation of Pak6 expression. Together, our data emphasise the relevance of DNMT1-dependent crosstalk with histone tail methylation for transcriptional control of genes like Pak6 required for proper cortical interneuron migration

DNA Methyltransferase 1 (DNMT1) Acts on Neurodegeneration by Modulating Proteostasis-Relevant Intracellular Processes

The limited regenerative capacity of neurons requires a tightly orchestrated cell death and survival regulation in the context of longevity, as well as age-associated and neurodegenerative diseases. Subordinate to genetic networks, epigenetic mechanisms, such as DNA methylation and histone modifications, are involved in the regulation of neuronal functionality and emerge as key contributors to the pathophysiology of neurodegenerative diseases. DNA methylation, a dynamic and reversible process, is executed by DNA methyltransferases (DNMTs). DNMT1 was previously shown to act on neuronal survival in the aged brain, whereby a DNMT1-dependent modulation of processes relevant for protein degradation was proposed as an underlying mechanism. Properly operating proteostasis networks are a mandatory prerequisite for the functionality and long-term survival of neurons. Malfunctioning proteostasis is found, inter alia, in neurodegenerative contexts. Here, we investigated whether DNMT1 affects critical aspects of the proteostasis network by a combination of expression studies, live cell imaging, and protein biochemical analyses. We found that DNMT1 negatively impacts retrograde trafficking and autophagy, with both being involved in the clearance of aggregation-prone proteins by the aggresome–autophagy pathway. In line with this, we found that the transport of GFP-labeled mutant huntingtin (HTT) to perinuclear regions, proposed to be cytoprotective, also depends on DNMT1. Depletion of Dnmt1 accelerated perinuclear HTT aggregation and improved the survival of cells transfected with mutant HTT. This suggests that mutant HTT-induced cytotoxicity is at least in part mediated by DNMT1-dependent modulation of degradative pathways

DNMT1 modulates interneuron morphology by regulating <i>Pak6</i> expression through crosstalk with histone modifications

<p>Epigenetic mechanisms of gene regulation, including DNA methylation and histone modifications, call increasing attention in the context of development and human health. Thereby, interactions between DNA methylating enzymes and histone modifications tremendously multiply the spectrum of potential regulatory functions. Epigenetic networks are critically involved in the establishment and functionality of neuronal circuits that are composed of gamma-aminobutyric acid (GABA)-positive inhibitory interneurons and excitatory principal neurons in the cerebral cortex. We recently reported a crucial role of the DNA methyltransferase 1 (DNMT1) during the migration of immature POA-derived cortical interneurons by promoting the migratory morphology through repression of <i>Pak6</i>. However, the DNMT1-dependent regulation of <i>Pak6</i> expression appeared to occur independently of direct DNA methylation. Here, we show that in addition to its DNA methylating activity, DNMT1 can act on gene transcription by modulating permissive H3K4 and repressive H3K27 trimethylation in developing inhibitory interneurons, similar to what was found in other cell types. In particular, the transcriptional control of <i>Pak6</i>, interactions of DNMT1 with the Polycomb-repressor complex 2 (PCR2) core enzyme EZH2, mediating repressive H3K27 trimethylations at regulatory regions of the <i>Pak6</i> gene locus. Similar to what was observed upon <i>Dnmt1</i> depletion, inhibition of EZH2 caused elevated <i>Pak6</i> expression levels accompanied by increased morphological complexity, which was rescued by siRNA-mediated downregulation of <i>Pak6</i> expression. Together, our data emphasise the relevance of DNMT1-dependent crosstalk with histone tail methylation for transcriptional control of genes like <i>Pak6</i> required for proper cortical interneuron migration.</p

EphrinA5 regulates cell motility by modulating Snhg15/DNA triplex-dependent targeting of DNMT1 to the Ncam1 promoter

Cell–cell communication is mediated by membrane receptors and their ligands, such as the Eph/ephrin system, orchestrating cell migration during development and in diverse cancer types. Epigenetic mechanisms are key for integrating external “signals”, e.g., from neighboring cells, into the transcriptome in health and disease. Previously, we reported ephrinA5 to trigger transcriptional changes of lncRNAs and protein-coding genes in cerebellar granule cells, a cell model for medulloblastoma. LncRNAs represent important adaptors for epigenetic writers through which they regulate gene expression. Here, we investigate a lncRNA-mediated targeting of DNMT1 to specific gene loci by the combined power of in silico modeling of RNA/DNA interactions and wet lab approaches, in the context of the clinically relevant use case of ephrinA5-dependent regulation of cellular motility of cerebellar granule cells. We provide evidence that Snhg15, a cancer-related lncRNA, recruits DNMT1 to the Ncam1 promoter through RNA/DNA triplex structure formation and the interaction with DNMT1. This mediates DNA methylation-dependent silencing of Ncam1, being abolished by ephrinA5 stimulation-triggered reduction of Snhg15 expression. Hence, we here propose a triple helix recognition mechanism, underlying cell motility regulation via lncRNA-targeted DNA methylation in a clinically relevant context