Scientific Focus

Peter Ebert, Elke Gabriel, Sugirtahn Sivalingam, Düsseldorf

Mareike Albert, Dresden

The neocortex is instrumental for higher cognitive functions in humans. During neocortex development, epigenetic mechanisms regulate developmental gene expression programs, which are key for proper brain function later in life. Human brain development is highly protracted, and during this long period of development and maturation, the brain is sensitive to external environmental stimuli, leading to positive or negative outcomes. Environmental factors that signal to the neural chromatin may have profound impacts on the development of the neocortex, with potentially long-lasting effects on brain function. In this proposal, we aim to investigate the effects of maternal exposure to different nutritional stimuli on neural chromatin and cell fate. This project aims to systematically investigate a large set of histone modifications with single-cell resolution using Epi-CyTOF in human cortical organoids exposed to different nutritional stimuli. Moreover, cell type-specific epigenomic and single-cell RNA-seq will identify the genes that are sensitive to epigenetic adaptations induced by nutritional stimuli. Functional studies using epigenetic inhibitors and epigenome editing will provide mechanistic insight and will help explore the potential compensation of neurodevelopmental outcomes after neurotoxin exposure. Taken together, this project is expected to shed light on how the neural chromatin integrates environmental stimuli provided by maternal nutrition and on how the altered epigenomes affect neural cell fate in the human developing neocortex.

Sreelakshmi Bindu Jyothikumar, Daniele Bano, Sandra Blaess, Oliver Brüstle, Bonn

Neurodevelopmental disorders (NDDs) affect 10-15% of children worldwide. Both genetic and environmental factors contribute the pathogenesis of NDDs, with maternal immune activation during pregnancy emerging as a major risk factor. The molecular mechanisms underlying gene- by-environment interactions in NDDs remain poorly studied. This project aims to investigate early- life neuroinflammatory events and their potential to induce lifelong epigenetic changes. The central hypothesis is that neuroinflammatory signals drive epigenetic remodelling in astrocytes, leading to a primed state more susceptible to future insults. We will focus on the ATP-dependent chromatin remodelling SWI/SNF/BAF complex, given its causative role in NDDs, and aim to elucidate its contribution to astrocyte reactivity under inflammatory conditions. Our assessments include cross-species analyses of the epigenetic transcriptional responses and the underlying chromatin landscapes that regulate neuroinflammation in iPSC-derived organoids and in vivo in transgenic mice.

Jovica Ninkovic, Stefan Stricker, Munich

Recent advancements in genome-wide and single-cell methodologies have highlighted extensive epigenomic variation across the many important cell types in the brain. Despite this progress, the specific chromatin modifications that are critically relevant to distinct cellular lineages remain poorly understood. This knowledge gap primarily stems from the limited availability of methods capable of concurrently manipulating multiple chromatin marks within well-characterized models of cell fate transitions in the mammalian brain. To overcome this issue, we are first characterizing epigenomic differences between four different astrocyte states, two susceptible to neuronal induction, two not; two derived from mouse brains, the other two differentiated from human iPSCs. Next, we will apply a novel strategy allowing efficient manipulation of chromatin accessibility and DNA de-methylation of multiple chromatin differences in well established models of astrocyte to neuron reprogramming. Following this experimental strategy will allow elucidating the role of chromatin dynamics for cell identity barriers and advance our understanding of brain cell lineage specification during neurodevelopmental processes.

Anthi C. Krontira, Aikaterina Nikolaidi, Magdalena Götz, Munich

Neurogenesis can be continued into adulthood in many if not most brain regions in vertebrates, while it is terminated in most brain regions in the adult mammalian brain. To understand the regulators of the neurogenesis-to-gliogenesis switch we performed an unbiased multiome analysis of radial glial cells at the peak of neurogenesis versus the transition to gliogenesis in two neighboring regions – one where neurogenesis largely terminates, the cerebral cortex, and one where it continues, the lateral ganglionic eminence. This showed a surprisingly large down-regulation of chromatin remodellers at the end of neurogenesis with rather few new ones up-regulated in the gliogenic phase. This encompasses a switch from Brg1 to Brm-containing SWI/SNF chromatin remodelling complexes and a cortex-specific expression of Setd2 H3-K36 methylation complexes at the peak of neurogenesis. Here we aim to perform a small targeted CRISPR-screen in vivo to determine the function of these and other regulated chromatin remodelling factors during neurogenesis and at the onset of gliogenesis. We then aim to determine the composition of the functionally relevant complexes using our proteomic expertise towards a molecular understanding of their specific function related to neurogenesis versus gliogenesis. We could complement this by a candidate approach understanding the function of the NURD complex, components of which we found largely downregulated at the onset of gliogenesis and that we additionally identified as a key interactor for the transcription factor TGIF2, a protein we have shown to control neurogenesis. 

André Reis, Tomohisa Toda, Erlangen

During development, neurons need to receive various inputs such as synaptic inputs to build a functional network. Upon stimulation, neurons are activated and incoming information is integrated and translated into the expression of activity-regulated genes (ARGs). ARGs regulate cell type-specific neuroplastic function and reorganize synaptic connectivity, therefore the tight regulation of neuronal states by ARG expression is fundamental for brain development.  We recently found that Nup153, one of nuclear pore complex proteins, represses ARGs at the basal state in developing neurons. Depletion of Nup153 de-represses ARG expression without additional stimulation. Accumulating evidence indicates that nuclear pore complex proteins work as a structural hub for chromatin and epigenetic regulators. Therefore, we hypothesize that Nup153 organizes neuronal state-specific epigenetic landscapes and regulates neuronal responsiveness by tuning ARG expression.

In the proposed project, by combining epigenomics and proteomics approaches, we will investigate how Nup153 and its epigenetic cofactors organize epigenetic states between two different neuronal states, basal versus active.

Understanding epigenetic mechanisms regulating distinct neuronal states will provide new insights in the regulation of plasticity during brain development. Nuclear pore complex proteins are also recognized as a target of neurological disorders. Therefore, the success of the proposed project will provide fundamental insights into the pathophysiological development of these disorders.

Moritz Mall, Heidelberg

Recent studies have underscored the importance of active gene repression, not just activation, in maintaining stable neuronal cell identities. While terminal selectors and their interactions with epigenetic co-activators are well-characterized for driving neuronal gene expression, comparatively little is known about how transcriptional repressors collaborate with repressive epigenetic machinery to silence unwanted gene programs lifelong. Contrary to the long-standing notion of stable epigenetic silencing, emerging evidence indicates that continuous, active repression is required to preserve the neuronal state, even after differentiation is complete. Age-associated alterations in neuronal gene expression have been linked to neurodegenerative diseases, suggesting that the stability of epigenetic repression may deteriorate over time. Our results show that MYT1L, a neuron-specific transcriptional repressor linked to brain disorders, interacts with repressive chromatin regulators (e.g., LSD1, HDAC) and is required to maintain neuronal fate and function in mature neurons in vivo. This interaction may be key to ensuring that non-neuronal or progenitor-like programs remain OFF, thus safeguarding neuronal identity and function across the lifespan. Investigating how repressive epigenetic complexes function in developing and mature–long-lived neurons by interaction with neuron-specific transcript factors can, therefore, provide unique insights into whether active repression is required to suppress unwanted plasticity and maintain neuronal identity. More generally, it will enable insights about the stability of cell fate and the role of active repression in this process. We will use induced human neurons and in vivo mouse models to address these questions, employing neuron-specific and developmentally timed Cre-mediated MYT1L deletions. By manipulating MYT1L and its interacting epigenetic modifiers at various stages of neuronal differentiation and maturation, we aim to identify how these factors cooperate to prevent fate destabilization. 

Oleksandra Pavlovska, Giacomo Masserdotti, Helmholtz Munich

Vivien Hoof, Fabian Müller, Julia M. Schulze-Hentrich, Saarbrücken

The formation of the mammalian brain is characterized by high cell type diversity and begins in the early stages of prenatal development through a series of crucial processes that produce the cells, form distinct anatomical regions, and fine-tune the intricate network of neural connections throughout the central nervous system. An equally intriguing aspect of neural development that is not well understood is the establishment of sex-specific characteristics that tailor physiological processes and certain behavioral patterns to be distinct between males and females. These distinctions also play a crucial role in influencing susceptibility to various conditions such as neurodevelopmental, neurodegenrative, and psychiatric disorders.
Despite advancements in brain imaging and transcriptome analysis that revealed sex dimorphism across brain regions and cell types, our understanding of these distinctions at the individual cell level, especially during development, remains incomplete. Furthermore, we still lack a thorough understanding of the underlying causes of sex-specific gene expression patterns and the influence of epigenetic mechanisms at chromatin level. Hence, we here propose a comprehensive dual strategy to address open questions on the intricate interactions between (epi)genetic and hormonal regulation. We will combine an i) experimental approach leveraging our expertise in mouse models and employing advanced single-cell multiome profiling of the transcriptome and epigenome with ii) computational approaches for identifying epigenome-transciptome linkages and gene- regulatory networks. By merging these complementary areas of expertise, we seek to gain deeper insights into how sex-dependent hormone level during development provide key signals to gene expression and the underlying chromatin (accessibility) in single cells. Our project aims to decipher the adaptation of the epigenome to hormonal cues during development and unravel the sex-dimorphic consequences such as masculinization versus non-masculinization of the brain in males and females at the nexus of genetics and hormones. Using organotypic brain slices, we then investigate the underlying mechanisms of the identified transcription factors and epigenetic regulators.
Finally, we will ensure that the computational methods developed in our project are accessible as user-friendly software tools and platforms for data exploration. This approach will promote sharing of both methods and data within EPIADAPT and extend to the wider research community.

Robert Schneider, Neuherberg

The aim of this project is to study the role of a key chromatin component, the linker histone H1, during differentiation. We aim to unravel the role of (i) histone H1 subtypes in pluripotent and linage-committed differentiated cells that are characterised by differences in their chromatin organisation and (ii) of distinct H1 subtypes in differently adapted epigenomes. Our results will contribute to understanding the functions of “the forgotten histone”, histone H1 in the dynamics of epigenomes, to clarifying the setting in which chromatin regulation drives development and to address questions of cause and consequences in biological processes. For this we will use a combination of gene expression and chromatin architecture profiling with targeted manipulations of H1 subtypes at different stages of neuronal differentiation.

Helene Kretzmer, Matthew Kraushar, Michael Robson, Berlin and Potsdam

The brain is built from many different types of neurons that arise from neural progenitors during development. The neocortex, a highly evolved brain region in mammals, contains an especially diverse array of neuronal subtypes. How these subtypes are specified remains unclear, but the timed regulation of gene expression in the nucleus plays a crucial role. Our project focuses on a critical developmental window when genome activity and organization undergo rapid changes that may influence neuronal fate. We hypothesize that these large-scale changes in nuclear structure and function act as a switch, reshaping gene expression programs to guide neuron identity. To address this, our SPP2502 project will combine cutting-edge technologies to track gene expression and genome organization at single-cell resolution, and experimentally manipulate nuclear architecture and activity in the neocortex in vivo. Our interdisciplinary team brings together expertise in neurodevelopment, genome regulation, and advanced bioinformatics. Together, we aim to reveal how precisely timed nuclear dynamics control neuronal fate during neocortex development.

Camila Fullio, Annalisa Izzo, Kasra Mokhtarzadeh Azar, Tanja Vogel, Freiburg

During cerebral cortex development, the histone methyltransferase Disruptor of telomeric silencing 1 like (DOT1L) conferring H3K79me plays a pivotal role controlling the balance between progenitor proliferation and differentiation. This role of DOT1L is evolutionary conserved, as loss-of-function (LOF) in both mice and humans result in smaller brains. Interestingly, in humans a gain-of-function (GOF) of DOT1L also associates with microcephaly. To fully appreciate DOT1L functions during brain development, especially in humans, we here aim to follow up on our central hypothesis, that different H3K79me states (H3K79me1, me2, and me3) confer different functionalities at different locations. We propose that a diverse set of mechanisms controls H3K79me states, by regulating DOT1L’s localization, activity, or its interactome. Molecularly, these processes can be controlled in part by signaling/phosphorylation, protein conformation and/or interacting partners. Experimentally, we will use a single-cell (sc)multiomics approach (scRNA-, scATAC-, scCUT&Tag) to untangle transcriptomic changes and the adapted epigenetic landscape during human brain development. We will investigate three human DOT1L mutations, one GOF and two LOF mutations, that we introduced into human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9. hiPSCs will be differentiated towards neural stem cells, neurons, and cortical organoids. We propose to use mass spectrometry to resolve protein complexes of DOT1L that are mediating canonical (H3K79me) and non-canonical functions of DOT1L, and posttranslational modifications of DOT1L. Thereby we aim to determine potential mechanisms of how DOT1L confers locus-specific H3K79me with different levels and states. To identify the upstream regulative layer, we will unravel signaling pathways that control DOT1L in regard to histone methylation activity or cell cycle progression.