3D chromatin organisation in direct neuronal reprogramming – reshaping the nucleus to mold new neurons

How transcription factors can rewire 3D genome architecture and how such regulation affects cell fate decisions is one of the fundamental questions in chromatin biology. Reprogramming of glial cells into neurons by a single neurogenic factor provides an excellent system to study this phenomenon and to determine the global changes in chromatin architecture which occur in direct neuronal reprogramming. To tackle this fascinating project Boyan Bonev, an expert in chromatin architecture and high resolution Hi-C and Magdalena Götz, who pioneered the glia-to-neuron reprogramming, propose to combine their unique expertise and dissect the relationship between 3D nuclear architecture, chromatin accessibility and gene expression during neuronal reprogramming. We first aim to determine the 3D genome organization genome-wide in astrocytes and induced neurons using high-resolution Hi-C. We will also exploit an in vitro model of astrocytes that are more resistant to reprogramming to determine if global changes in chromatin compaction and long-range interactions underlie this lack of plasticity. We will then explore how transcriptional changes (RNA-seq data have been generated already) correspond to changes in chromatin looping and examine by Hi-ChIP to which extent direct binding of the reprogramming factor (Neurogenin2) is involved in re-shaping chromatin architecture. Furthermore, using genome engineering to tether Ngn2 to specific loci and delete Ngn2-bound enhancers, we will determine if Ngn2 binding is necessary and/or sufficient to cause chromatin looping. In the last part of the proposal, we will examine if YY1 is important for formation of cell type specific regulatory interactions during neuronal reprogramming and use ChIP-MS and locus-specific proteomics approach to identify other factors relevant for rewiring the chromatin during glia-to-neuron reprogramming. Taken together, this project will not only yield unprecedented insights into how a single TF can reorganize 3D chromatin architecture, but also help to improve this process to perfect the induced neurons. 

Regulation of mammalian genome architecture and mobility

In this project, we plan to investigate the architecture and mobility of the mammalian genome. We and others have shown that different levels of organization of the genome can be visualized during duplication of the genome as replicons and clusters thereof, likely corresponding to DNA loops and Mbp topologically associated domains (TADs), respectively. This will now allow us to investigate how different epigenetic states and different subnuclear spatial localization patterns impact genome architecture and mobility. We will additionally compare data from different cell types and mammalian species, pluripotent versus somatic cells, and will probe potential regulators using cells deficient on known epigenetic/chromatin factors or pharmacological inhibition. Finally, we will investigate whether genome mobility is affected by genome metabolism. Novel computational methods for automated image analysis based on deep learning will be developed for accurate quantification of the chromatin higher-order structures and their mobility based on microscopy data at different resolution levels. This work should provide insights on the organization and mobility of the mammalian genome and how this regulates or is regulated by genomic processes. 

Assessing the Function and Dynamics of Spatial Genome Architecture during Embryogenesis

Gene expression is initiated through the action of enhancer elements, which are dispersed throughout the genome, often at great distances from the target genes they regulate.  How enhancers in one part of a chromosome can impart regulatory information to another distal region is a long-stranding question in genome regulation.  Despite having direct implications for development, evolution and disease, the underlying mechanism and realtime dynamics of enhancer-promoter topologies remain unknown.  To address this, we will take an interdisciplinary approach, combining genetic dissection in both cis and trans, quantitative single cell imaging, genome engineering, and state-of-the-art live imaging during embryogenesis.  Many of the general principles underlying genome regulation are highly conserved from Drosophila to humans.  We will leverage the ease of genome engineering in Drosophila, and the wealth of information about developmental enhancers to functionally dissect the inherent properties of genome architecture.   

This proposal has three complementary aims: (1) To dissect the requirement of genome architecture for enhancer function, we will genetically delete all interacting regions within three chromatin domains and assess their functional impact on genome architecture, gene expression and embryonic development in homozygous embryos.  (2) To understand how genome architecture is first established during development, we will deplete different factors from early embryos in trans.  The combination of aims 1 and 2 will provide one of the most comprehensive functional dissections of the role of 3D genome architecture during embryogenesis to date.  (3) To measure the dynamics and order of events in genome regulation, we will engineer embryos with tagged enhancers, promoters and nascent transcripts at two loci to quantify the dynamics of distal enhancer-promoter interactions during embryogenesis and determine how that relates to functional output (i.e. nascent transcription).  The combined results from all three aims will provide unique functional, quantitative and dynamic insights, providing the missing data to move from correlation to causation in genome regulation.  

Understanding how changes in spatial chromatin organization cause defective gene expression in acute myeloid leukemia with cohesin mutations

Multiple large-scale sequencing projects have identified a large number of mutations recurrently acquired in acute myeloid leukemia (AML). Mutations often cause defective gene regulation and consequently AML subtypes can be recognized based on unique gene expression signatures. Recently it has been shown that mutations may not only occur in coding regions of genes, but also in elements that regulate their expression. Enhancer dislocation as well as mutations that either generate or destroy enhancers have been reported in cancer. In this application I propose to investigate other mechanisms of altered gene regulation by changes in enhancer-to-promoter association, i.e. in AML with mutations in cohesin genes.  Members of the cohesin complex have been reported to be mutated in ~15% of AML patients. Since cohesin plays a critical role in the alignment and stabilization of replicated chromosomes, one would expect that mutations in genes encoding for cohesin members are particularly found in leukemias with severe chromosomal defects. However, the opposite appears to be true, as most AML patients with mutations in members of the cohesin complex have a normal karyotype. Therefore these mutant protein members are likely to drive tumorigenesis through other functions of cohesin, i.e. via regulation of three-dimensional chromatin organization, looping and insulation of topologically associated domains. Cohesin consequently shapes genomewide gene expression patterns, regulating cell-type specific gene sets that are essential for healthy hematopoietic development. Cohesin complexes carry one of the two versions of the SA subunit, namely STAG1 (SA1) or STAG2 (SA2), respectively. SA1-cohesin complex has been reported to be particularly involved in stabilization of TADs, via the interaction with CTCF. SA2- cohesin complexes preferentially support interactions between celltype specific enhancers and promoters. These SA2 interactions, may either involve CTCF proteins or they act via the Mediator complex, of which MED12 is key in human hematopoietic progenitor cells (HSPCs). Mutations have been reported in several cohesin members in AML, in particular in STAG1, STAG2 or RAD21. We hypothesize that these mutations affect the cohesin complex formation, the chromatin structure and consequently enhancer-to-promoter interactions, which will cause changes in gene expression. In this proposal we will study the potential differences and functions between the two variant cohesin complexes in HSPCs as well as the consequences of their specific downregulation in spatial chromatin architecture and gene expression. Furthermore we will study the consequences of mutations in cohesin members on chromatin structure and gene expression in cells from patients with AML. The results obtained from this project will reveal fundamental insights into the effect of mutated SA1- and SA-2 cohesin complexes on 3D chromatin folding and gene regulation in primary AML cells.

Single molecule imaging of architectural proteins during zebrafish embryo development

The organization of chromatin in the nucleus of a cell is closely interlinked with functions such as gene expression, replication and DNA repair. In interphase, two important levels of organization exist within chromosome territories, separation of chromatin in active and inactive compartments and a network of self-associating chromatin loops. These loops are involved in transcription regulation by controlling the interaction between enhancer and promoter regions of chromatin. They arise and dissolve dynamically through the interplay of architectural proteins and their cofactors. How binding of architectural proteins to chromatin evolves in the course of early embryo development to mediate chromatin architecture is unclear. In this project, we want to study the evolution of interactions between architectural proteins and chromatin during early embryogenesis. In many model organisms such as Drosophila and zebrafish, this developmental period embraces the activation of zygotic transcription. We plan to record the kinetic behaviour of single architectural proteins by single molecule fluorescence imaging in live, developing zebrafish embryos during the time period of transcription activation. Thus we expect to obtain new insights into the functional interplay of chromatin architecture, gene transcription and architectural proteins.

Measuring genome organization during stem cell differentiation with super-resolution microscopy

The establishment, maintenance and change of cellular identities during development and differentiation is controlled by complex signaling pathways that include interactions of cellular factors and epigenetic modifications. There is growing evidence that in addition to the well-studied DNA and histone modifications also spatial genome architecture might contribute to the overall epigenetic information content that defines the identity and potential of individual cells. We now want to systematically investigate changes in genome organization during early development where changes in genome-wide transcription are accompanied by specific epigenetic changes. With defined stem cell culture systems, we want to recapitulate the defined steps from naïve pluripotency to primed pluripotency and subsequent cellular differentiation. We will focus on the genome organization of the Nanog pluripotency gene cluster and measure changes in condensation levels and long-range interactions between regulatory elements (enhancers and promoters) during stem cell differentiation using refined fluorescent hybridization protocols and live cell measurements with super-resolution microscopy. This microscopic approach does not reach the molecular resolution of conformation capture methods (like e.g. HiC) but provides physical distances and single cell resolution. Moreover, automated high-throughput microscopy enables the identification of rare cell populations and functional links with morphological features and physiological states. We will introduce specific mutations to dissect the role of cis-acting DNA sequence elements in regulating the activity and spatial organization of the Nanog pluripotency gene cluster. In parallel we will study trans-acting epigenetic factors (DNMTs, TETs and HMT) and their role in local genome condensation, folding and activity.  Our study should complement other methodological approaches of this priority program and should help to elucidate the role and regulation of spatial genome architecture during early development and cellular differentiation.

Nuclear landscape of HIV-1 integration in microglia – unexplored HIV-1 reservoirs

The introduction of combinatorial antiretroviral therapy (cART) has significantly improved the management of HIV-1 infection as well as the health and life expectancy of HIV-1 infected individuals. While viral loads can be efficiently controlled by cART, transcriptionally silenced, but replication competent virus persists integrated into the host genome, creating long-lasting reservoirs in different anatomical sites throughout the body. These long-lived reservoirs in memory T cells, macrophages and brain microglia, are unreachable by current treatments and impede a functional HIV-1 cure . Where the virus is hidden in the human genome has functional consequences for the viral fate . Our previous work on T cells, major sanctuaries of latent HIV-1, has started exploring the link between 3D host genome organization and HIV-1 integration site selection. We showed that HIV-1 does not position its genome randomly but prefers the outer spatial shells, associated with the open chromatin domains underneath the nuclear pore complex (NPC) . More recently we discovered that HIV-1 commonly targets clusters of cell type specific genes with super-enhancer (SE) regulatory elements (Lucic et al doi: doi.org/10.1101/287896).  

Whereas  several  studies report on the mechanisms of chromatin mediated transcriptional silencing of HIV-1 in microglia, almost nothing is known about the integration sites of the virus in these cells. Here we propose to study HIV-1 integration and latency in microglia through a genomics view-point and to investigate DNA features and chromatin factors that could contribute to integration and silencing events in HIV-1 life cycle. We propose to identify the integration sites of HIV-1, to define the chromatin signatures and to map the genome contacts with respect to the integrated viral genome. Lusic lab will focus on the cell biology and genomics of viral infection, while the Herrmann group will contribute to the project by analysing these complex genomics data and generating an overall picture of chromatin profiles and genomic contacts relevant for the virus in microglial cells. We will join forces with other members of the SPP proposal, and we will collaborate directly with Vassilis Roukos (IMB Mainz) and Irina Solovei (LMU Munich) to visualize HIV-1 and genomic regions where it integrates in microglia by fluorescence in situ hybridization (FISH) and high-throughput microscopy.

Structuring the genome through phase-separated transcriptional condensates

Much recent work indicates that the 3D organization of the genome plays essential roles in the control of gene expression programs that underlie the cellular identity of all cell types in complex metazoans. Here we propose a model that gene expression in turn has a significant contribution to structuring the genome. We have recently discovered that gene transcription involves a liquid-liquid phase separation event that underlies the formation of transcriptional condensates of unusual features. Phase separation is typically driven by multivalent, low affinity interactions between intrinsically disordered regions (IDRs) in proteins, and numerous transcription factors that occupy enhancer elements contain sequence portions that resemble IDRs. In Aim 1, we will investigate the hypothesis that transcription factor IDRs are able to drive the formation of nuclear condensates that facilitate long distance DNA interactions in mammalian cells. Furthermore, human genetics data indicate that a large number of human developmental disorders are caused by mutations in transcriptional regulators, and that these mutations tend to occur in the predicted IDRs of those transcriptional regulators. In Aim 2, we will test the hypothesis that such diseaseassociated IDR mutations impair the ability of transcriptional regulators to form nuclear condensates, and consequently disrupt long-range DNA interactions. Our studies will provide new insights into the molecular processes that regulate genome structure, and into the mechanistic relationship between genome structure and gene expression. The results of this work we believe will ultimately facilitate the design of strategies that interfere with IDR-driven genome structuring as a therapeutic approach.

Determining TADs as conserved regulatory units in vertebrate heart development

How the information for complex spatiotemporal gene expression patterns is encoded in the genome remains a fundamental question in biology. Major advances in recent years have highlighted the importance of the 3D chromatin structure for gene regulation and identified topologically associating domains (TADs) as conserved spatial units of the genome. Interestingly, TADs overlap with gene regulatory landscapes and large syntenic blocks, raising the possibility that these domains reflect a conserved functional unit of the genome. This poses the question whether TADs are able to function independently of their genome of origin.  Here, I propose to study the relationship between conserved TADs and gene regulation using vertebrate heart development as a model system. The hearts of birds and mammals share many general features but have evolved independently over the past 250 million years. I will study the conservation of gene regulatory mechanims in vertebrate heart development by comparing 3D-chromatin architecture and epigenetic landscapes in chicken and mouse heart development.  In a second phase, I will take this comparison one step further and experimentally test the functional conservation of TADs. Using a combination of genome engineering and BAC-transgenesis, I will insert entire chicken TADs into the mouse genome. I will then test if and how the 3D-chromatin architecture, epigenetic landscapes, and ultimately the biological output of the transgenic chicken TADs produce a transgene that is functional in the exogenous genome.  The results of this study will elucidate to which degree DNA sequence determines chromatin folding and how homologous regulatory landscapes are able to fold and function if transplanted into species separated by over 250 million years of evolution.

Evolution of 3D chromatin architecture: The role of CTCF across taxa

The three-dimensional organization of the genome is developmentally dynamic and has been shown to critically affect gene regulation. In animals, this spatial organization is reflected by intricate interactions that bring genes and regulatory elements separated widely in the linear genome into close physical proximity and by sequestering sections of the genome into megabase-scale topologically associated domains. Recently, the transcriptional repressor CCCTC-binding factor (CTCF) has emerged as a key player in mediating 3D genome organization due to its capacity to dimerize in vivo in an orientation-dependent manner. CTCF knock out causes loss of genome topology and lethality. Though CTCF is deeply conserved among the bilaterian clade, mounting evidence demonstrates that the property of CTCF to interconnect distant genomic regions has been acquired at some point during the evolution of the chordates, concomitant with the expansion of the non-coding regulatory genome. It remains unresolved how CTCF promotes genome topology in vertebrates, while its primary function in insects, for example, remains refined to interactions and gene regulation on the short-distance scale.  We propose an innovative multi-species approach using organisms with distinct roles of CTCF in 3D chromatin organization to functionally dissect its divergent modes, as well as the mechanisms that mediate CTCF function in flies, mice, and ascidians. Using CRISPR/Cas genomic editing, we will exchange CTCF proteins between species in vivo to assess its function in different regulatory contexts and its impact on spatial genome organization and transcriptional programs. Using innovative proteomic stratagems, we will then dissect species- and context-specific CTCF protein-protein interactions to then directly test regulatory mode requirements. The proposed project will advance our understanding of the evolutionary origins of 3D chromatin organization, its functional importance for developmental gene regulation, and the molecular mechanisms by which divergent CTCFs act. 

Modification of 3D genome architecture and gene expression at the Fgf8 locus by transposable elements and structural variations

The genome of vertebrates consists mainly of non-coding sequence of which more than half is of repetitive in nature. In previous studies we were able to show that structural variants (SVs) can result in gene misexpression and disease by altering the 3D conformation of chromosomes. I here propose that repetitive elements can interfere with 3D genome folding thereby inducing ectopic contacts over TAD-boundaries with subsequent misexpression and disease and that this mechanism can produce similar phenotypes as genomic rearrangements by SVs. We will investigate this hypothesis by studying the pathology of SVs at the Fgf8 locus that are causal for split-hand-foot-malformation (SHFM) in humans. A SHFM phenotype is also caused by retrotransposon (MusD) insertions at the same locus in the mouse mutant dactylaplasia (Dac). We will use CRISPR/Cas9 genome editing to re-engineer the human SVs in mice to study their effect on gene regulation and limb development. The pathology of the MusD insertion in the Dac mutant will be studied with a detailed expression analysis in dac limb buds using expression profiling and single cell RNA sequencing. In addtition, we will investigate histone modification and CTCF binding and perform capture HiC from Dac/Dac embryos as well as the SHFM rearrangements to investigate their effect on chromatin modification and configuration. In a next step we will manipulate the Dac genome in ES cell generated from Dac/Dac embryos in order to rescue the Dac phenotype. We will study the effect of MusD transposable elements (TEs) genome wide by 4C in suceptible (129) vs. non suceptible (C57B6) strains to identify regions in which active TEs interfer with neighboring regions thereby changing 3D genome architecture. At the same time we aim at identifying the molecular pathology of duplications at the human FGF8 locus and unravel, why the mouse and the human phenotypes are so similar. This study will not only provide insight into the regulatory effect TEs might have and how they interfere with gene regulation, it will also tell us how similar phenotypes, in this case SHFM, can arise from different pathologies. Lessons learned from the Dac mutation and the human SHFM locus can be transferred to other human diseases advancing other studies into the causes of malformation in genetic disease with unknown cause and/or unusual inheritance.

Exploring the contribution of transcription to native chromatin looping and genome conformation

The precise spatiotemporal control of gene expression is critical for cell identity and cell homeostasis. We now understand that this control is exerted also via the three-dimensional organization of the genome. Thus, the converging and conflicting dynamic forces that act on chromosomes in order to regulate their architecture are of utmost importance in understanding gene regulation. A number of transcription factors and “structural“ proteins has been extensively studied for the contribution to genomic architecture. However, an enzyme that is essential for life and is a powerful molecular machine central to chromatin processes, the RNA polymerase, has not been analyzed in detail to date. It is, therefore, necessary to bridge this gap-of-knowledge and study the direct impact of the different RNA polymerases on chromatin dynamics and vice versa, to finally unveil the full 3D regulatory repertoire of mammalian cells. This requires generation of mammalian cell lines with specific, inducible, and reversible degradation of RNA polymerase II and/or III, and subsequently the implementation of our “native” chromosome conformation capture technology in conjunction with a novel and truly integrative pipeline for in silico analysis on the basis of unsupervised “deep” machine learning. We expect that this project will resolve the debate on the contribution of active transcription to chromatin looping, and lay the ground for identifying new paths via which mammalian genomes are organized and regulated in a spatiotemporal manner.

Cell-state specific 3D genome architecture in heterogeneous cell populations of the brain

Accurate levels of gene expression during development and in environmental responses are mediated through specific three-dimensional (3D) contacts between non-coding regulatory regions and their target genes. Defects in the spatial relationship of regulatory elements to their target genes are linked to disease, and are increasingly important to understand complex neurological disorders, often associated with deregulation of chromatin factors. However, efforts to study 4D genome architecture in the nervous system have been limited to the use of bulk tissues, cells differentiated in vitro or neurons dissociated from the brain. Thus, it remains a major challenge to dissect the 4D nucleome alterations that accompany activation processes in specific neurons, and how they relate with homeostatic responses during neuronal activation and in circadian processes.  In this proposal, we aim to understand the relationship between the 4D genome and gene expression in specific cells of the brain, and its deregulation in neurodevelopmental disorders associated with Shank3 mutations, which are risk factors for Autism Spectrum Disorders (ASD). Further to its synaptic roles, Shank3 shuttles to the nucleus where it modulates gene expression in response to neuronal activity. First, we will determine 3D genome topologies in specialized pyramidal neurons in the hippocampus of wildtype and mutant Shank3 mice, to investigate Shank3 genomic targets and mechanisms of action. Second, we will develop a novel technology to dissect quantitative relationships between 3D folding patterns and cellular levels of nuclear factors, an essential step towards finer dissection of 3D genome folding mechanisms in heterogeneous tissues such as the brain.

Dissecting the role of chromosome genome organization in chromosome fragility and the formation of leukemia driving translocations

Processes that traverse DNA are associated with dramatic changes in DNA topology. These changes occur in the context of the chromatin and must be coordinated with processes that shape chromosome organization per se, such as the formation of chromatin loops and the higher-order chromosome structure. Accumulating topological stress is dissipated at strategic genomic locations, such as promoters and chromatin loop boundaries, by the action of enzymes called topoisomerases. Type II topoisomerases (TOP2), incise both strands of DNA to release supercoiling, unknot, and decatenate DNA. Abortive TOP2 actions induced by the presence of a widely used class of chemotherapeutics, called topoisomerase poisons, however, stabilize TOP2s on DNA, promoting chromosome breakage within recurrent fragile sites that often form chromosome fusions that drive secondary, therapyrelated leukemias. The contribution of cellular processes in triggering the conversion of stabilized TOP2 to DNA breaks that promote the formation of oncogenic translocations remains elusive.  Driven by our preliminary results, we propose that transcription and processes that shape chromosome folding are essential contributors to chromosome fragility that form oncogenic fusions. Our aim, therefore, is to directly assess the effect of the 3D chromosome organization to two different aspects of the formation of recurrent fusions: (1) the role of chromatin loop dynamics to chromosome fragility at recurrent translocation hot spots and (2) the role of spatial genome organization to the chromosome-end synapsis, a crucial step of the translocation process that dictates the choice of partners. We will directly assess the effects of chromatin loop dynamics to TOP2-induced genomic instability by using state-of-the-art next-generation sequencing approaches to profile DNA breaks across the genome, in cellular systems allowing the formation or elimination of chromatin loops, in a controlled fashion. We will also directly evaluate a causal relationship of the 3D nuclear architecture with translocation formation by combining high-throughput imaging methodologies we have developed to probe chromosome breakage, synapsis and fusions, with precision genome editing to perturb gene activity and chromosome structure at single cell resolution. We anticipate that our proposed work will set 3D chromatin folding as a major contributor to the cellular aetiology of recurrent oncogenic fusions.

Identification of functional genetic variants affecting allele-specific chromatin conformation using Genome Architecture Mapping

The identification of the effects of somatic and germline genetic variation is one of the most pressing challenges in genomics with far-reaching implications for health and disease. Traditional approaches have focused on the genome-wide association of variants with changes in gene expression (GWAS), which have revolutionised our understanding of human disease. Despite their success, GWAS approaches suffer from two major drawbacks. Firstly, while the effects of some genetic variants might directly be visible in expression levels, in many cases functionality of these variants is determined by intermediate phenotypes, such as epigenetic modifications and chromatin interactions, which are frequently ignored. Secondly, GWAS approaches are only feasible for common variants with a relatively high recurrence of more than 1% minor allele frequency in the population. In cancer, which is characterised by ubiquitous somatic variation with low to no recurrence, these approaches must rely on aggregation of somatic mutations in genomic windows, which severely limits detection power.  

The aim of this proposal is to develop new computational approaches for identifying functional genetic variation that affects chromatin conformation and promoter – enhancer interactions. To overcome sample noise and increase detection power we propose to derive allele-specific chromatin contact maps from Genome Architecture Mapping (GAM), a novel ligationfree genome-wide experimental technique developed by our collaboration partner Ana Pombo at the MDC. We will use GAM for the reconstruction of parental haplotypes to derive haplotype-specific contact maps and develop machine learning algorithms that predict differential chromatin contacts from their sequence context.  

This novel predictor will in turn be used to predict the effects of somatic genetic variants from the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium, in which we are currently investigating genometranscriptome interactions. Using these predicted chromatin contact changes as an intermediate phenotype, we will derive a novel way of associating multiple somatic variants in their sequence context with changes in allele-specific expression, thereby overcoming the limitations of traditional GWAS approaches in somatic association studies. This work will shed new light on the effects of somatic genetic variation on chromatin state, allow us to identify functional somatic regulatory variants and predict new regulatory drivers of cancer onset and progression.

Spatial organization of transcribed genes in mammalian cells

Knowledge pertaining to the fine molecular mechanisms of transcriptional activation and regulation, including co- and post-transcriptional processes, is rapidly expanding. Similarly, the importance of global genomic arrangement, illustrated by the spatial segregation of transcriptionally active euchromatin from inactive heterochromatin, is widely appreciated and studied. However, our knowledge regarding an intermediate level of transcriptional organization, relating to the spatial arrangement of individual transcribed genes, is surprisingly limited. 

This proposal describes my recent discovery of the phenomenon of Transcription Loops (TLs), structures formed by highly expressed and strongly decondensed genes with RNA polymerase II complexes moving along the gene axis. The phenomenon has been observed with two long genes, thyroglobulin and titin, expressed in thyrocytes and muscle cells, respectively. TLs dynamically change neighboring chromosomal loci by separating flanking sequences, modifying the structure of harboring chromosome territories and protruding into the nuclear space due to their intrinsic rigidity. I speculate that this rigidity is caused by a dense decoration of the gene axis with multiple elongating polymerases with attached voluminous nascent RNPs. The preliminary data obtained so far leads me to hypothesize that TL formation is one of the universal principles of eukaryotic gene expression that has not been appreciated until now due to the resolution limits of light microscopy and/or due to the low expression of studied genes.

To reinforce and demonstrate the generality of the above conclusions derived from the study of two genes, I plan to (i) search for more examples of TLs; (ii) experimentally inhibit or induce TLs of long genes; (iii) experimentally truncate long genes or elongate short genes thereby converting them into irresolvable or resolvable TLs, respectively; (iv) obtain more information about the fine structure of TLs on both microscopic and chromatin levels; and (v) elucidate the mechanisms of TL formation by building a polymer model based on parameters inferred from microscopy and Hi-C maps. In addition, since high thyroglobulin expression is essential for thyroid physiology and the phenomenon of the thyroglobulin TL is potentially important for medicine, I plan to further investigate the thyroglobulin TLs, performing an evolutionary study of the gene in other vertebrate groups and examining the possible regulation of thyroglobulin expression by T3/T4 hormones levels and mRNA regulation through intron retention. 

The project will (i) provide new insights into the spatial organization of expressed genes, (ii) elucidate the role of transcription in shaping the nuclear architecture, and (iii) present a new model for the spatial dynamics of transcription in eukaryotes.

Position effects in the 3D genome as the cause of neurodevelopmental disorders

Structural variants (SVs) have the potential to disrupt the complex threedimensional (3D) chromatin organization of the genome. This may lead to the repositioning of topologically associating domains (TAD) boundaries and the relocation of enhancer elements in other compartments causing misexpression and disease. So far most cases of TAD-disruption described are associated with extremely rare skeletal phenotypes or cancers. The role of spatial genome architecture in more common phenotypes such as intellectual disability or neurodevelopmental disorders remains poorly understood. Considering that neurodevelopmental disorders are very frequent with a prevalence of up to 1% and often caused by SVs makes it is important to study this disease group in context of the 3D position effects.  The overall aim of the proposed project is to investigate the role of SVs that cause position effects in the 3D genome as a new pathomechanism for neurodevelopmental disorders.  However, several key technical and analytical challenges hinder progress in studying the role of non-coding variation and TAD-disruption in neurodevelopmental disorders: affected tissues and cells of patients are usually not available for functional studies and very few animal models exits since neurological phenotypes are much harder to investigate in animal models. Current pehnotypoing technologies lack the throughput and resolution to obtain a global view of the dynamic molecular processes underway in the diverse and rapidly expanding populations and subpopulations of neurons during development.  To addressed these technical challenges and limitations we propose three experimental approaches:   Work package 1: We aim to generate CRISPR/Ca9 based mouse models of SVs that change the spatial genome architecture and cause neurodevelopmental disorders. We will investigate inter-TAD-SVs that cause autosomal dominant demyelinating leukodystrophy and intra-TADs-deletions associated with severe autism.  Work package 2: We aim to use chromosome conformation capture technologies in mouse models and patients cells to evaluate the effect of SVs causing neurodevelopmental disorders in the 3D genome. We will study inter-TAD-SVs and intra-TADs-deletions.  Work package 3: We aim to use single cell-RNA-seq as a phenotyping tool for mouse models of neurodevelopmental disorders to identify previously overlooked cell type specific phenotypes and misexpression. Sc-RNA-seq provides the necessary resolution to investigate transcriptional changes, molecular states, and trajectories of neuronal cells during development and disease.  Our study will contribute to a better understanding of genotype-phenotype correlations of 3D position effects associated with neurodevelopmental disorders. We also will create an important scientific resources for the study of these very rare diseases, which will ultimately result in better family counselling and patient care.

Functional Characterization and Heterogeneity of the DLBCL and AML 3D Genomes

Chromatin conformation constitutes a fundamental level of eukaryotic genome regulation. However, our ability to examine its biological function and role in disease has been hindered by the large amounts of starting material required to perform current experimental approaches, limiting the examination in primary diseased tissues. As a result, we currently lack an understanding of how chromatin conformation is affected in diseased cells. Recently, we have developed a low input Hi-C method suitable for the direct examination of chromatin architecture in diseased tissue from patients, and we have demonstrated its applicability in proof-of-principle examinations of chromatin conformation in B-cells from a diffuse large B-cell lymphoma (DLBCL) patient. These experiments have resulted in interesting and valuable observations such as: i) the unbiased characterisation of patientspecific structural variation across the genome; ii) the detection of large regions with loss of heterozygosity; and, iii) unexpectedly, a global trend for the appearance of new conformational features, such as TADs, in the disease sample. In this proposal, we now aim to build on these experimental and computational methods to discover the fundamental principles and functional consequence of chromatin organisation changes in blood malignancies. In Aim 1, we will produce genome-wide chromatin conformation maps for a small cohort of DLBCL and acute myeloid leukaemia (AML) patients and a set of healthy donors as a control. These datasets will allow us to characterise the chromatin conformation heterogeneity between these samples and to identify recurrent changes in chromatin organisation in disease. In Aim 2, we will develop a computational analysis framework for the systematic and statistical analysis of chromatin architecture differences between any set of samples. These novel methods will allow us to perform an unbiased ranking of the chromatin conformation differences between disease and control samples as well as to automatically identify structural variation events. In Aim 3, we will generate functional data examining the transcriptional state of these samples, their level of chromatin accessibility and the binding state of architectural binding proteins, such as CTCF. This will allow us to evaluate the functional role of changes in chromatin conformation across samples and to pinpoint the molecular mechanisms driving these. Overall, this integrated, multidisciplinary approach will result in a deep characterisation of the changes in chromatin organisation in these malignancies and their functional implication in gene expression. In addition, our results will provide fundamental insight into how and where aberrations in chromatin architecture occur in disease, which will be an invaluable asset in future to devise personalised therapeutic intervention strategies.

From fibers to the sea of nuclesomes: Computer simulations of the regulation of the spatial structure of Mbp chromatin domains in the nucleus

In the nucleus of eukaryotes DNA is wrapped around histone proteins forming cylindrical units called nucleosomes. Nucleosomes are connected by linker DNA and form a bead-on-a-string like structure termed chromatin. In vitro chromatin adopts a fiber-like structure with a diameter of 30 nm while in general in vivo no such structure is observed. In the proposed research we will apply coarse-grained computer simulations of chromatin to elucidate conditions and properties of this folding, its regulation and the consequences of the changes. Linker DNA and the nucleosomes are modelled by cylindrical units interacting by elastic and electrostatic forces. Metropolis Monte Carlo and replica exchange methods are used to sample an ensemble of configurations in equilibrium. The conditions of high nucleosome density as in the nucleus will be mimicked in the simulations by modelling long nucleosome chains in the range of 1.5 Mbp and larger applying periodic boundary conditions. The spacing of the nucleosomes will be based on synthetic and real nucleosome positions from experiments. Fundamental spatial properties of the folding of higher order models will be extracted from these simulations. The model will be extended by potentials describing the loop-forming proteins cohesin and CTCF. The analysis of data from simulations will elucidate the distribution of contact probabilities as observed e.g. in chromosome conformation capture techniques and explain changes of the spatial structure by nucleosome repositioning and/or CTCF/cohesin depletion connected with gene regulation. Data from ATACSeq and Hi-C from healthy and malign cells provides information about transcription factor binding, chromatin accessibility and spatial structure. We will combine these data in models of large domains of chromatin, so called TADS, to elucidate cis- and trans-regulation mechanisms of genes and the deregulation of genes in malign cells.