Targeting and dynamics of gene repression during stem cell differentiation

The identity and function of different cellular subtypes critically depend on their unique set of expressed genes. Gene expression programs and their changes during development are mainly controlled by sequence-specific DNA binding factors. It has recently become clear that chromatin modifications are important regulators of these processes. While there are several chromatin-based pathways that correlate with gene repression, their exact role in silencing remains elusive. Moreover, for many repressive chromatin modifications a complete picture of the genomic distribution and its dynamics during development is lacking. Finally, it is still unclear how these genomic patterns of repressive chromatin marks are established. For my PhD work, I set out to address these questions by studying the targeting of H3K9me2 and DNA methylation during cellular differentiation. Our analysis revealed that H3K9me2 is highly abundant in embryonic stem cells and occurs in large domains that occupy more than half of the genome. H3K9me2 marks chromatin outside of transcribed, active or polycomb regulated sites, possibly keeping it in a repressed state. Importantly, abundance of H3K9me2 increases only slightly during neuronal differentiation, with a localized gain occurring at gene bodies of transcribed genes. By gene expression profiling we further show that the transcriptome complexity is very similar in stem cells and derived post-mitotic neurons. These data are in contrast to a previously suggested model which states that the pluripotent state of stem cells is accompanied by a global reduction in heterochromatin and a concomitant higher proportion of transcription. Together with results from other groups our data rather indicate that repressive chromatin is abundant in stem cells and upon differentiation gets redistributed only locally and not globally. It has been suggested that such a localized increase of repression at gene regulatory regions helps stabilizing lineage choices and differentiation processes. In order to investigate how chromatin-based repression pathways are targeted to gene regulatory sites, we focused on DNA methylation, a modification whose catalysis and epigenetic propagation are well understood. By site-specific sequence integration experiments we show that 1 kb promoter elements are sufficient to recapitulate endogenous DNA methylation patterns in stem cells and their dynamic changes upon differentiation, in a process that is independent of transcription. In stem cells, promoters are protected from DNA methylation by small sequence elements that we termed methylation determining regions (MDRs). Protection from DNA methylation by MDRs depends on a combination of DNA binding motifs, which get recognized by transcription factors such as RFX2. It has been speculated before that establishment of an unmethylated promoter state is facilitated by proteins that recognize unmethylated CpGs. While not excluding a role in maintenance, our data suggest that CpG-richness alone is not sufficient for initiation of this chromatin state. Remarkably, no additional sequence besides an MDR is needed to recapitulate differentiation-induced de novo methylation. Moreover, MDRs are able to protect neighboring sequences from DNA methylation in stem cells and from de novo methylation during differentiation. These results imply that one possible way of differentiation-induced de novo methylation could involve reduced binding of factors that protect from DNA methylation. In summary, H3K9me2 and DNA methylation occupy per default most the genome, even in cells with a high developmental potential. Accordingly, cellular differentiation is accompanied by focal, rather than global changes in repressive chromatin modifications. In the case of DNA methylation, such local changes at gene regulatory sites are determined by the underlying sequence and likely involve binding of transcription factors that protect from DNA methylation

SIMULATION-BASED EVALUATION OF RESERVATION MECHANISMS FOR THE TIME WINDOW ROUTING METHOD

Automated warehouses operated by a fleet of robots offer great flexibility, since fleet size can be adjusted easily to throughput requirements. Furthermore, they provide higher redundancy compared to common solutions for automated storage and retrieval systems.On the other hand, these systems require more complex control strategies to run robustly and efficiently. Special routing and deadlock handling strategies are necessary to avoid blocking and collisions among the robots.In this contribution, we focus on the time window routing method, an approach for avoiding deadlocks byreserving routes in advance. We present and discussdifferent reservation mechanisms that are evaluated bythe means of simulation.Automated warehouses operated by a fleet of robots offer great flexibility, since fleet size can be adjusted easily to throughput requirements. Furthermore, they provide higher redundancy compared to common solutions for automated storage and retrieval systems. On the other hand, these systems require more complex control strategies to run robustly and efficiently. Special routing and deadlock handling strategies are necessary to avoid blocking and collisions among the robots. In this contribution, we focus on the time window routing method, an approach for avoiding deadlocks by reserving routes in advance. We present and discuss different reservation mechanisms that are evaluated by the means of simulation

Methylation-Dependent and -Independent Genomic Targeting Principles of the MBD Protein Family

SummaryTo gain insight into the cellular readout of DNA methylation, we established a strategy for systematically profiling the genome-wide distribution of chromatin-interacting factors. This enabled us to create genomic maps for the methyl-CpG-binding domain (MBD) family of proteins, including disease-relevant mutants, deletions, and isoforms. In vivo binding of MBD proteins occurs predominantly as a linear function of local methylation density, requiring functional MBD domains and methyl-CPGs. This interaction directs specificity of MBD proteins to methylated, CpG-dense, and inactive regulatory regions. In contrast, binding to unmethylated sites varies between MBD proteins and is mediated via alternative domains or protein-protein interactions. Such targeting is exemplified by NuRD-complex-mediated tethering of MBD2 to a subset of unmethylated, active regulatory regions. Interestingly, MBD3 also occupies these sites, but like MBD2, binding is independent of the presence of hydroxymethylation. These functional binding maps reveal methylation-dependent and -independent binding modes and revise current models of DNA methylation readout through MBD proteins

Product ban versus risk management by setting emission and technology requirements: The effect of different regulatory schemes taking the use of trichloroethylene in Sweden and Germany as an example

This report highlights the opportunities inherent in smart regulatory measures to effectively reduce risks related to hazardous substance emissions and exposure, and underscores the danger of simplistic and ineffective policy. The example of different regulatory approaches used in Germany and Sweden to regulate the use of trichloroethylene was taken as the basis for the study. During the 1990s, due to environmental, health and safety considerations, the use of trichloroethylene in Europe was a subject of broad concern. As a consequence, the use of trichloroethylene became regulated through multiple approaches, such as labelling, handling regulations and performance standards. Since that time the absolute emissions of trichloroethylene in Europe have been decreasing consistently in all member states. These results were achieved by various regulatory measures governing the use of trichloroethylene in industrial applications that have been introduced by individual Member States. However, given the implementation responsibility at Member State level not all member States have implemented the same set of regulatory measures. In Germany, for example, the use of trichloroethylene is regulated through strict technical standards for equipment and emissions that has required companies to replace existing old machines with the state-of-the-art equipment. In Sweden a general ban on trichloroethylene use was introduced in 1996, which however eventually evolved into an exemption permit system for companies that found no alternative to degreasing with trichloroethylene. --

Two- and three-input TALE-based AND logic computation in embryonic stem cells

Biological computing circuits can enhance our ability to control cellular functions and have potential applications in tissue engineering and medical treatments. Transcriptional activator-like effectors (TALEs) represent attractive components of synthetic gene regulatory circuits, as they can be designed de novo to target a given DNA sequence. We here demonstrate that TALEs can perform Boolean logic computation in mammalian cells. Using a split-intein protein-splicing strategy, we show that a functional TALE can be reconstituted from two inactive parts, thus generating two-input AND logic computation. We further demonstrate three-piece intein splicing in mammalian cells and use it to perform three-input AND computation. Using methods for random as well as targeted insertion of these relatively large genetic circuits, we show that TALE-based logic circuits are functional when integrated into the genome of mouse embryonic stem cells. Comparing construct variants in the same genomic context, we modulated the strength of the TALE-responsive promoter to improve the output of these circuits. Our work establishes split TALEs as a tool for building logic computation with the potential of controlling expression of endogenous genes or transgenes in response to a combination of cellular signals

The changing epidemiology of human monkeypox-A potential threat? A systematic review

Monkeypox, a zoonotic disease caused by an orthopoxvirus, results in a smallpox-like disease in humans. Since monkeypox in humans was initially diagnosed in 1970 in the Democratic Republic of the Congo (DRC), it has spread to other regions of Africa (primarily West and Central), and cases outside Africa have emerged in recent years. We conducted a systematic review of peer-reviewed and grey literature on how monkeypox epidemiology has evolved, with particular emphasis on the number of confirmed, probable, and/or possible cases, age at presentation, mortality, and geographical spread. The review is registered with PROSPERO (CRD42020208269). We identified 48 peer-reviewed articles and 18 grey literature sources for data extraction. The number of human monkeypox cases has been on the rise since the 1970s, with the most dramatic increases occurring in the DRC. The median age at presentation has increased from 4 (1970s) to 21 years (2010-2019). There was an overall case fatality rate of 8.7%, with a significant difference between clades-Central African 10.6% (95% CI: 8.4%- 13.3%) vs. West African 3.6% (95% CI: 1.7%- 6.8%). Since 2003, import- and travel-related spread outside of Africa has occasionally resulted in outbreaks. Interactions/activities with infected animals or individuals are risk behaviors associated with acquiring monkeypox. Our review shows an escalation of monkeypox cases, especially in the highly endemic DRC, a spread to other countries, and a growing median age from young children to young adults. These findings may be related to the cessation of smallpox vaccination, which provided some cross-protection against monkeypox, leading to increased human-to-human transmission. The appearance of outbreaks beyond Africa highlights the global relevance of the disease. Increased surveillance and detection of monkeypox cases are essential tools for understanding the continuously changing epidemiology of this resurging disease

Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates