Regulation and readout of mammalian DNA methylation

The mesmerizing phenotypical and functional diversity of mammalian cell types is to a large extent attributed to epigenetic signals that work together with the DNA sequence to determine gene expression programs. DNA methylation is one of the most important types of epigenetic signals and its paramount role was recognized in early genetic studies. Still, even after decades of active research, a comprehensive understanding of the mechanisms that regulate the chromatin targeting and activity of DNA methyltransferases has not been achieved. In this work, three main directions of research were undertaken, with the ultimate goal of shedding mechanistic and methodological insights into the generation and maintenance of DNA methylation patterns. In the first project of this thesis, a combination of biochemical and cellular experiments was used to assess the cellular role of the putative chromatin remodeler HELLS, an essential cofactor of DNA methyltransferases. By employing chromatin fractionation assays and microscopy-based techniques, I could show that the ATPase activity of HELLS is necessary for the high nuclear mobility of the protein and its ability to get released from compacted chromatin sites. In addition, the H3K9me3 pathway was also found to play an important role in the exchange of HELLS at heterochromatin. Taken together, this work provides the first evidence for a role of ATP hydrolysis in the association between HELLS and chromatin and hints at a model where the fast exchange of HELLS at repetitive DNA sequences might enhance the local recruitment of epigenetic enzymes, such as DNA methyltransferases (DNMTs). This could subsequently lead to the local stabilization of silencing complexes at heterochromatin. In the second project of this thesis, the putative interaction between the de novo DNA methyltransferase DNMT3A and the 5mC-reading protein MeCP2 was addressed. By building on previous data from our laboratory, which documented a direct interaction between the TRD domain of MeCP2 and the ADD domain of DNMT3A, causing an inhibition of DNMT3A activity in vitro, I could show that these proteins also interact in the mouse brain and the inhibitory effect of this interaction is also observed in stable cells lines overexpressing MeCP2. Furthermore, by using conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, I could elucidate the mechanism of the inhibition of DNMT3A by MeCP2. Accordingly, I found that MeCP2 stabilizes an allosterically closed conformation of DNMT3A, an effect that could be successfully relieved by addition of unmodified histone H3. These results were supported by whole genome bisulfite brain methylome analysis of a Mecp2 knockout mouse model. Collectively, the findings derived from this project offer unprecedented insights into the regulation of DNMT3A activity and propose a model in which the enzyme is under the combined control of MeCP2 and H3 tail modifications. Accordingly, depending on the modification status of the H3 tail at target sites, MeCP2 can act as either a repressor or activator of DNA methylation. Finally, in the third project of this thesis, the focus was placed on the development and application of a novel method that would enable for the first time the locus-specific visualization of epigenetic modifications in living mammalian cells. This urgent and unmet technological need was solved by developing a set of modular fluorescence complementation-based epigenetic biosensors for live cell microscopy applications. In these tools, the high DNA sequence specificity of engineered anchor proteins such as ZFs, TALEs, and CRISPR/Cas9 proteins, was combined with the great versatility of chromatin reading domains as natural detector modules of DNA methylation and histone 3 lysine 9 trimethylation. With this approach, I could detect both of these marks for the first time, at defined, endogenous DNA sequences in different mouse and human cell lines. Furthermore, I could follow the changes in the levels of these epigenetic modifications with locus-specific resolution after treatment with epigenetic inhibitors or the induction of epigenetic enzymes. It is anticipated that either in their present form or in combination with the ongoing developments in genomic targeting and microscopy technologies, these tools will greatly improve our understanding of how specific epigenetic signals, like DNA methylation, are set, erased and maintained during embryonic development or onset of disease. Taken together, the results of this doctoral thesis demonstrate how a synergistic use of biochemical and cellular methods allows to derive deep insights into the epigenetic signaling network centered around the regulation of mammalian DNA methylation

Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing target epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset

Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2

Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3( DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tailmodifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation

An ATPase-deficient variant of the SNF2 family member HELLS shows altered dynamics at pericentromeric Heterochromatin

The HELLS (helicase, lymphoid specific, also known as lymphoid-specific helicase) protein is related to the SNF2 (sucrose non-fermentable 2) family of chromatin remodeling ATPases. It is required for efficient DNA methylation in mammals, particularly at heterochromatin-located repetitive sequences. In this study, we investigated the interaction of HELLS with chromatin and used an ATPase-deficient HELLS variant to address the role of ATP hydrolysis in this process. Chromatin fractionation experiments demonstrated that, in the absence of the ATPase activity, HELLS is retained at the nuclear matrix compartment, defined in part by lamin B1. Microscopy studies revealed a stronger association of the ATPase-deficient mutant with heterochromatin. These results were further supported by fluorescence recovery after photobleaching measurements, which showed that, at heterochromatic sites, wild-type HELLS is very dynamic, with a recovery half-time of 0.8 s and a mobile protein fraction of 61%. In contrast, the ATPase-deficient mutant displayed 4.5-s recovery half-time and a reduction in the mobile fraction to 30%. We also present evidence suggesting that, in addition to the ATPase activity, a functional H3K9me3 signaling pathway contributes to an efficient release of HELLS from pericentromeric chromatin. Overall, our results show that a functional ATPase activity is not required for the recruitment of HELLS to heterochromatin, but it is important for the release of the enzyme from these sites

Numerical models development for unidirectional air flow diffusers with lobed and circular orifices

In order to achieve more realistic boundary conditions on the inlet of a ventilation system it is necessary to study the influences of the air diffuser orifices geometry on the airflow distribution in the enclosure. Integrating these orifices directly in a real scale air diffuser for a numerical study will result in a huge computational grid which will translate in huge computational resources and a much larger calculation time. The solution, in this case, was the numerical simulation of the airflow through small parts of the studied air diffuser. Later, the numerical results will be implemented as boundary conditions in the unidirectional diffuser of a numerical simulation that represents a real scale operating room (OR). In the current study two diffusers with different orifices were studied, one having circular („O”) and the other one lobbed („+”) orifices. The initial numerical model had 25 orifices on the diffuser, but because of the very large numerical grid resulted for the initial meshes (>35 million tetrahedral cells), a solution with only 4 orifices was chosen for this study. A mesh independency study was made for these two types of air diffusers. The numerical studies were made using RANS method, with SST k-ω turbulence model in steady state conditions. The numerical results obtained with the first step models showed very good agreement with the PIV stereoscopic experimental measurements

Indoor Air Pollution with Fine Particles and Implications for Workers’ Health in Dental Offices: A Brief Review

(1) Background: Indoor air pollution can affect the well-being and health of humans. Sources of indoor pollution with particulate matter (PM) are outdoor particles and indoor causes, such as construction materials, the use of cleaning products, air fresheners, heating, cooking, and smoking activities. In 2017, according to the Global Burden of Disease study, 1.6 million people died prematurely because of indoor air pollution. The health effects of outdoor exposure to PM have been the subject of both research and regulatory action, and indoor exposure to fine particles is gaining more and more attention as a potential source of adverse health effects. Moreover, in critical situations such as the current pandemic crisis, to protect the health of the population, patients, and staff in all areas of society (particularly in indoor environments, where there are vulnerable groups, such as people who have pre-existing lung conditions, patients, elderly people, and healthcare professionals such as dental practitioners), there is an urgent need to improve long- and short-term health. Exposure to aerosols and splatter contaminated with bacteria, viruses, and blood produced during dental procedures performed on patients rarely leads to the transmission of infectious agents between patients and dental health care staff if infection prevention procedures are strictly followed. On the other hand, in the current circumstances of the pandemic crisis, dental practitioners could have an occupational risk of acquiring coronavirus disease as they may treat asymptomatic and minimally symptomatic patients. Consequently, an increased risk of SARS-CoV-2 infection could occur in dental offices, both for staff that provide dental healthcare and for other patients, considering that many dental procedures produce droplets and dental aerosols, which carry an infectious virus such as SARS-CoV-2. (2) Types of studies reviewed and applied methodology: The current work provides a critical review and evaluation, as well as perspectives concerning previous studies on health risks of indoor exposure to PM in dental offices. The authors reviewed representative dental medicine literature focused on sources of indoor PM10 and PM2.5 (particles for which the aerodynamic diameter size is respectively less than 10 and 2.5 μm) in indoor spaces (paying specific attention to dental offices) and their characteristics and toxicological effects in indoor microenvironments. The authors also reviewed representative studies on relations between the indoor air quality and harmful effects, as well as studies on possible indoor viral infections acquired through airborne and droplet transmission. The method employed for the research illustrated in the current paper involved a desk study of documents and records relating to occupational health problems among dental health care providers. In this way, it obtained background information on both the main potential hazards in dentistry and infection risks from aerosol transmission within dental offices. Reviewing this kind of information, especially that relating to bioaerosols, is critical for minimizing the risk to dental staff and patients, particularly when new recommendations for COVID-19 risk reduction for the dental health professional community and patients attending dental clinics are strongly needed. (3) Results: The investigated studies and reports obtained from the medical literature showed that, even if there are a wide number of studies on indoor human exposure to fine particles and health effects, more deep research and specific studies on indoor air pollution with fine particles and implications for workers’ health in dental offices are needed. As dental practices are at a higher risk for hazardous indoor air because of exposure to chemicals and microbes, the occupational exposures and diseases must be addressed, with special attention being paid to the dental staff. The literature also documents that exposure to fine particles in dental offices can be minimized by putting prevention into practice (personal protection barriers such as masks, gloves, and safety eyeglasses) and also keeping indoor air clean (e.g., high-volume evacuation, the use of an air-room-cleaning system with high-efficiency particulate filters, and regularly maintaining the air-conditioning and ventilation systems). These kinds of considerations are extremely important as the impact of indoor pollution on human health is no longer an individual issue, with its connections representing a future part of sustainability which is currently being redefined. These kinds of considerations are extremely important, and the authors believe that a better situation in dentistry needs to be developed, with researchers in materials and dental health trying to understand and explain the impact of indoor pollution on human health

Analysis of the substrate specificity of the SMYD2 protein lysine methyltransferase and discovery of novel non-histone substrates

The SMYD2 protein lysine methyltransferase methylates various histone and non-histone proteins and is overexpressed in several cancers. Using peptide arrays, we investigated the substrate specificity of the enzyme, revealing a recognition of leucine (or weaker phenylalanine) at the -1 peptide site and disfavor of acidic residues at the +1 to +3 sites. Using this motif, novel SMYD2 peptide substrates were identified, leading to the discovery of 32 novel peptide substrates with a validated target site. Among them, 19 were previously reported to be methylated at the target lysine in human cells, strongly suggesting that SMYD2 is the protein lysine methyltransferase responsible for this activity. Methylation of some of the novel peptide substrates was tested at the protein level, leading to the identification of 14 novel protein substrates of SMYD2, six of which were more strongly methylated than p53, the best SMYD2 substrate described so far. The novel SMYD2 substrate proteins are involved in diverse biological processes such as chromatin regulation, transcription, and intracellular signaling. The results of our study provide a fundament for future investigations into the role of this important enzyme in normal development and cancer

Indoor Air Pollution with Fine Particles and Implications for Workers’ Health in Dental Offices: A Brief Review

(1) Background: Indoor air pollution can affect the well-being and health of humans. Sources of indoor pollution with particulate matter (PM) are outdoor particles and indoor causes, such as construction materials, the use of cleaning products, air fresheners, heating, cooking, and smoking activities. In 2017, according to the Global Burden of Disease study, 1.6 million people died prematurely because of indoor air pollution. The health effects of outdoor exposure to PM have been the subject of both research and regulatory action, and indoor exposure to fine particles is gaining more and more attention as a potential source of adverse health effects. Moreover, in critical situations such as the current pandemic crisis, to protect the health of the population, patients, and staff in all areas of society (particularly in indoor environments, where there are vulnerable groups, such as people who have pre-existing lung conditions, patients, elderly people, and healthcare professionals such as dental practitioners), there is an urgent need to improve long- and short-term health. Exposure to aerosols and splatter contaminated with bacteria, viruses, and blood produced during dental procedures performed on patients rarely leads to the transmission of infectious agents between patients and dental health care staff if infection prevention procedures are strictly followed. On the other hand, in the current circumstances of the pandemic crisis, dental practitioners could have an occupational risk of acquiring coronavirus disease as they may treat asymptomatic and minimally symptomatic patients. Consequently, an increased risk of SARS-CoV-2 infection could occur in dental offices, both for staff that provide dental healthcare and for other patients, considering that many dental procedures produce droplets and dental aerosols, which carry an infectious virus such as SARS-CoV-2. (2) Types of studies reviewed and applied methodology: The current work provides a critical review and evaluation, as well as perspectives concerning previous studies on health risks of indoor exposure to PM in dental offices. The authors reviewed representative dental medicine literature focused on sources of indoor PM10 and PM2.5 (particles for which the aerodynamic diameter size is respectively less than 10 and 2.5 μm) in indoor spaces (paying specific attention to dental offices) and their characteristics and toxicological effects in indoor microenvironments. The authors also reviewed representative studies on relations between the indoor air quality and harmful effects, as well as studies on possible indoor viral infections acquired through airborne and droplet transmission. The method employed for the research illustrated in the current paper involved a desk study of documents and records relating to occupational health problems among dental health care providers. In this way, it obtained background information on both the main potential hazards in dentistry and infection risks from aerosol transmission within dental offices. Reviewing this kind of information, especially that relating to bioaerosols, is critical for minimizing the risk to dental staff and patients, particularly when new recommendations for COVID-19 risk reduction for the dental health professional community and patients attending dental clinics are strongly needed. (3) Results: The investigated studies and reports obtained from the medical literature showed that, even if there are a wide number of studies on indoor human exposure to fine particles and health effects, more deep research and specific studies on indoor air pollution with fine particles and implications for workers’ health in dental offices are needed. As dental practices are at a higher risk for hazardous indoor air because of exposure to chemicals and microbes, the occupational exposures and diseases must be addressed, with special attention being paid to the dental staff. The literature also documents that exposure to fine particles in dental offices can be minimized by putting prevention into practice (personal protection barriers such as masks, gloves, and safety eyeglasses) and also keeping indoor air clean (e.g., high-volume evacuation, the use of an air-room-cleaning system with high-efficiency particulate filters, and regularly maintaining the air-conditioning and ventilation systems). These kinds of considerations are extremely important as the impact of indoor pollution on human health is no longer an individual issue, with its connections representing a future part of sustainability which is currently being redefined. These kinds of considerations are extremely important, and the authors believe that a better situation in dentistry needs to be developed, with researchers in materials and dental health trying to understand and explain the impact of indoor pollution on human health

Experimental investigation of thermal vehicular environment during the summer season

International audienceThermal comfort evaluation for vehicle occupants is very complicated due to the transient nature and non-uniformity of the vehicle interior. The thermal sensation of an automobile occupant is affected by the surrounding environment. Furthermore, the existing standard was developed for steady state and controlled conditions and it utilizes three evaluation indices, some of which are not adapted for this complex environment. In this article, the three standardized indices are compared in terms of thermal comfort, for a passenger vehicle in summer season. The results show that the mean values of the PMV/PPD model calculated at a single point with Comfort Sense equipment are far from the TSV mean values which were collected in questionnaires, while the t(eq) index which was calculated with an advanced thermal manikin are closer to the TSV comfort votes. This may be explained by the fact that the TSV and t(eq) consider the sensation for each body part at the local level. For a correct evaluation of the thermal comfort in non-uniform and transient environments like in vehicles, it is not enough to measure a single point