Uncovering the mechanisms and information content of CpG-resolved DNA methylation programming during hematopoietic differentiation

DNA methylome remodeling is an essential molecular mechanism underlying all stages of hematopoietic differentiation. However, current datasets only cover a fraction of the genome and are often limited to specific hematopoietic cell types. A comprehensive, genome-wide atlas of the DNA methylation dynamics during hematopoietic differentiation is still missing. Preliminary evidence suggests that the single-cell landscape of the hematopoietic stem and progenitor cell (HSPC) compartment is characterized by a structured continuum of epigenetically-defined cell states. Significant advances in charting this epigenetic state manifold have recently been achieved for the chromatin accessibility and histone modification layers. However, despite its potential importance, the landscape of single-cell DNA methylome states in the HSPC compartment remains largely unexplored. This project aimed to comprehensively map the genome-wide DNA methylation dynamics during hematopoietic differentiation and leverage this atlas as a reference to analyze the single-cell DNA methylome landscape in the HSPC compartment and among mature hematopoietic cells. The functional importance and rich information content of differentially methylated regions (DMRs) are well-established. However, the DNA methylation layer inherently possesses the capability to encode information at CpG resolution. The role and extent of differentially methylated CpG (DMCpG) programming within DMR regions is largely unexplored. This project therefore aimed to evaluate the role and mechanisms of DMCpG programming during hematopoietic differentiation. Using high-coverage tagmentation-based whole-genome bisulfite sequencing data for 25 hematopoietic populations, I have compiled a genome-wide, dual-layer DMR/DMCpG atlas, which maps, annotates, and integrates DMR and DMCpG programming during hematopoietic differentiation. Loss of stemness was associated with lineage-independent gain of DNA methylation, while lineage specification was accompanied by hierarchical DNA methylation dynamics, characterized by unidirectional loss of DNA methylation. Different DMCpGs within focal DMR intervals were often distinctly programmed and thus contained heterogeneous information content. In particular, most of the DMRs were seeded and progressively expanded through subsequent programming of specific DMCpGs at different stages of differentiation. Mature hematopoietic cells exhibited systematic seed DMCpG hypomethylation in DMRs associated with alternative cell fates. This seed hypomethylation likely represents epigenetic memory of alternative fate explorations in progenitor cells. Collectively, these findings suggest a hierarchical model of DNA methylation programming, in which information is encoded through DMR programming and through DMCpG programming within DMR regions. This model represents a significant extension of the commonly accepted paradigm of regional DNA methylation programming. Using the dual-layer DMR/DMCpG atlas as a reference, single-cell methylome states for 312 HSPCs, as well as for a total of 136 mature B cells, T cells, CFU-Es, and monocytes, could be dissected with high resolution. The HSPC compartment was characterized by a structured continuum of single-cell DNA methylome states. Multiple lines of evidence suggested that differentiation starts from apex HSCs possessing a lineage-naive DNA methylome state. Exit from the apex HSC state was initiated by balanced, multi-lineage DMR seeding. This early DMR programming was strictly restricted to specific DMR seeding regions, which often comprised only one or two DMCpGs. This contrasts with the conventional paradigm that functionally relevant DMRs always contain at least several DMCpGs. Further differentiation within the HSPC compartment was accompanied by continuous, gradually more lineage-specific accumulation of hypomethylation, leading to progressive DMR expansion. The dual-layer DMR/DMCpG atlas provides an essential resource for studying the epigenetic regulation of the hematopoietic differentiation process and serves as a valuable reference for the analysis of single-cell bisulfite sequencing data. This work highlights the highly-resolved, progressive, and stable nature of DNA methylome remodeling during hematopoietic differentiation and reveals several aspects of the structure and information content of the DNA methylome layer which go beyond the currently accepted paradigms. It appears likely that the DNA methylome remodeling mechanisms active in other differentiation systems and related processes, such as tumor evolution, share the same principles of hierarchical DNA methylation programming with CpG resolution. However, in many systems, the information content of the DNA methylome may be convoluted by a combination of this programming mechanism and other programming mechanisms characterized by stochastic regional accumulation of DNA methylation alterations. The analysis strategies presented in this work provide a basis for the further development of computational methods capable of dissecting the rich but complex information content of the DNA methylome with high resolution

Construction and commissioning of a collinear laser spectroscopy setup at TRIGA Mainz and laser spectroscopy of magnesium isotopes at ISOLDE (CERN) (07.10.2010)

Collinear laser spectroscopy has been used as a tool for nuclear physics for more than 30 years. The unique possibility to extract nuclear properties like spins, radii and nuclear moments in a model-independent manner leads to important physics results to test the predictive power of existing nuclear models. rnThis work presents the construction and the commissioning of a new collinear laser spectroscopy experiment TRIGA-LASER as a part of the TRIGA-SPEC facility at the TRIGA research reactor at the University of Mainz. The goal of the experiment is to study the nuclear structure of radioactive isotopes which will be produced by neutron-induced fission near the reactor core and transported to an ion source by a gas jet system. rnThe versatility of the collinear laser spectroscopy technique will be exploited in the second part of this thesis. The nuclear spin and the magnetic moment of the neutron-deficient isotope Mg-21 will be presented, which were measured by the detection of the beta-decay asymmetry induced by nuclear polarization after optical pumping. A combination of this detection method with the classical fluorescence detection is then used to determine the isotope shifts of the neutron-rich magnesium isotopes from Mg-24 through Mg-32 to study the transition to the ''island of inversion''.Kollineare Laserspektroskopie ist seit über 30 Jahren ein wichtiges Instrument für die Untersuchung von Kerneigenschaften. Die einzigartige Möglichkeit, Kernstrukturgrößen wie Spins, Radien und Kernmomente modellunabhängig zu extrahieren, gibt den gewonnenen Daten ein besonders großes Gewicht beim Test der Vorhersagekraft existierender Kernmodelle. rnIn dieser Arbeit werden der Aufbau und Testmessungen eines neuen Experiments für kollineare Laserspektroskopie TRIGA-LASER am TRIGA Forschungsreaktor der Universität Mainz vorgestellt. TRIGA-LASER ist eines von zwei Experimentzweigen der TRIGA-SPEC Anlage. Ziel des Laserexperiments ist die Untersuchung von radioaktiven Isotopen, welche durch neutroneninduzierte Spaltung nahe des Reaktorkerns produziert und durch ein Gas-Jet Transportsystem zur Ionequelle geführt werden. rnDie Vielseitigkeit der von uns angewendeten experimentellen Methode wird im zweiten Teil dieser Arbeit ausgenutzt. Wir präsentieren den Kernspin und das magnetische Moment des neutronenarmen Isotops Mg-21, welche wir durch den Nachweis der beta-Zerfalls-Asymmetrie nach optischem Pumpen bestimmt haben. Die Kombination dieser Nachweismethode mit dem klassischen optischen Nachweis wird dann verwendet, um die Isotopieverschiebungen der neutronenreichen Kerne von Mg-24 bis Mg-32 zu bestimmen. Dies dient der Untersuchung der ''island of Inversion''