The Architecture And Dynamics Of Gene Regulatory Networks Directing Cell-Fate Choice During Murine Hematopoiesis

Mammals produce hundreds of billions of new blood cells every day througha process known as hematopoiesis. Hematopoiesis starts with stem cells that develop into all the different types of cells found in blood by changing their genome-wide gene expression. The remodeling of genome-wide gene expression can be primarily attributed to a special class of proteins called transcription factors (TFs) that can activate or repress other genes, including genes encoding TFs. TFs and their targets therefore form recurrent networks called gene regulatory networks (GRNs). GRNs are crucial during physiological developmental processes, such as hematopoiesis, while abnormalities in the regulatory interactions of GRNs can be detrimental to the organisms. To this day we do not know all the key compo-nents that comprise hematopoietic GRNs or the complete set of their regulatory interactions. Inference of GRNs directly from genetic experiments is low throughput and labor intensive, while computational inference of comprehensive GRNs is challenging due to high processing times. This dissertation focuses on deriving the architecture and the dynamics of hematopoietic GRNs from genome-wide gene expression data obtained from high-resolution time-series experiments. The dissertation also aims to address the technical challenge of speeding up the process of GRN inference. Here GRNs are inferred and modeled using gene circuits, a data-driven method based on Ordinary Differential Equations (ODEs). In gene circuits, the rate of change of a gene product depends on regulatory influences from other genes encoded as a set of parameters that are inferred from time-series data. A twelve-gene GRN comprising genes encoding key TFs and cytokine receptors involved in erythrocyte-neutrophil differentiation was inferred from a high-resolution time-series dataset of the in vitro differentiation of a multipotential cell line. The inferred GRN architecture agreed with prior empirical evidence and pre- dicted novel regulatory interactions. The inferred GRN model was also able to predict the outcome of perturbation experiments, suggesting an accurate inference of GRN architecture. The dynamics of the inferred GRN suggested an alternative explanation to the currently accepted sequence of regulatory events during neutrophil differentiation. The analysis of the model implied that two TFs, C/EBPα and Gfi1, initiate cell-fate choice in the neutrophil lineage, while PU.1, believed to be a master regulator of all white-blood cells, is activated only later. This inference was confirmed in a single-cell RNA-Seq dataset from mouse bone marrow, in which PU.1 upregulation was preceded by C/EBPα and Gfi1 upregulation. This dissertation also presents an analysis of a high-temporal resolution genome-wide gene expression dataset of in vitro macrophage-neutrophil differentiation. Analysis of these data reveal that genome-wide gene expression during differentiation is highly dynamic and complex. A large-scale transition is observed around 8h and shown to be related to wide-spread physiological remodeling of the cells. The genes associated by myeloid differentiation mainly change during the first 4 hours, implying that the cell-fate decision takes place in the first four hours of differentiation. The dissertation also presents a new classification-based model-training technique that addresses the challenge of the high computational cost of inferring GRNs. This method, called Fast Inference of Gene Regulation (FIGR), is demonstrated to be two orders magnitude faster than global non-linear optimization techniques and its computational complexity scales much better with GRN size. This work has demonstrated the feasibility of simulating relatively large realistic GRNs using a dynamical and mechanistically accurate model coupled to high-resolution time series data and that such models can yield novel biological insight. Taken together with the macrophage-neutrophil dataset and the computationally efficient GRN inference methodology, this work should open up new avenues for modeling more comprehensive GRNs in hematopoiesis and the broader field of developmental biology

Design Principles Of Mammalian Transcriptional Regulation

Transcriptional regulation occurs via changes to different biochemical steps of transcription, but it remains unclear which steps are subject to change upon biological perturbation. Single cell studies have revealed that transcription occurs in discontinuous bursts, suggesting that features of such bursts like burst fraction (what fraction of time a gene spends transcribing RNA) and burst intensity could be points of transcriptional regulation. Both how such features might be regulated and the prevalence of such modes of regulation are unclear. I first used a synthetic transcription factor to increase enhancer-promoter contact at the β -globin locus. Increasing promoter- enhancer contact specifically modulated the burst fraction of β -globin in both immortalized mouse and primary human erythroid cells. This finding raised the question of how generally important the phenomenon of burst fraction regulation might be, compared to other modes of regulation. For example, biochemical studies have suggested that stimuli predominantly affect the rate of RNA polymerase II (Pol II) binding and the rate of Pol II release from promoter-proximal pausing, but the prevalence of these modes of regulation compared to changes in bursting had not been examined. I combined Pol II ChIP-seq and single cell transcriptional measurements to reveal that an independently regulated burst initiation step is required before polymerase binding can occur, and that the change in burst fraction produced by increased enhancer-promoter contact was caused by an increased burst initiation rate. Using a number of global and targeted transcriptional regulatory perturbations, I showed that biological perturbations regulated both burst initiation and polymerase pause release rates, but seemed not to regulate polymerase binding rate. Our results suggest that transcriptional regulation primarily acts by changing the rates of burst initiation and polymerase pause release

Exploiting natural and induced genetic variation to study hematopoiesis

PUZZLING WITH DNA Blood cell formation can be studied by making use of natural genetic variation across mouse strains. There are, for example, two mouse strains that do not only differ in fur color, but also in average life span and more specifically in the number of blood-forming stem cells in their bone marrow. The cause of these differences can be found in the DNA of these mice. This DNA differs slightly between the two mouse strains, making some genes in one strain just a bit more or less active compared to those same genes in the other strain. The aim of part I of this thesis was to study the influence of genetic variation on gene expression and how this might explain the specific characteristics of the mouse strains. One of the findings in this study was that the influence of genetic variation on gene expression is strongly cell-type-dependent. Additionally, blood cell formation can be studied by introducing genetic variation into the system. In part II of this thesis genetic variation was introduced into mouse blood-forming stem cells by letting random DNA sequences or “barcodes” integrate into the DNA of these cells. Thereby, these cells were provided with a unique and identifiable label that was heritable from mother- to daughter cell. In this manner the fate of blood-forming stem cells and their progeny could be tracked following transplantation in mice. This technique is very promising for monitoring blood cell formation in future clinical gene therapy studies in humans. PUZZELEN MET DNA Bloedvorming kan bestudeerd worden door gebruik te maken van natuurlijke genetische variatie tussen muizenstammen. Zo bestaan er bijvoorbeeld twee muizenstammen die niet alleen verschillen in vachtkleur, maar ook in gemiddelde levensduur en meer specifiek in het aantal bloedvormende stamcellen dat zich in hun beenmerg bevindt. De oorzaak van deze verschillen kan gevonden worden in het DNA van deze muizen. Dat DNA verschilt net iets tussen de twee muizenstammen, waardoor sommige genen in de ene stam actiever of juist minder actief zijn dan diezelfde genen in de andere stam. In deel I van dit proefschrift is onderzocht hoe genetische variatie de expressie van genen beïnvloedt en hoe dit de specifieke eigenschappen van de muizenstammen zou kunnen verklaren. Er is onder andere gevonden dat de invloed van genetische variatie op de expressie van genen sterk celtype-afhankelijk is. Daarnaast kan bloedvorming bestudeerd worden door genetische variatie te introduceren in het systeem. In deel II van dit proefschrift is genetische variatie in bloedvormende stamcellen van muizen geïntroduceerd door random DNA volgordes of “barcodes” te laten integreren in het DNA van deze cellen. Dit resulteert erin dat elke cel voorzien wordt van een uniek label dat overgegeven wordt van moeder- op dochtercel. De DNA volgorde van het label kan gelezen worden met behulp van een zogenaamde sequencing techniek. Op deze manier kan het lot van bloedvormende stamcellen en hun nakomelingen gevolgd worden na transplantatie in muizen. Deze techniek is zeer veelbelovend voor het monitoren van bloedvorming in toekomstige klinische gentherapie studies in de mens.

Developmental dynamics of transcription and genome architecture

Abstract Regulation of gene expression is necessary for the control of complex developmental processes. The genome has to shape in specific conformations to fit inside the nucleus and to tether specific regulatory elements to their target genes. Although the linear composition of many genomes is largely known, their three dimensional (3D) organization and dynamics are largely unknown. Hence, in order to unravel gene regulation, it is necessary to understand the chromatin structure and organization. Furthermore, developmental procedures are controlled by complex combinatorial transcription factor (TF) networks. Hence, unveiling those networks will provide a better insight towards understanding those developmental procedures. The work described in this thesis aims to study the genome conformation/interactome and their effect on gene regulation and to unveil the role of transcription factor proteins (TFs) in complex developmental processes

Early fate decisions in hematopoietic stem and progenitor cells. Through the lens of genomic and functional assays.

Hematopoietic stem cells (HSCs) are rare cells on top of the differentiation hierarchy of hematopoiesis. HSCs are unique in their combined capacity to differentiate into all mature blood lineages and self-renew to maintain the HSC pool. Based on classical models of hematopoiesis in mouse, the self-renewal potential of HSCs is gradually and step-wise lost during the transition from long term (LT)-HSCs to multipotent progenitors (MPPs) accompanied by upregulated expression of the cell-surface marker FMS-like tyrosine kinase 3 (Flt3). The Flt3+ multipotent progenitors serve as developmental intermediates for hematopoietic lineage priming. Notably, the 25% highest Flt3+ cells, known as lymphoid-primed MPPs (LMPPs), have been defined as restricted, lymphoid primed cells with decreased megakaryocyte and erythroid (MegE) priming. However, recent single cell RNA sequencing (scRNA-seq) studies question the step-wise model of HSC differentiation and instead suggest a continuum model of the early hematopoietic hierarchy, where the first differentiation events occur in a low-primed cloud of HSPCs without sharply defined gene expression programs. In this model, no transition of different lineages from MPPs with intermediate gene expression occur, instead these progenitors are largely comprised of uni-lineage-primed cells. The overall aim of this thesis is to investigate how cellular-fate options emerge in the cloud of hematopoietic stem/progenitor cells (HSPCs), at what stage the multipotency gives way to lineage priming, and how this stage can be detected. For this aim, single-cell (sc) chromatin accessibility (ATAC-seq), scRNA-seq and sc-qPCR analysis were employed extensively to identify the transition of HSCs to lineage restricted multipotent progenitor cells and functionally validated using in vivo and in vitro assay.In paper I, scATAC-seq was used to map the accessibility of 571 transcription factor (TF)-binding motifs as a measure of lineage priming along the Flt3 differentiation axis. The resulting data identified a transition point of highly lineage-primed cells within the continuum of HSPCs where self-renewal and multipotency was lost and lineage commitment initiated. This transition point is characterized by down-regulation of CD9 and up-regulation of Flt3 cell surface expression. Within the Flt3 intermediate population (Flt3int), LSKFlt3intCD9high cells display co-incidental stem and multi-lineage primed chromatin states while the downstream LSKFlt3intCD9low contain an LMPP-like program. Also, this priming seems to initiate in the epigenome without being starkly reflected in the transcriptome. In order to validate the genomic data from the aforementioned analysis, we established in vitro culture systems to functionally examine the differentiation fates of cells at a clonal level (Paper II). The result confirms that LSKFlt3intCD9high cells generated more multilineage progeny compared to clones within the LSKFlt3intCD9low fraction. It has been shown extensive changes in heterogeneity of human hematopoietic cells with age. For example increase HSCs frequency and myeloid output while lymphoid output is decreased. However human immunophenotypic changes associated with aging have received little attention. To this end we in paper III examined CD9 cell surface expression in correlation with molecular programs and functional features of human HSPCs throughout life and in leukemia. Interestingly, only a small fraction of HSPCs expressed CD9 in neonatal hematopoiesis and in young adult bone marrow while CD9 expression substantially increased during situations of myeloid and megakaryocytic biased hematopoiesis, such as during ageing or in chronic myeloid leukemia (CML). Thus, CD9 represents an HSC marker for myeloid-biased hematopoiesis

Dynamic Modeling of the JAK2/STAT5 Signal Transduction Pathway to Dissect the Specific Roles of Negative Feedback Regulators

Erythropoietin (Epo) acts as the key regulator of red blood cell development in mammals. During erythropoiesis, Epo initiates the JAK2/STAT5 signal transduction pathway that elicits pro-survival signals in erythroid progenitor cells. Therefore, the tight regulation of JAK2/STAT5 signaling is crucial for the fine-tuned balance of erythrocyte production. Recently, several factors regulating Epo-induced JAK2/STAT5 signaling have been identified. However, their relative contribution in controlling the dynamic behavior of JAK2/STAT5 signaling is poorly understood. To elucidate the specific roles of these negative regulators in attenuating the pathway, data-based mathematical modeling was employed. In this study, standardized protocols were established facilitating the generation of quantitative time-resolved data of Epo-induced JAK2/STAT5 pathway activation in primary erythroid progenitor cells and the hematopoietic cell line BaF3-EpoR, which is a frequently used model system to study EpoR signaling. For the fine-tuned overexpression of negative regulators in hematopoietic cells, an inducible Tet-On retroviral vector system was developed. Systematic comparison of stoichiometries and activation dynamics of Epo-induced JAK2/STAT5 signaling in CFU-E and BaF3-EpoR cells revealed fundamental differences between both cell types, emphasizing the importance of the use of primary cells in the investigation of EpoR signaling. Genome-wide expression profiling identified potential feedback regulators of Epo-induced JAK2/STAT5 signaling in CFU-E cells. To dissect the complex roles of negative regulators that employ different mechanisms to attenuate JAK2/STAT5 signaling, a data-based dynamic pathway model was established. Calibration of the mathematical model was performed using multiple experimental data sets of Epo-induced JAK2/STAT5 signaling monitored under different conditions. The estimated parameters were fully identifiable and displayed small confidence intervals, which are required for accurate simulations. Comprehensive model analysis identified the rapid recruitment of the phosphatase SHP-1 as major mechanism controlling the early-phase kinetics of pathway activation, while the two transcriptionally induced regulators SOCS3 and CIS were elucidated as modulators of the STAT5 steady-state phosphorylation level. Furthermore, global sensitivity analysis uncovered the concentrations of SHP-1 and JAK2 as well as the parameter SOCS3 expression as critical to control the integral signal strength of nuclear phosphorylated STAT5, which is proportionally linked to the survival of erythroid progenitor cells. In conclusion, by combining mathematical modeling with experimental data, the crucial regulators enabling the tight control of Epo-induced JAK2/STAT5 signaling were elucidated. The detailed understanding of the molecular processes and regulatory mechanisms of Epo-induced signaling during normal erythropoiesis can be further exploited to gain insights into alterations promoting erythroleukemia and related malignant hematopoietic diseases

Engineering of 3D mesenchymal tissues for bone regeneration and hematopoiesis modeling

The bone organ has two main functions in the adult. a) It provides mechanical support and protects organs, while b) the bone marrow hosts hematopoiesis, a process ensuring lifelong production and renewal of the blood tissue. Therefore, the general aim of my thesis is to engineer mesenchymal tissues able to support bone healing and bone marrow functions. Their recapitulation in vitro by using primary hMSCs (or mesenchymal cell lines) could: (i) help fulfilling a clinical need in bone regenerative medicine by providing engineered off-the-shelf coated extracellular matrix (ECM) with tunable compositions, and (ii) provide an animal-free model to study bone biology and hematology in health and disease. These aims are addressed through a combination of hMSCs and 3D perfusion bioreactor systems

Development of a bio-inspired in silico-in vitro platform: towards personalised healthcare through optimisation of a bone-marrow mimicry bioreactor

Human red blood cell production, or erythropoiesis, occurs within bone marrow. Living animal and human cadaver models have demonstrated the marrow production of red blood cells is a spatially-complex process, where cells replicate, mature, and migrate between distinct niches defined by biochemical nutrient access, supportive neighboring cells, and environmental structure. Unfortunately, current research in understanding normal and abnormal human production of blood takes place in petri dishes and t-flasks as 2D liquid suspension cultures, neglecting the role of the marrow environment for blood production. The culture of blood on marrow-mimetic 3D biomaterials has been used as a laboratory model of physiological blood production, but lacks characterization. In this work, a 3D biomaterial platform is developed and to capture the in vivo blood production process and manufacture red blood cells from human umbilical cord blood. First ceramic hollow fibres were designed and tested to be incorporated and perfused in a 3D porous scaffold bioreactor to mimic marrow structure, provide a better expansion of cell numbers, a better diffusion of nutrients, and allow for the continuous, non-invasive harvest of small cells in comparison to static, unperfused biomaterials. Quantitative 3D image analysis tools were developed to spatially assess bioreactor distributions and associations of and between different cell types. Using these tools, the bioreactor distribution of red blood cell production were characterized within niches in collaboration with supportive, non-blood cell types and designed miniaturised, parallelised mini-bioreactors to further explore bioreactor capabilities. This thesis presents a hollow fibre bioreactor able to produce blood cells alongside supportive cells at 1,000-fold higher cell densities with 10-fold fewer supplemented factor than flask cultures, without serum, with one cell source, and continuously harvest enucleate red blood cell product to provide a physiologically-relevant model for cell expansion protocols.Open Acces

CHO microRNA engineering is growing up : recent successes and future challenges

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines