Deciphering Effects of Idh1-R132H Mutations on the Regulation of Hematopoietic Differentiation

The hematopoietic system is a highly versatile regenerative tissue, in which hematopoietic stem cells drive the life-long production of multiple mature blood cell types. During hematopoietic differentiation, the regulation of genome-wide epigenetic patterns of histone modification or DNA methylation marks is an essential process orchestrating cell identities, lineage decisions and developmental cell fates. In acute myeloid leukemia, mutations frequently affect direct and indirect epigenetic regulators and modifiers such as isocitrate dehydrogenase 1 (IDH1) or DNA methyltransferase 3 alpha (DNMT3A), and result in disturbed epigenetic landscapes and differentiation patterns. Here, IDH1 mutations promote oncogenic transformation through the de novo production of the metabolite D2-hydroxyglutarate, which induces a genome and epigenome instability by inhibiting multiple histone and DNA demethylases. Yet, molecular details of how IDH1 mutations alter characteristics of individual hematopoietic cell types remain poorly understood. In the course of this thesis, combinatorial mouse models carrying specific Idh1-R132H and DNMT3A-R882H mutations, which frequently co-occur in acute myeloid leukemia patients, were extensively characterized. By integrating phenotypic readouts in combination with latest advances in high-throughput single-cell RNA-sequencing approaches, cooperativity and impact of these mutations on individual cell types of the hematopoietic system were delineated and gene regulatory networks which are altered upon the expression of an Idh1-R132H or a DNMT3A-R882H mutation were identified. At a phenotypic level, neither an Idh1-R132H mutation alone nor in combination with a DNMT3A-R882H mutation resulted in the development of myeloid malignancies, suggesting a restricted oncogenic potential of these mutations and additional intrinsic or extrinsic factors to be required for further malignant transformation. However, Idh1-R132H mutated hematopoietic stem cells displayed increased engraftment and reconstitution potential during serial transplantations and featured aberrant expression of genes associated with DNA damage and DNA repair. Furthermore, both Idh1-R132H single-mutant and Idh1-R132H DNMT3A-R882H double-mutant mice displayed aberrant differentiation patterns predominantly affecting the myelo-monocytic lineage, culminating in a favored monocytic cell fate and increased monocyte and monocyte progenitor counts in the bone marrow. By employing a multi-layered single-cell transcriptome analysis of nearly all cell types within the hematopoietic compartment, differentiation trajectories from hematopoietic stem cells towards mature differentiated cells were reconstructed and underlying molecular defects characterized. Pseudotime-inferred myeloid lineage trajectories revealed an aberrant lineage specification in particular for Idh1-R132H DNMT3A-R882H double-mutated myeloid progenitor cells, resembling a differentiation arrest at the stage of common myeloid progenitors and an ineffective hematopoietic differentiation as seen in myelodysplastic syndromes. At the molecular level, this aberrant population was characterized by an altered metabolic signature and elevated Myc signaling, which is involved in the regulation of terminal myeloid differentiation. Importantly, we could correlate this transcriptome-defined population to a surface marker-defined population, allowing the prospective isolation of these cells for further investigation. Independent of a DNMT3A-R882H mutation, the expression of an Idh1-R132H mutation resulted in the deregulation of several key regulatory factors which either orchestrate monocyte and macrophage development or their activation upon inflammatory stimuli. In line with this, monocyte progenitor cells displayed elevated interferon signaling levels, suggesting that a proinflammatory environment is a common characteristic of an Idh1-R132H mutated hematopoietic compartment and could contribute to leukemic transformation upon additional events. In summary, the experimental framework presented in this thesis enhanced our understanding of how IDH1-R132H mutations alone or in combination with a DNMT3A-R882H mutation in patients synergistically drive leukemia initiation and progression. The identified molecular characteristics will be of benefit in designing treatment strategies for patients carrying IDH1-R132H and DNMT3A-R882H mutations and can be used as a resource when studying these mutations in the context of altered physiological conditions and upon additional extrinsic stimuli

Differential IRF8 Transcription Factor Requirement Defines Two Pathways of Dendritic Cell Development in Humans

The formation of mammalian dendritic cells (DCs) is controlled by multiple hematopoietic transcription factors, including IRF8. Loss of IRF8 exerts a differential effect on DC subsets, including plasmacytoid DCs (pDCs) and the classical DC lineages cDC1 and cDC2. In humans, cDC2-related subsets have been described including AXL+ SIGLEC6+ pre-DC, DC2 and DC3. The origin of this heterogeneity is unknown. Using highdimensional analysis, in vitro differentiation, and an allelic series of human IRF8 deficiency, we demonstrated that cDC2 (CD1c+ DC) heterogeneity originates from two distinct pathways of development. The lymphoidprimed IRF8hi pathway, marked by CD123 and BTLA, carried pDC, cDC1, and DC2 trajectories, while the common myeloid IRF8lo pathway, expressing SIRPA, formed DC3s and monocytes. We traced distinct trajectories through the granulocyte-macrophage progenitor (GMP) compartment showing that AXL+ SIGLEC6+ pre-DCs mapped exclusively to the DC2 pathway. In keeping with their lower requirement for IRF8, DC3s expand to replace DC2s in human partial IRF8 deficiency

Mapping the transcriptional landscape of haematopoietic stem and progenitor cells

Maintenance of the blood system requires balanced cell-fate decisions of haematopoietic stem and progenitor cells (HSPCs). Individual haematopoietic stem cells (HSCs) decide between self-renewal and differentiation and can generate all mature cell types. Cell-fate decisions are made at the single-cell level and are governed by regulatory networks. Dysregulation in this balanced process could lead to serious blood disorders such as leukaemia; therefore, it is important to understand how individual cells make these cell-fate decisions. To investigate HSPC populations, 1,654 cells were profiled by single-cell RNA-sequencing. Index sorting made it possible to sort HSPCs using broad sorting gates and retrospectively assign them to common HSPC populations, retaining all information about specific functionally pure populations while also capturing any intermediate cells normally excluded by conventional gating. Reconstruction of differentiation trajectories revealed dynamic expression changes associated with early lineage differentiation from HSCs. This transcriptional atlas of HSPC differentiation was further used to identify candidate genes for a CRISPR screen investigating genes implicated in HSC biology. These candidate gene perturbations were interrogated for changes in the expression of the HSC marker EPCR, as well as changes in apoptosis and lineage output. Transcription factors play a key role in regulating cell-fate decisions and operate within organized regulatory programs. To study relationships between transcription factors in HSPC populations, qRT-PCR was used to profile the expression of 41 genes, including 31 transcription factors, in HSPCs at the single-cell level. This approach confirmed known aspects of haematopoiesis and made deeper investigation of HSPC heterogeneity possible. Regulatory networks were reconstructed using Boolean network inference models and recapitulated differentiation of HSCs towards megakaryocyte–erythrocyte progenitors and lymphoid-primed multipotent progenitors. By comparing these two models, a rule specific to the megakaryocyte-erythrocyte progenitor network was identified, in which GATA2 positively regulated Nfe2 and Cbfa2t3h. This was subsequently validated using transcription factor binding profiles and in vitro luciferase assays using a model cell line. Overall, the work presented in this thesis confirmed known aspects of HSPC biology using single-cell gene expression analysis and demonstrated how in silico approaches can be used to guide in vitro and in vivo investigations. In addition, the single-cell RNA-sequencing data was developed into an intuitive web interface that can be used to visualise the gene expression for any gene of choice at single-cell resolution across the HSPC atlas, providing a powerful resource for the haematopoietic community.My funding for the CIMR 4 year programme was provided by the Medical Research Council (MRC)

Micromodel Study on Colloid Retention and Mobilization under different Geo-Chemical Conditions during Single and Two-Phase Flow

Understanding the transport of colloids and colloid-facilitated transport of contaminants is essential for efficient cleanup and remediation processes. Various factors and mechanisms contributing to their retention in the porous media have been studied indirectly through laboratory column breakthrough analysis and directly using visualization studies. Micromodels are analogs to porous media that allow the real-time visualization of pore-scale processes that occur at highly controllable physio-chemical conditions in the laboratory scale. In this thesis, we used a glass micromodel with representative geometry to observe the pore-scale mechanisms during colloid retention and mobilization experiments in a saturated and unsaturated porous media. The focus of this research was to investigate the colloid retention mechanisms under different physio-chemical conditions such as variable colloid type, solution ionic strength, and solution pH. Various colloid retention sites in unsaturated porous media were identified from the captured images and videos during drainage (using CO2 gas ) in a saturated micromodel. Quantitative analysis of colloid mobilization was performed using image-processing algorithms on a Representative Elementary Area (REA) image of the micromodel before and after drainage. This study also investigated colloid mobilization from AWI during imbibition in porous media. The impact of colloid hydrophobicity on mobilization was observed in a micromodel. The visual findings explained with the theoretical conceptualization of the forces acting on a colloid at AWSI. The colloid reattachment on SWI found during the dissolution of the gas bubble for hydrophilic colloids due to their greater capillary potential. Whereas, the lifting-capillary forces on hydrophobic colloids resulted in aggregation of excess colloids on AWI. This study also examined the retention and release of colloids under the influence of perturbations in flow rate and solution chemistry. The retention of three different types of colloids (i.e., favorable, unfavorable, and medium favorable conditions) was observed visually in a micromodel. The pore-scale visualizations reveal the impact of colloid deposition profile on colloid release with an increase in flow rate and solution pH as well as a decrease in solution ionic strength. The results from this study show the dependence of favorability of interaction conditions on colloid deposition profile as well as the colloid release during hydro-chemical perturbations in saturated porous media. This dissertation accompanied by supplementary material showing video images of the illustrated processe

Dissecting the development of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are an immune subset specialized in the production of Type I Interferons (IFNs). Conventional dendritic cells (cDCs) originate mostly from a common dendritic cell progenitor (CDP), whereas pDCs have been shown to develop from both CDPs as well as common lymphoid progenitors (CLPs). In contrast to the current literature, we here show that pDCs mostly differentiate from an IL-7R expressing lymphoid progenitor. IL-7R+ progenitors can be subdivided into three distinct subsets based on the expression of SiglecH and Ly6D: double negative (DN), Ly6D+ single positive (SP) and double positive (DP) progenitors. Each of these subsets identifies a specific developmental stage along the pDC lineage, where commitment by IL-7R+ progenitors is achieved upon expression of Ly6D and SiglecH (DP pre-pDCs). Further, RNA sequencing analysis of IL-7R+ lymphoid progenitor subsets revealed the transcriptional landscape of pDC development along the lymphoid branch, where high expression of the transcription factor IRF8 marks pDC commitment and anticipates the increase of TCF4 levels. The transcriptional signature of DP pre-pDCs correlates with the lineage potential assessed in vitro, in which DP pre-pDCs are fully committed to the pDC lineage. Moreover, single cell RNA sequencing on bone marrow and splenic pDCs revealed pDC heterogeneity in both tissues and further supported the dual origin of pDC from myeloid and lymphoid precursors. While all pDCs have the potential to secrete Type I IFNs and have high expression levels of pDC-specific transcript, only myeloid-derived pDCs share with cDCs the capacity to process and present antigen, suggesting that functional specification is directly linked to developmental origin

Heterogeneous Chip Multiprocessor: Data Representation, Mixed-Signal Processing Tiles, and System Design

With the emergence of big data, the need for more computationally intensive processors that can handle the increased processing demand has risen. Conventional computing paradigms based on the Von Neumann model that separates computational and memory structures have become outdated and less efficient for this increased demand. As the speed and memory density of processors have increased significantly over the years, these models of computing, which rely on a constant stream of data between the processor and memory, see less gains due to finite bandwidth and latency. Moreover, in the presence of extreme scaling, these conventional systems, implemented in submicron integrated circuits, have become even more susceptible to process variability, static leakage current, and more. In this work, alternative paradigms, predicated on distributive processing with robust data representation and mixed-signal processing tiles, are explored for constructing more efficient and scalable computing systems in application specific integrated circuits (ASICs). The focus of this dissertation work has been on heterogeneous chip multi-processor (CMP) design and optimization across different levels of abstraction. On the level of data representation, a different modality of representation based on random pulse density modulation (RPDM) coding is explored for more efficient processing using stochastic computation. On the level of circuit description, mixed-signal integrated circuits that exploit charge-based computing for energy efficient fixed point arithmetic are designed. Consequently, 8 different chips that test and showcase these circuits were fabricated in submicron CMOS processes. Finally, on the architectural level of description, a compact instruction-set processor and controller that facilitates distributive computing on System-On-Chips (SoCs) is designed. In addition to this, a robust bufferless network architecture is designed with a network simulator, and I/O cells are designed for SoCs. The culmination of this thesis work has led to the design and fabrication of a heterogeneous chip multi- processor prototype comprised of over 12,000 VVM cores, warp/dewarp processors, cache, and additional processors, which can be applied towards energy efficient large-scale data processing

The Missouri Miner, September 13, 1995

https://scholarsmine.mst.edu/missouri_miner/3702/thumbnail.jp

The Role of Myc and AP-1 Transcription Factors in the Development and Function of the Dendritic Cell Lineage

Dendritic cells (DCs) orchestrate immune responses to foreign and self proteins by capturing, processing and presenting antigens to naïve CD4+ and CD8+ T cells in specialized regions of lymphoid organs. Consequently, DCs function in many disparate infectious settings, during which their activation can result in both pathogenic and protective responses. The diverse properties of DCs manifest through the actions of a limited set of lineage-specifying DNA-binding proteins called transcription factors (TFs). The precise molecular program controlled by these developmentally important TFs (such as Irf8 and Batf3) is largely unknown because most studies to date have been largely descriptive. We have identified bona fide targets of BATF3 and IRF8 by comparative microarray analysis and chromatin immunoprecipitation followed by sequencing (ChIP-Seq). These targets provided insights into the specialized processes carried out by DCs and may also represent future pathways for manipulation during the development of vaccines. We characterized the stage-specific actions of BATF3 in the development of CD8+ DCs, a critical initiator of cellular immunity to intracellular pathogens and tumors. Batf3 induction occurs after the expression of Irf8 in the precursor to classical DCs (pre-cDC), in which BATF3 acts subsequent to an Irf8-dependent lineage commitment step to induce the terminal maturation and survival of CD8+ DCs. Genomic regions bound by IRF8 and BATF3 were identified by ChIP-Seq, and notably, the proximal promoters of CD8+ DC-specific genes showed IRF8 and BATF3 co-occupancy. Furthermore, we evaluated IRF8 binding in BATF3-deficient cells to identify BATF3-independent targets. One such candidate was the Myc homolog Mycl1 (L-Myc). Analysis by quantitative real-time PCR (qPCR) of the hematopoietic compartment revealed that Mycl1 expression is restricted to pDCs and cDCs. To evaluate the significance of L-Myc activity in dendritic cells, we generated a new knock-in mouse model of the Mycl1 locus by replacing the first coding exon with an in-frame GFP cassette. Analysis of heterozygous mice (Mycl1+/gfp) revealed that Mycl1 expression is restricted to dendritic cells. Interestingly, induction of Mycl1 occurs at the CDP to pre-cDC transition concurrent with the loss of Myc (c-Myc) from DCs. Although dispensable for the development of DCs, L-Myc supports the growth and survival of dendritic cells. Moreover, following activation of DCs with pathogen associated molecular patterns (PAMPs) or activating cytokines, L-Myc protein levels either remained constant or increased, suggesting that growth-promoting circumstances are intricately linked to levels of L-Myc. Lastly, L-Myc deficient DCs primed antigen-specific responses poorly and were incapable of supporting the intracellular growth of Listeria monocytogenes. Collectively, these findings suggest that the switch from Myc to L-Myc expression represents a strategy of growth in the face of disparate inflammatory signals experienced during infections. L-Myc may therefore represent a therapeutic target for selective inhibition or augmentation of immune responses driven by dendritic cel

Doctor of Philosophy

dissertationIn recent years, a number of trends have started to emerge, both in microprocessor and application characteristics. As per Moore's law, the number of cores on chip will keep doubling every 18-24 months. International Technology Roadmap for Semiconductors (ITRS) reports that wires will continue to scale poorly, exacerbating the cost of on-chip communication. Cores will have to navigate an on-chip network to access data that may be scattered across many cache banks. The number of pins on the package, and hence available off-chip bandwidth, will at best increase at sublinear rate and at worst, stagnate. A number of disruptive memory technologies, e.g., phase change memory (PCM) have begun to emerge and will be integrated into the memory hierarchy sooner than later, leading to non-uniform memory access (NUMA) hierarchies. This will make the cost of accessing main memory even higher. In previous years, most of the focus has been on deciding the memory hierarchy level where data must be placed (L1 or L2 caches, main memory, disk, etc.). However, in modern and future generations, each level is getting bigger and its design is being subjected to a number of constraints (wire delays, power budget, etc.). It is becoming very important to make an intelligent decision about where data must be placed within a level. For example, in a large non-uniform access cache (NUCA), we must figure out the optimal bank. Similarly, in a multi-dual inline memory module (DIMM) non uniform memory access (NUMA) main memory, we must figure out the DIMM that is the optimal home for every data page. Studies have indicated that heterogeneous main memory hierarchies that incorporate multiple memory technologies are on the horizon. We must develop solutions for data management that take heterogeneity into account. For these memory organizations, we must again identify the appropriate home for data. In this dissertation, we attempt to verify the following thesis statement: "Can low-complexity hardware and OS mechanisms manage data placement within each memory hierarchy level to optimize metrics such as performance and/or throughput?" In this dissertation we argue for a hardware-software codesign approach to tackle the above mentioned problems at different levels of the memory hierarchy. The proposed methods utilize techniques like page coloring and shadow addresses and are able to handle a large number of problems ranging from managing wire-delays in large, shared NUCA caches to distributing shared capacity among different cores. We then examine data-placement issues in NUMA main memory for a many-core processor with a moderate number of on-chip memory controllers. Using codesign approaches, we achieve efficient data placement by modifying the operating system's (OS) page allocation algorithm for a wide variety of main memory architectures

REGULATION OF THE HEMATOPOIETIC SELF-RENEWAL AND LINEAGE CHOICE: ROLE OF PBX1 AND MICRO-RNAS

Hematopoiesis is a highly regulated process (Orkin and Zon, 2008). For a constant supply of short-lived terminally differentiated blood cells, and for rapid response to hematopoietic stresses, HSCs are endowed with the ability to continuously provide more mature progenitors while properly maintaining their pool size, without exhaustion, throughout life. This equilibrium is finely maintained on one hand by precisely balancing self-renewal and differentiation in HSCs, and on the other hand by regulating proliferation and differentiation also of their downstream progenitors, including lineage choice. Thus, understanding mechanisms of regulation of self-renewal versus differentiation of HSCs and downstream progenitors is a central issue in stem cell field, since tiny alteration of these mechanisms may lead to hematopoietic failure and disease. Pbx1 is a transcription factor that positively regulates post-natal HSC quiescence. Its absence in HSCs causes an excessive proliferation that leads to their exhaustion, indicating a profound self-renewal defect, and premature myeloid differentiation at the expenses of lymphoid differentiation (Ficara et al., 2008). However, the precise molecular mechanisms through which Pbx1 exerts its function in HSCs and its role in progenitor biology are two still unexplored issues. In particular, it is not known whether Pbx1 function is also mediated by micro-RNAs, crucial new players in the regulation of proliferation and differentiation in several tissues, including the hematopoietic system. Taking advantage of Pbx1 conditional KO mice, in this study, we demonstrate that Pbx1 functions as a brake on cell differentiation not only in HSCs but also in multi-potent and myeloid-restricted progenitors, to maintain progenitor reservoirs and lymphoid potential. In absence of Pbx1, both myeloid and lymphoid progenitors are able to differentiate into mature progeny but with higher efficiency and premature kinetic for the myeloid lineage and a decreased efficiency in the lymphoid (and erythroid) lineage. Pbx1 acts also upstream to lineage restricted progenitors, affecting lineage choice of multipotent progenitors (MPPs) by restraining myeloid differentiation and allowing lymphoid differentiation. Moreover, we show that in the absence of Pbx1 HSCs display an altered micro-RNA profile, which resembles the normal MPP profile, suggesting a role for miRNAs in maintaining HSC identity. Pbx1-null HSCs and MPPs show specific lists of DE miRNAs, with substantial overlaps with the normal HSC-to-MPP transition. Combining miRNA data with transcriptional profile data of the same populations (Ficara et al., 2008) allowed searching for miRNA predicted targets whose change in expression inversely correlates with those of mRNAs. This analysis, coupled with extensive bioinformatics studies (promoter analysis, co-targeting and GSEA) allowed selection of very few candidate miRNAs to be further studied for their specific role in maintaining HSC self-renewal, and their possible regulation by Pbx1. In addition to a list of Pbx1-dependent miRNAs in HSCs and MPPs, we also analyzes for the first time miRNAs characteristic of the normal transition from HSCs to the first MPP stage, which represent an important finding for understanding miRNAs physiologically involved at the apex of hematopoietic system. Overall, this analysis set the basis for the discovery of miRNAs involved in the regulation of self-renewal versus differentiation of HSCs