Dissecting RNA biology in hematopoietic stem cells: The long non-coding RNA Meg3 is dispensable for hematopoiesis and alternative polyadenylation orchestrates hematopoietic stem cell function

Hematopoietic stem cells (HSCs) reside at the top of a tightly regulated and hierarchically organized differentiation cascade. They are multipotent and able to self-renew, thereby ensuring life-long replenishment of mature blood cells. In-depth Omics analyses within the hematopoietic hierarchy, including analysis of HSCs and multipotent progenitor (MPP) cells, revealed not only differential expression of protein-coding genes, but also HSC-specific splicing variants and expression of long non-coding RNAs (lncRNAs) upon HSC commitment (Cabezas-Wallscheid et al., 2014; Klimmeck et al., 2014; Luo et al., 2015). Functional analyses revealed that non-coding RNAs and regulation of RNA biogenesis are crucial for HSC function and potency. In this thesis, the role of the lncRNA maternally expressed gene 3 (Meg3) in hematopoiesis is studied. In addition, I introduce the RNA regulatory mechanism alternative polyadenylation (APA) as a mechanism controlling HSC/MPP biology in homeostasis and during replicative stress response by extensive in silico, in vitro and in vivo analyses. Furthermore, I generated an inducible knockout mouse model, targeting the prominent APA regulator poly(A) binding protein 1 (Pabpn1). Thereby, I aimed to dissect the general role of lncRNAs and RNA regulatory mechanisms in hematopoiesis. Project 1: The role of the lncRNA Meg3 in HSCs The tumor suppressor lncRNA Meg3 is encoded in the imprinted Dlk1-Meg3 locus and expressed from the maternally inherited allele (Zhou et al., 2012). We and others found Meg3 to be highly and specifically expressed in the HSC compartment compared to MPP cells. In this thesis, I crossed an inducible Meg3 flox mouse model (Klibanski et al., unpublished) to MxCre mice, generating MxCre Meg3 mat flox/pat wt mice. Cre-induction deleted the maternal allele in the hematopoietic compartment and thereby completely abrogated the expression of Meg3 and its associated miRNA cluster in HSCs. Extensive in vivo and in vitro analyses of adult mice harboring a Meg3-deficient blood system surprisingly did not reveal any impairment of hematopoiesis or stem cell function. In addition, I performed serial transplantation assays to investigate the functional capacity of Meg3-deficient HSCs. Again, knockout cells did not exhibit altered blood contribution, even upon tertiary transplantation. Imprinting of the Dlk1- Meg3 locus has recently been reported to regulate fetal liver HSC function (Qian et al., 2016). To analyze effects of the hematopoiesis-specific Meg3 knockout in the developing embryo, we generated VavCre Meg3 mat flox/pat wt mice. Cre+ offspring were born and developed normally. In- depth analysis of adult animals revealed loss of Meg3 expression in HSCs, but again no hematopoietic impairments were detected. Next, I performed interferon-mediated stimulation in MxCre Meg3 mat flox/pat wt mice. During both activation and recovery phase, Meg3-deficient adult HSCs responded highly similar compared to controls. Taken together, my work shows that the highly expressed, imprinted lncRNA Meg3 is dispensable for the function of HSCs during adulthood and embryonic development. In the adult system, loss of Meg3 does not impair the performance in serial reconstitution assays or response to stress mediators. (Sommerkamp et al., 2019) Project 2: HSC function, differentiation and activation are regulated by APA The majority of mammalian genes have multiple different polyadenylation sites. The RNA editing mechanism APA controls the selection of these sites, thereby altering 3’-UTR length and isoform expression. Thus, APA modulates RNA stability, localization, protein output and even protein localization (Di Giammartino et al., 2011; Tian and Manley, 2017). So far, the role of APA in the regulation of the adult HSC/MPP compartment has not been studied. In this thesis, I show that the APA regulator Pabpn1 is essential for HSCs, as knockdown (KD) of Pabpn1 led to decreased HSC function in vitro and in vivo. To analyze the prevalence of APA at the top of the hematopoietic hierarchy, I established an ultra-low input 3’-Seq approach and performed analysis of HSCs, MPP cells and HSCs activated by inflammation. Bioinformatic analysis revealed dynamic APA patterns in numerous genes between HSCs and MPPs as well as during inflammation-induced HSC stress response. We observed global 3’-UTR shortening both upon HSC differentiation towards MPPs and HSC activation. Further, 3’-Seq analysis of Pabpn1 KD cells revealed that PABPN1 regulates APA in the HSC/MPP compartment. We observed an APA-mediated glutaminase (Gls) isoform switch upon exit of HSCs from quiescence. Gls isoform switching led to enhanced relative expression of the highly active GLS GAC isoform and overall increased GLS protein levels. In line, small molecule-mediated inhibition of glutaminolysis in vitro enhanced HSC maintenance by limiting proliferation. I could show that Gls isoform switching leading to increased glutaminolysis is mediated by the APA regulator NUDT21 and in turn is required for proper HSC function. KD of Nudt21 led to inhibition of Gls isoform switching, impaired HSC function and a partial block in HSC differentiation. In summary, my results install differential employment of APA and associated glutamine metabolism adaptations as novel layers in the regulation of the HSC-controlled hematopoietic hierarchy. (Sommerkamp et al., under revision) Project 3: Generation of Pabpn1flox mice To enable in-depth in vivo analysis of the role of APA in different tissues and disease settings in the future, we generated an inducible Pabpn1flox mouse model. Here, we used the Easi- CRISPR approach (Miura et al., 2018; Quadros et al., 2017) to generate transgenic animals. I performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency in vivo. Two different crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, were injected into one of the pronuclei of zygotes together with a long single-stranded DNA (ssDNA) template. By extensive genotyping, I identified 3 of 17 offspring animals to be correctly targeted. Homozygous offspring mice were successfully bred and can be used in the future to determine functionality of the Pabpn1flox mouse model and to functionally asses the role of APA in different biological settings

THE MECHANISM OF CYTOKINE SYNERGY INDUCED BY COMBINATORIAL TLR ACTIVATION

Ph.DDOCTOR OF PHILOSOPH

PTH1R Signaling in Osteoblasts Stimulated with Functionally Selective Ligands: Phosphoproteomics Reveals Unique Signaling Networks

The past 20 years have seen G-protein coupled receptor (GPCR) theory advance significantly. Receptors are now thought of as adopting multiple conformations in a state of dynamic equilibrium. The study of GPCR biased agonism has emerged from this changing concept of receptors and introduced the field to “pluridimensional efficacy.” It is thought that a single readout of efficacy is no longer sufficient and multiple parameters of efficacy must be measured in drug screens to improve the ability to predict in vivo effects. While several GPCRs have multiple cognate ligands that elicit functionally-selective responses, the present study focused on biased signaling of the parathyroid hormone receptor (PTH1R). Parathyroid hormone (PTH) maintains serum calcium and is a key regulator of bone remodeling. Human PTH1-34 (Forteo) is the only FDA approved drug used for treatment of osteoporosis that acts via its anabolic actions on osteoblasts. However, the therapeutic utilization of PTH1-34 is limited by its catabolic effects, mediated in part by protein kinase A, which after two years culminate in net bone resporption through the activation of osteoclasts by RANKL. The experimental, biased ligand of the PTH1R, bovine parathyroid hormone residues 7-34 with D-Trp12 and Tyr34 (bPTH(7-34)), does not exhibit the catabolic effects of PTH1-34. In vivo administration of the conventional ligand, PTH 1-34, and the biased ligand, bPTH(7-34), for eight weeks increased bone mineral density (BMD) in mice. The anabolic effect of bPTH(7-34) in vivo was lost in β-arrestin deficient mice, revealing a dependence on β-arrestin mediated signaling. Further analysis of osteoblast and osteoclast number, transcript expression, and the generation of second messengers revealed the anabolic effect of each ligand was achieved by different mechanisms. To elucidate the unique, proximal signaling events activated by acute stimulation of the PTH1R with the biased agonist, this study focused on characterization and comparison of the phosphorylation-mediated signaling profiles of osteoblasts stimulated with each osteogenic agonist. Relative changes in phosphorylation were measured using a SILAC-based phosphoproteomic screen following acute stimulation of MC3T3- E1 preosteoblast cells with hPTH(1-34) or bPTH(7-34) for 5 minutes. The experiments were performed in proliferating preosteoblasts (Day 0) and differentiating osteoblasts (Day 10). Over ten thousand sites of phosphorylation were observed. Regulated phosphosites and phosphoproteins were examined for putative kinase activity, targeted signaling pathways, and biological processes. Differences were observed in the kinases stimulated by each agonist. For example, bPTH(7-34) treatment activated MAPK1 and increased phosphorylation of downstream substrates, while phosphorylation of predicted MAPK1 substrates were decreased with hPTH(1-34) activation. While both drugs regulated phosphorylation of proteins in signaling pathways involving GPCR signaling (PLC, MTOR, Rho GTPases); Ingenuity Pathway Analysis (IPA) also revealed discrete signaling networks engaged by each drug. PTH (1-34) treatment yielded regulated proteins involved in cytoskeletal dynamics and the Wnt/β- catenin pathway, whereas bPTH(7-34) treatment modulated pathways related to survival (ATM, CDKs, and p70S6K) and transcription (Jak/Stat, and PPARα). Cell-based assays confirmed hPTH(1-34) and bPTH(7-34) both confer resistance to etoposide-induced apoptosis and bPTH(7-34) increases proliferation in MC3T3-E1 cells. At the biological process level, both ligands modulated proteins involved in cell survival, migration, growth, and bone metabolism. Comparison of regulated phosphoproteins at two time points during osteogenic differentiation unexpectedly revealed that the bPTH(7-34) gave a more robust effect in proliferating preosteoblasts, whereas hPTH(1-34) stimulated more sites of phosphorylation in differentiating osteoblasts. This observation indicates the differential effects of each agonist may result from changes in signaling mediators that are expressed at these two time points. While the PTH receptor was present at both time points, β-arrestin was more highly expressed in proliferating preosteoblasts

The Use of Catalytically Dead Cas9 to Identify Key Transcription Regulators in Triple Negative Breast Cancer

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer cases. It tends to be aggressive, high-grade and poorly differentiated tumour with poor clinical outcome. Lack of expression of oestrogen, progesterone receptors, and human epidermal growth factor receptor 2 make TNBC patients ineligible to hormonal therapy. For these reasons the identification of novel clinical targets still remains a priority. With the advances of multiomics, many of the genes transcriptionally upregulated in TNBC have been identified, but how they are disregulated is still unknown: the understanding of how this works at a transcriptional level could contribute to the development of a novel therapeutic approach. We report here a new methodology to identify key transcription regulators that we applied to investigate the expression of highly expressed genes in TNBC: our approach combines RIME proteomics with CRISPR/Cas9 technology. In brief, we targeted putative regulatory regions of differentially regulated genes in TNBC compared to other subtypes of breast cancer using a catalytically dead version of the Cas9 protein (dCas9). In particular, we focused on transcription regulators like FOXC1, NFIB and NFE2L3. We then performed RIME proteomics to identify which proteins are in close proximity to dCas9 and thus potentially bound to these putative regulatory regions. In addition, we developed a novel, statistical approach to analyse these particular proteomic datasets based on the relative abundance of the protein of interest, and a powerful ranking method to identify biologically and therapeutic meaningful candidates. Through this process, we identified three putative regulatory proteins, MTA2, CDK1 and CDK6, bound to all three loci. In here, we reported how their knockdown, performed by shRNA, directly affects the expression of the investigated genes in a panel of TNBC cell lines, and their oncogenic capacity in vitro. These results demonstrate the importance of these transcription regulators for TNBC biology, and they highlight the necessity of a deeper understanding of their roles in gene expression regulation

Global and Local Regulation of Gene Expression in the Human Brain

Neuropsychiatric disorders are behavioral conditions marked by intellectual, social, or emotional deficits that can be linked to diseases of the nervous system. Autism spectrum disorder (ASD), schizophrenia (SCZ), bipolar disorder (BP), major depressive disorder (MDD), and attention deficit and hyperactivity disorder (ADHD) are common, heritable diseases each with a prevalence exceeding 1% of the population, none of which can be characterized by discernable anatomical or neurological pathologies. Genetic association studies have identified mutations in hundreds of genes that contribute to risk for at least one of these disorders, and have shown that a substantial fraction of the genetic liability is shared between many of these neuropsychiatric diseases. It has long been hoped that with enough genetic evidence we will identify the biological pathways, developmental time points, and brain regions that, when disrupted, give rise to neuropsychiatric disorders. However, the cellular and functional complexity of the human brain, as well as the genetic complexity of neuropsychiatric disease, make it difficult to search for such convergence. In this thesis, I investigate global and local transcriptional regulation within and across 12 regions of the human brain in order to investigate the regional specificity of neuropsychiatric disorders. I develop novel bioinformatics methods – ranging from data processing to network construction – to identify whether the transcriptional regulation of a set of genes is shared or specific. I hypothesize that local, region-specific transcriptional regulation corresponds directly to cell types and processes that are specific to, or far more prevalent in, a given region; that cross-regional transcriptional regulation corresponds to cell types that show little heterogeneity across brain regions; and that genetic disruption of region-specific transcriptional programs results in regional susceptibility. I use a systems-biology approach to summarize transcriptional regulation into reproducibly co-expressed gene sets (“co-expression modules”), which can be analyzed statistically to identify common functions, pathways, and cell types. I then integrate data from genetic association studies to ascertain gene sets conferring outsized risk for neuropsychiatric disorders, thereby implicating the corresponding pathways for further investigation in disease etiology. Finally, I use the network structure itself to investigate the genetic architecture of ASD and SCZ in terms of omnigenics and network polygenics. Chapter 1 presents the biological background for the studies and summarizes some of the major studies of neuropsychiatric disorders along with their principal methods and conclusions. In chapter 2, utilizing my multi-regional co-expression approach, I identify 12 brain-wide, 114 region-specific, and 50 cross-regional co-expression modules. Nearly 40% of expressed genes fall into brain-wide modules and correspond to major cell classes and conserved biological processes, while region-specific modules comprise 25% of expressed genes and correspond to region-specific cell types. The detailed study in chapter 3 demonstrates that neuropsychiatric risk concentrates in both brain wide and multi-regional modules, implicating major core cell types in disease etiology but not region-specific susceptibility. Chapter 4 presents a new and more general framework for defining genetic networks. Using this framework, I show that the network pattern of ASD-associated rare loss-of-function mutations, as well as the large number of significant targets for trans master regulators in BP and SCZ, support a classical polygenic architecture with thousands of directly causal genes. These results suggest that a nontrivial component of risk for neuropsychiatric disease comes from the global polygenic disruption of neuronal function and neuronal maturation

Plasticity in gene expression programmes of dendritic cells responding to antigens.

Dendritic cells (DCs) are professional antigen presenting cells whose function is to initiate and shape an appropriate adaptive immune response. This requires an ability to distinguish differences between whole pathogens, in order to orchestrate effective downstream immunological outcomes. However, cellular re-programming of DC functions during these events are not well understood. A paradigm of dendritic cell biology is that DCs have two modes of function that relate to their differentiation states. An immature DC functions as an immune sentinel, to monitor and interrogate its surroundings for pathogens. Encounter with such stimuli results in a process termed "maturation", where DCs acquire the properties of effective antigen presenting cells. However, this process of differentiation is complex. In this thesis, gene expression profiling of DCs exposed to pathogen components has revealed three distinct phases of maturation, with statistically significant expression of subsets of genes characterising these phases. Transcriptional regulation of the signalling pathways involving p38 and ERK MAP kinases important to DC function were identified. Specific inhibitors of p38 and ERK confirmed their differential role in DC maturation, with p38 activity being necessary for the initiation of DC maturation, whilst ERK activity persists to maintain DC survival. Concurrent with the core maturation process is the DCs' ability to differentially respond to pathogens. Gene expression analysis of DCs exposed to whole viruses supports the model of DC plasticity to different pathogenic stimuli. Using exploratory cluster analysis and a novel vector algebra method, core and pathogen-specific gene expression programmes were identified. The programmes involving the differential regulation of cytokines were confirmed at the transcript level and at the protein level. Together these data show that DCs mature to effective antigen presenting cells via an orchestrated pattern of at least three gene expression programmes. Superimposed on this core maturation response are pathogen-specific transcriptional programmes. Therefore, we conclude that DCs can translate different pathogenic stimuli into core DC maturation and pathogen-specific responses that together shape an appropriate adaptive immune response