Small molecule screen in zebrafish and HSC expansion

The zebrafish (Danio rerio) has emerged as a valuable model organism that is amenable for large-scale chemical and genetic screens. The ability of zebrafish to produce large quantities of synchronized, externally fertilized, transparent embryos makes them ideal for screens, which often are not possible in mammalian models. Signaling pathways important for hematopoiesis are well conserved between zebrafish and mammals, making many targets identified in zebrafish screens applicable to mammals. Hematopoiesis in zebrafish occurs in two waves: the primitive or embryonic wave and the definitive or adult wave. Definitive hematopoietic stem cells arise in the aorta-gonad-mesonephros region (AGM) and express conserved markers such as runx1 and c-myb that allow for the detection of stem cells by whole-mount in situ hybridization (WISH). In this protocol, we will discuss a chemical screen in zebrafish embryos to detect compounds that expand or deplete hematopoietic stem cells (HSCs) in vivo. This type of screen represents a powerful tool to study HSCs in zebrafish

CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB activation by TNFR family members

Familial cylindromatosis is an autosomal dominant predisposition to tumours of skin appendages called cylindromas. Familial cylindromatosis is caused by mutations in a gene encoding the CYLD protein of previously unknown function1. Here we show that CYLD is a deubiquitinating enzyme that negatively regulates activation of the transcription factor NF-κB by specific tumour-necrosis factor receptors (TNFRs). Loss of the deubiquitinating activity of CYLD correlates with tumorigenesis. CYLD inhibits activation of NF-κB by the TNFR family members CD40, XEDAR and EDAR in a manner that depends on the deubiquitinating activity of CYLD. Downregulation of CYLD by RNA-mediated interference augments both basal and CD40-mediated activation of NF-κB. The inhibition of NF-κB activation by CYLD is mediated, at least in part, by the deubiquitination and inactivation of TNFR-associated factor 2 (TRAF2) and, to a lesser extent, TRAF6. These results indicate that CYLD is a negative regulator of the cytokine-mediated activation of NF-κB that is required for appropriate cellular homeostasis of skin appendages

Thymocyte-specific truncation of the deubiquitinating domain of CYLD impairs positive selection in a NF-kappaB essential modulator-dependent manner

The cylindromatosis tumor suppressor gene (Cyld) encodes a deubiquitinating enzyme (CYLD) with immunoregulatory function. In this study, we evaluated the role of Cyld in T cell ontogeny by generating a mouse (Cyld(Delta9)) with a thymocyte-restricted Cyld mutation that causes a C-terminal truncation of the protein and reciprocates catalytically inactive human mutations. Mutant mice had dramatically reduced single positive thymocytes and a substantial loss of peripheral T cells. The analyses of polyclonal and TCR-restricted thymocyte populations possessing the mutation revealed a significant block in positive selection and an increased occurrence of apoptosis at the double-positive stage. Interestingly, in the context of MHC class I and II restricted TCR transgenes, lack of functional CYLD caused massive deletion of thymocytes that would have been positively selected, which is consistent with an impairment of positive selection. Biochemical analysis revealed that Cyld(Delta9) thymocytes exhibit abnormally elevated basal activity of NF-kappaB and JNK. Most importantly, inactivation of NF-kappaB essential modulator fully restored the NF-kappaB activity of Cyld(Delta9) thymocytes to physiologic levels and rescued their developmental and survival defect. This study identifies a fundamental role for functional CYLD in establishing the proper threshold of activation for thymocyte selection by a mechanism dependent on NF-kappaB essential modulator

Bloody zebrafish: Novel methods in normal and malignant hematopoiesis

Hematopoiesis is an optimal system for studying stem cell maintenance and lineage differentiation under physiological and pathological conditions. In vertebrate organisms, billions of differentiated hematopoietic cells need to be continuously produced to replenish the blood cell pool. Disruptions in this process have immediate consequences for oxygen transport, responses against pathogens, maintenance of hemostasis and vascular integrity. Zebrafish is a widely used and well-established model for studying the hematopoietic system. Several new hematopoietic regulators were identified in genetic and chemical screens using the zebrafish model. Moreover, zebrafish enables in vivo imaging of hematopoietic stem cell generation and differentiation during embryogenesis, and adulthood. Finally, zebrafish has been used to model hematopoietic diseases. Recent technological advances in single-cell transcriptome analysis, epigenetic regulation, proteomics, metabolomics, and processing of large data sets promise to transform the current understanding of normal, abnormal, and malignant hematopoiesis. In this perspective, we discuss how the zebrafish model has proven beneficial for studying physiological and pathological hematopoiesis and how these novel technologies are transforming the field

Lymphocyte-Specific Function of the DNA Plymerase Epsilon Subunit Pole3 Revealed by Neomorphic Alleles

Immunodeficiencies are typically caused by loss-of-function mutations in lymphocyte-specific genes. Occasionally, mutations in ubiquitous general-purpose factors, including those affecting essential components of the DNA polymerase epsilon (POLE) holoenzyme, have cell-type-specific consequences. POLE3, one of the four components of the POLE holoenzyme, features a histone fold domain and a unique acidic C terminus, making it a particularly attractive candidate mediating cell type-specific activities of POLE. Mice lacking Pole3 survive up to late embryonic stages, indicating that this subunit is dispensable for DNA replication. The phenotypes of viable hypomorphic and neomorphic alleles are surprisingly tissue restricted and reveal a stage-specific function of the histone fold domain of Pole3 during T and B cell development. Gradual introduction of positively charged residues into the acidic C terminus leads to peripheral lymphopenia of increasing severity. Our findings serve as a paradigm to understand the molecular basis of cell-type-specific non-replicative functions of the ubiquitous POLE complex

NF-kappaB is essential for induction of CYLD, the negative regulator of NF-kappaB: evidence for a novel inducible autoregulatory feedback pathway

The transcription factor NF-κB regulates genes involved in inflammatory and immune responses, tumorigenesis, and apoptosis. In contrast to the pleiotropic stimuli that lead to its positive regulation, the known signaling mechanisms that underlie the negative regulation of NF-κB are very few. Recent studies have identified the tumor suppressor CYLD, loss of which causes a benign human syndrome called cylindromatosis, as a key negative regulator for NF-κB signaling by deubiquitinating tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2, TRAF6, and NEMO (NF-κB essential modulator, also known as IκB kinase γ). However, how CYLD is regulated remains unknown. The present study revealed a novel autoregulatory feedback pathway through which activation of NF-κB by TNF-α and bacterium nontypeable Haemophilus influenzae (NTHi) induces CYLD that in turn leads to the negative regulation of NF-κB signaling. In addition, TRAF2 and TRAF6 appear to be differentially involved in NF-κB-dependent induction of CYLD by TNF-α and NTHi. These findings provide novel insights into the autoregulation of NF-κB activation

Truncation of the Deubiquitinating Domain of CYLD in Myelomonocytic Cells Attenuates Inflammatory Responses

The cylindromatosis tumor suppressor (CYLD) is a deubiquitinating enzyme that has been implicated in various aspects of adaptive and innate immune responses. Nevertheless, the role of CYLD in the function of specific types of immune cells remains elusive. In this report we have used conditional gene targeting in mice to address the role of the deubiquitinating activity of CYLD in the myelomonocytic lineage. Truncation of the deubiquitinating domain of CYLD specifically in myelomonocytic cells impaired the development of lethal LPS-induced endotoxic shock and the accumulation of thioglycollate-elicited peritoneal macrophages. Our data establish CYLD as a regulator of monocyte-macrophage activation in response to inflammatory stimuli and identify it as a potential target for therapeutic intervention in relevant inflammatory disorders in humans

TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages

Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB–Irg1–itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32–BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB’s role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo