Mis-expression of GATA6 re-programs cell fate during early hematopoiesis.

The traditional view of hematopoiesis is that myeloid cells derive from a common myeloid progenitor (CMP), whereas all lymphoid cell populations, including B, T, and natural killer (NK) cells and possibly plasmacytoid dendritic cells (pDCs), arise from a common lymphoid progenitor (CLP). In Max41 transgenic mice, nearly all B cells seem to be diverted into the granulocyte lineage. Here, we show that these mice have an excess of myeloid progenitors, but their CLP compartment is ablated, and they have few pDCs. Nevertheless, T cell and NK cell development proceeds relatively normally. These hematopoietic abnormalities result from aberrant expression of Gata6 due to serendipitous insertion of the transgene enhancer (Eµ) in its proximity. Gata6 mis-expression in Max41 transgenic progenitors promoted the gene-regulatory networks that drive myelopoiesis through increasing expression of key transcription factors, including PU.1 and C/EBPa. Thus, mis-expression of a single key regulator like GATA6 can dramatically re-program multiple aspects of hematopoiesis.

A Human Homozygous HELQ Missense Variant Does Not Cause Premature Ovarian Insufficiency in a Mouse Model.

Disruption of meiosis and DNA repair genes is associated with female fertility disorders like premature ovarian insufficiency (POI). In this study, we identified a homozygous missense variant in the HELQ gene (c.596 A>C; p.Gln199Pro) through whole exome sequencing in a POI patient, a condition associated with disrupted ovarian function and female infertility. HELQ, an enzyme involved in DNA repair, plays a crucial role in repairing DNA cross-links and has been linked to germ cell maintenance, fertility, and tumour suppression in mice. To explore the potential association of the HELQ variant with POI, we used CRISPR/Cas9 to create a knock-in mouse model harbouring the equivalent of the human HELQ variant identified in the POI patient. Surprisingly, Helq knock-in mice showed no discernible phenotype, with fertility levels, histological features, and follicle development similar to wild-type mice. Despite the lack of observable effects in mice, the potential role of HELQ in human fertility, especially in the context of POI, should not be dismissed. Larger studies encompassing diverse ethnic populations and alternative functional approaches will be necessary to further examine the role of HELQ in POI. Our results underscore the potential uncertainties associated with genomic variants and the limitations of in vivo animal modelling.

Differential in vivo roles of Mpl cytoplasmic tyrosine residues in murine hematopoiesis and myeloproliferative disease.

Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.

ACAD10 is not required for metformin's metabolic actions or for maintenance of whole-body metabolism in C57BL/6J mice.

AIM: Acyl-coenzyme A dehydrogenase family member 10 (ACAD10) is a mitochondrial protein purported to be involved in the fatty acid oxidation pathway. Metformin is the most prescribed therapy for type 2 diabetes; however, its precise mechanisms of action(s) are still being uncovered. Upregulation of ACAD10 is a requirement for metformin's ability to inhibit growth in cancer cells and extend lifespan in Caenorhabditis elegans. However, it is unknown whether ACAD10 plays a role in metformin's metabolic actions. MATERIALS AND METHODS: We assessed the role for ACAD10 on whole-body metabolism and metformin action by generating ACAD10KO mice on a C57BL/6J background via CRISPR-Cas9 technology. In-depth metabolic phenotyping was conducted in both sexes on a normal chow and high fat-high sucrose diet. RESULTS: Compared with wildtype mice, we detected no difference in body composition, energy expenditure or glucose tolerance in male or female ACAD10KO mice, on a chow diet or high-fat, high-sucrose diet (p ≥ .05). Hepatic mitochondrial function and insulin signalling was not different between genotypes under basal or insulin-stimulated conditions (p ≥ .05). Glucose excursions following acute administration of metformin before a glucose tolerance test were not different between genotypes nor was body composition or energy expenditure altered after 4 weeks of daily metformin treatment (p ≥ .05). Despite the lack of a metabolic phenotype, liver lipidomic analysis suggests ACAD10 depletion influences the abundance of specific ceramide species containing very long chain fatty acids, while metformin treatment altered clusters of cholesterol ester, plasmalogen, phosphatidylcholine and ceramide species. CONCLUSIONS: Loss of ACAD10 does not alter whole-body metabolism or impact the acute or chronic metabolic actions of metformin in this model.

Mind bomb 2 limits inflammatory dermatitis in Sharpin mutant mice independently of cell death.

Skin inflammation is a complex process implicated in various dermatological disorders. The chronic proliferative dermatitis (cpd) phenotype driven by the cpd mutation (cpdm) in the Sharpin gene is characterized by dermal inflammation and epidermal abnormalities. Tumour necrosis factor (TNF) and caspase-8-driven cell death causes the pathogenesis of Sharpincpdm mice; however, the role of mind bomb 2 (MIB2), a pro-survival E3 ubiquitin ligase involved in TNF signaling, in skin inflammation remains unknown. Here, we demonstrate that MIB2 antagonizes inflammatory dermatitis in the context of the cpd mutation. Surprisingly, the role of MIB2 in limiting skin inflammation is independent of its known pro-survival function and E3 ligase activity. Instead, MIB2 enhances the production of wound-healing molecules, granulocyte colony-stimulating factor, and Eotaxin, within the skin. This discovery advances our comprehension of inflammatory cytokines and chemokines associated with cpdm pathogenesis and highlights the significance of MIB2 in inflammatory skin disease that is independent of its ability to regulate TNF-induced cell death.

Genome-wide CRISPR screening identifies a role for ARRDC3 in TRP53-mediated responses.

Whole-genome screens using CRISPR technologies are powerful tools to identify novel tumour suppressors as well as factors that impact responses of malignant cells to anti-cancer agents. Applying this methodology to lymphoma cells, we conducted a genome-wide screen to identify novel inhibitors of tumour expansion that are induced by the tumour suppressor TRP53. We discovered that the absence of Arrestin domain containing 3 (ARRDC3) increases the survival and long-term competitiveness of MYC-driven lymphoma cells when treated with anti-cancer agents that activate TRP53. Deleting Arrdc3 in mice caused perinatal lethality due to various developmental abnormalities, including cardiac defects. Notably, the absence of ARRDC3 markedly accelerated MYC-driven lymphoma development. Thus, ARRDC3 is a new mediator of TRP53-mediated suppression of tumour expansion, and this discovery may open new avenues to harness this process for cancer therapy.

Trabid patient mutations impede the axonal trafficking of adenomatous polyposis coli to disrupt neurite growth.

ZRANB1 (human Trabid) missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterized these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organization and neural development. Zranb1 knock-in mice harboring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalized causing impaired organization of the cytoskeleton that underlie the neuronal and developmental phenotypes.

Cleavage of cFLIP restrains cell death during viral infection and tissue injury and favors tissue repair.

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. Cellular FLICE-like inhibitory protein (cFLIP) is a crucial regulator of cell death and a substrate of Caspase-8. However, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we found an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to severe acute respiratory syndrome coronavirus (SARS-CoV)-induced lethality, impaired skin wound healing, and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to tumor necrosis factor(TNF)-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on glutamine-469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress to ensure the execution of repair programs.

Deletion of the transcriptional regulator TFAP4 accelerates c-MYC-driven lymphomagenesis.

Many lymphoid malignancies arise from deregulated c-MYC expression in cooperation with additional genetic lesions. While many of these cooperative genetic lesions have been discovered and their functions characterised, DNA sequence data of primary patient samples suggest that many more do exist. However, the nature of their contributions to c-MYC driven lymphomagenesis have not yet been investigated. We identified TFAP4 as a potent suppressor of c-MYC driven lymphoma development in a previous genome-wide CRISPR knockout screen in primary cells in vivo [1]. CRISPR deletion of TFAP4 in Eµ-MYC transgenic haematopoietic stem and progenitor cells (HSPCs) and transplantation of these manipulated HSPCs into lethally irradiated animals significantly accelerated c-MYC-driven lymphoma development. Interestingly, TFAP4 deficient Eµ-MYC lymphomas all arose at the pre-B cell stage of B cell development. This observation prompted us to characterise the transcriptional profile of pre-B cells from pre-leukaemic mice transplanted with Eµ-MYC/Cas9 HSPCs that had been transduced with sgRNAs targeting TFAP4. This analysis revealed that TFAP4 deletion reduced expression of several master regulators of B cell differentiation, such as Spi1, SpiB and Pax5, which are direct target genes of both TFAP4 and MYC. We therefore conclude that loss of TFAP4 leads to a block in differentiation during early B cell development, thereby accelerating c-MYC-driven lymphoma development.

Deletion of Gpatch2 does not alter Tnf expression in mice.

The cytokine TNF has essential roles in immune defence against diverse pathogens and, when its expression is deregulated, it can drive severe inflammatory disease. The control of TNF levels is therefore critical for normal functioning of the immune system and health. We have identified GPATCH2 as a putative repressor of Tnf expression acting post-transcriptionally through the TNF 3' UTR in a CRISPR screen for novel regulators of TNF. GPATCH2 is a proposed cancer-testis antigen with roles reported in proliferation in cell lines. However, its role in vivo has not been established. We have generated Gpatch2-/- mice on a C57BL/6 background to assess the potential of GPATCH2 as a regulator of Tnf expression. Here we provide the first insights into Gpatch2-/- animals and show that loss of GPATCH2 affects neither basal Tnf expression in mice, nor Tnf expression in intraperitoneal LPS and subcutaneous SMAC-mimetic injection models of inflammation. We detected GPATCH2 protein in mouse testis and at lower levels in several other tissues, however, the morphology of the testis and these other tissues appears normal in Gpatch2-/- animals. Gpatch2-/- mice are viable, appear grossly normal, and we did not detect notable aberrations in lymphoid tissues or blood cell composition. Collectively, our results suggest no discernible role of GPATCH2 in Tnf expression, and the absence of an overt phenotype in Gpatch2-/- mice warrants further investigation of the role of GPATCH2.

Survey of activation-induced genome architecture reveals a novel enhancer of Myc.

The transcription factor Myc is critically important in driving cell proliferation, a function that is frequently dysregulated in cancer. To avoid this dysregulation Myc is tightly controlled by numerous layers of regulation. One such layer is the use of distal regulatory enhancers to drive Myc expression. Here, using chromosome conformation capture to examine B cells of the immune system in the first hours after their activation, we reveal a previously unidentified enhancer of Myc. The interactivity of this enhancer coincides with a dramatic, but discrete, spike in Myc expression 3 h post-activation. However, genetic deletion of this region, has little impact on Myc expression, Myc protein level or in vitro and in vivo cell proliferation. Examination of the enhancer deleted regulatory landscape suggests that enhancer redundancy likely sustains Myc expression. This work highlights not only the importance of temporally examining enhancers, but also the complexity and dynamics of the regulation of critical genes such as Myc.

Generation of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance.

CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.

Tankyrase-mediated ADP-ribosylation is a regulator of TNF-induced death.

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose-binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

ZC3H12C expression in dendritic cells is necessary to prevent lymphadenopathy of skin-draining lymph nodes.

The role of RNA-binding proteins of the CCCH-containing family in regulating proinflammatory cytokine production and inflammation is increasingly recognized. We have identified ZC3H12C (Regnase-3) as a potential post-transcriptional regulator of tumor necrosis factor expression and have investigated its role in vivo by generating Zc3h12c-deficient mice that express green fluorescent protein instead of ZC3H12C. Zc3h12c-deficient mice develop hypertrophic lymph nodes. In the immune system, ZC3H12C expression is mostly restricted to the dendritic cell (DC) populations, and we show that DC-restricted ZC3H12C depletion is sufficient to cause lymphadenopathy. ZC3H12C can regulate Tnf messenger RNA stability via its RNase activity in vitro, and we confirmed the role of Tnf in the development of lymphadenopathy. Finally, we found that loss of ZC3H12C did not impact the outcome of skin inflammation in the imiquimod-induced murine model of psoriasis, despite Zc3h12c being identified as a risk factor for psoriasis susceptibility in several genome-wide association studies. Our data suggest a role for ZC3H12C in DC-driven skin homeostasis.

The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal.

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac), and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used 2 complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1-null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow 2 to 6 weeks after Hbo1 deletion. Hbo1-deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors. The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-, and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1, and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

The ubiquitylation of IL-1ß limits its cleavage by caspase-1 and targets it for proteasomal degradation.

Interleukin-1ß (IL-1ß) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in a variety of other inflammatory diseases. Therefore, IL-1ß activity must be fine-tuned to enable anti-microbial responses whilst limiting collateral damage. Here, we show that precursor IL-1ß is rapidly turned over by the proteasome and this correlates with its decoration by K11-linked, K63-linked and K48-linked ubiquitin chains. The ubiquitylation of IL-1ß is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1ß cleavage by caspase-1. IL-1ß K133 is modified by ubiquitin and forms a salt bridge with IL-1ß D129. Loss of IL-1ß K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1ß. Accordingly, Il1bK133R/K133R mice have increased levels of precursor IL-1ß upon inflammasome priming and increased production of bioactive IL-1ß, both in vitro and in response to LPS injection. These findings identify mechanisms that can limit IL-1ß activity and safeguard against damaging inflammation.

Type 1 conventional dendritic cell fate and function are controlled by DC-SCRIPT.

The functional diversification of dendritic cells (DCs) is a key step in establishing protective immune responses. Despite the importance of DC lineage diversity, its genetic basis is not fully understood. The transcription factor DC-SCRIPT is expressed in conventional DCs (cDCs) and their committed bone marrow progenitors but not in plasmacytoid DCs (pDCs). We show that mice lacking DC-SCRIPT displayed substantially impaired development of IRF8 (interferon regulatory factor 8)-dependent cDC1, whereas cDC2 numbers increased marginally. The residual DC-SCRIPT-deficient cDC1s had impaired capacity to capture and present cell-associated antigens and to secrete IL-12p40, two functional hallmarks of this population. Genome-wide mapping of DC-SCRIPT binding and gene expression analyses revealed a key role for DC-SCRIPT in maintaining cDC1 identity via the direct regulation of cDC1 signature genes, including Irf8 Our study reveals DC-SCRIPT to be a critical component of the gene regulatory program shaping the functional attributes of cDC1s.

The NK cell granule protein NKG7 regulates cytotoxic granule exocytosis and inflammation.

Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4+ and CD8+ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8+ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4+ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.