Disruption of the ZFP574-THAP12 complex suppresses B cell malignancies in mice.

Despite the availability of life-extending treatments for B cell leukemias and lymphomas, many of these cancers remain incurable. Thus, the development of new molecular targets and therapeutics is needed to expand treatment options. To identify new molecular targets, we used a forward genetic screen in mice to identify genes required for development or survival of lymphocytes. Here, we describe Zfp574, an essential gene encoding a zinc finger protein necessary for normal and malignant lymphocyte survival. We show that ZFP574 interacts with zinc finger protein THAP12 and promotes the G1-to-S-phase transition during cell cycle progression. Mutation of ZFP574 impairs nuclear localization of the ZFP574-THAP12 complex. ZFP574 or THAP12 deficiency results in cell cycle arrest and impaired lymphoproliferation. Germline mutation, acute gene deletion, or targeted degradation of ZFP574 suppressed Myc-driven B cell leukemia in mice, but normal B cells were largely spared, permitting long-term survival, whereas complete lethality was observed in control animals. Our findings support the identification of drugs targeting ZFP574-THAP12 as a unique strategy to treat B cell malignancies.

Viable mutations of mouse midnolin suppress B cell malignancies.

In a genetic screen, we identified two viable missense alleles of the essential gene Midnolin (Midn) that were associated with reductions in peripheral B cells. Causation was confirmed in mice with targeted deletion of four of six MIDN protein isoforms. MIDN was expressed predominantly in lymphocytes where it augmented proteasome activity. We showed that purified MIDN directly stimulated 26S proteasome activity in vitro in a manner dependent on the ubiquitin-like domain and a C-terminal region. MIDN-deficient B cells displayed aberrant activation of the IRE-1/XBP-1 pathway of the unfolded protein response. Partial or complete MIDN deficiency strongly suppressed Eµ-Myc-driven B cell leukemia and the antiapoptotic effects of Eµ-BCL2 on B cells in vivo and induced death of Sp2/0 hybridoma cells in vitro, but only partially impaired normal lymphocyte development. Thus, MIDN is required for proteasome activity in support of normal lymphopoiesis and is essential for malignant B cell proliferation over a broad range of differentiation states.

Dynamic molecular architecture and substrate recruitment of cullin3-RING E3 ligase CRL3KBTBD2.

Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3-RING E3 ubiquitin ligase (CRL3). CRL3KBTBD2 forms multimers, which disassemble into dimers upon substrate binding (CRL3KBTBD2-p85α) and/or neddylation by the activator NEDD8 (CRL3KBTBD2~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.

E3 ubiquitin ligase Herc3 deficiency leads to accumulation of subretinal microglia and retinal neurodegeneration.

Activated microglia have been implicated in the pathogenesis of age-related macular degeneration (AMD), diabetic retinopathy, and other neurodegenerative and neuroinflammatory disorders, but our understanding of the mechanisms behind their activation is in infant stages. With the goal of identifying novel genes associated with microglial activation in the retina, we applied a semiquantitative fundus spot scoring scale to an unbiased, state-of-the-science mouse forward genetics pipeline. A mutation in the gene encoding the E3 ubiquitin ligase Herc3 led to prominent accumulation of fundus spots. CRISPR mutagenesis was used to generate Herc3-/- mice, which developed prominent accumulation of fundus spots and corresponding activated Iba1 + /CD16 + subretinal microglia, retinal thinning on OCT and histology, and functional deficits by Optomotory and electrophysiology. Bulk RNA sequencing identified activation of inflammatory pathways and differentially expressed genes involved in the modulation of microglial activation. Thus, despite the known expression of multiple E3 ubiquitin ligases in the retina, we identified a non-redundant role for Herc3 in retinal homeostasis. Our findings are significant given that a dysregulated ubiquitin-proteasome system (UPS) is important in prevalent retinal diseases, in which activated microglia appear to play a role. This association between Herc3 deficiency, retinal microglial activation and retinal degeneration merits further study.

Essential role of MFSD1-GLMP-GIMAP5 in lymphocyte survival and liver homeostasis.

We detected ENU-induced alleles of Mfsd1 (encoding the major facilitator superfamily domain containing 1 protein) that caused lymphopenia, splenomegaly, progressive liver pathology, and extramedullary hematopoiesis (EMH). MFSD1 is a lysosomal membrane-bound solute carrier protein with no previously described function in immunity. By proteomic analysis, we identified association between MFSD1 and both GLMP (glycosylated lysosomal membrane protein) and GIMAP5 (GTPase of immunity-associated protein 5). Germline knockout alleles of Mfsd1, Glmp, and Gimap5 each caused lymphopenia, liver pathology, EMH, and lipid deposition in the bone marrow and liver. We found that the interactions of MFSD1 and GLMP with GIMAP5 are essential to maintain normal GIMAP5 expression, which in turn is critical to support lymphocyte development and liver homeostasis that suppresses EMH. These findings identify the protein complex MFSD1-GLMP-GIMAP5 operating in hematopoietic and extrahematopoietic tissues to regulate immunity and liver homeostasis.

Essential requirement for IER3IP1 in B cell development.

In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for aberrant immune function, we identified animals with low percentages of B220+ cells in the peripheral blood. The causative mutation was in Ier3ip1, encoding immediate early response 3 interacting protein 1 (IER3IP1), an endoplasmic reticulum membrane protein mutated in an autosomal recessive neurodevelopmental disorder termed Microcephaly with simplified gyration, Epilepsy and permanent neonatal Diabetes Syndrome (MEDS) in humans. However, no immune function for IER3IP1 had previously been reported. The viable hypomorphic Ier3ip1 allele uncovered in this study, identical to a reported IER3IP1 variant in a MEDS patient, reveals an essential hematopoietic-intrinsic role for IER3IP1 in B cell development and function. We show that IER3IP1 forms a complex with the Golgi transmembrane protein 167A and limits activation of the unfolded protein response mediated by inositol-requiring enzyme-1α and X-box binding protein 1 in B cells. Our findings suggest that B cell deficiency may be a feature of MEDS.

OVOL2 sustains postnatal thymic epithelial cell identity.

Distinct pathways and molecules may support embryonic versus postnatal thymic epithelial cell (TEC) development and maintenance. Here, we identify a mechanism by which TEC numbers and function are maintained postnatally. A viable missense allele (C120Y) of Ovol2, expressed ubiquitously or specifically in TECs, results in lymphopenia, in which T cell development is compromised by loss of medullary TECs and dysfunction of cortical TECs. We show that the epithelial identity of TECs is aberrantly subverted towards a mesenchymal state in OVOL2-deficient mice. We demonstrate that OVOL2 inhibits the epigenetic regulatory BRAF-HDAC complex, specifically disrupting RCOR1-LSD1 interaction. This causes inhibition of LSD1-mediated H3K4me2 demethylation, resulting in chromatin accessibility and transcriptional activation of epithelial genes. Thus, OVOL2 controls the epigenetic landscape of TECs to enforce TEC identity. The identification of a non-redundant postnatal mechanism for TEC maintenance offers an entry point to understanding thymic involution, which normally begins in early adulthood.

The dual lipid desaturase/hydroxylase DEGS2 controls phytoceramide levels necessary to counter intestinal inflammation.

Intestinal immunity is dependent on barrier function to maintain quiescence. The mechanisms for the maintenance of this barrier are not fully understood. Delta 4-desaturase, sphingolipid 2 (DEGS2) is a lipid desaturase and hydroxylase that catalyzes the synthesis of ceramide and phytoceramide from dihydroceramide. Using a forward genetic approach, we found and validated a mutation in Degs2 as causative of increasing susceptibility to colitis and altering the phytoceramide balance in the colon. DEGS2 is expressed in the intestinal epithelium, and the colitis phenotype is dependent on the non-hematopoietic compartment of the mouse. In the absence of DEGS2, the colon lacks phytoceramides and accumulates large amounts of the precursor lipid dihydroceramide. In response to dextran sodium sulfate (DSS)-induced colitis, colonic epithelial cells in DEGS2-deficient mice had increased cell death and decreased proliferation compared to those in wild-type mice. These findings demonstrate that DEGS2 is needed to maintain epithelial integrity, protect against DSS-induced colitis and maintain lipid balance in vivo.

Trans-Golgi protein TVP23B regulates host-microbe interactions via Paneth cell homeostasis and Goblet cell glycosylation.

A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.

Forward genetic screening using fundus spot scale identifies an essential role for Lipe in murine retinal homeostasis.

Microglia play a role in the pathogenesis of many retinal diseases. Fundus spots in mice often correlate with the accumulation of activated subretinal microglia. Here we use a semiquantitative fundus spot scoring scale in combination with an unbiased, state-of-the-science forward genetics pipeline to identify causative associations between chemically induced mutations and fundus spot phenotypes. Among several associations, we focus on a missense mutation in Lipe linked to an increase in yellow fundus spots in C57BL/6J mice. Lipe-/- mice generated using CRISPR-Cas9 technology are found to develop accumulation of subretinal microglia, a retinal degeneration with decreased visual function, and an abnormal retinal lipid profile. We establish an indispensable role of Lipe in retinal/RPE lipid homeostasis and retinal health. Further studies using this new model will be aimed at determining how lipid dysregulation results in the activation of subretinal microglia and whether these microglia also play a role in the subsequent retinal degeneration.

Obesity caused by an OVOL2 mutation reveals dual roles of OVOL2 in promoting thermogenesis and limiting white adipogenesis.

Using random germline mutagenesis in mice, we identified a viable hypomorphic allele (boh) of the transcription-factor-encoding gene Ovol2 that resulted in obesity, which initially developed with normal food intake and physical activity but decreased energy expenditure. Fat weight was dramatically increased, while lean weight was reduced in 12-week-old boh homozygous mice, culminating by 24 weeks in massive obesity, hepatosteatosis, insulin resistance, and diabetes. The Ovol2boh/boh genotype augmented obesity in Lepob/ob mice, and pair-feeding failed to normalize obesity in Ovol2boh/boh mice. OVOL2-deficient mice were extremely cold intolerant. OVOL2 is essential for brown/beige adipose tissue-mediated thermogenesis. In white adipose tissues, OVOL2 limited adipogenesis by blocking C/EBPα engagement of its transcriptional targets. Overexpression of OVOL2 in adipocytes of mice fed with a high-fat diet reduced total body and liver fat and improved insulin sensitivity. Our data reveal that OVOL2 plays dual functions in thermogenesis and adipogenesis to maintain energy balance.

Loss of immunity-related GTPase GM4951 leads to nonalcoholic fatty liver disease without obesity.

Obesity and diabetes are well known risk factors for nonalcoholic fatty liver disease (NAFLD), but the genetic factors contributing to the development of NAFLD remain poorly understood. Here we describe two semi-dominant allelic missense mutations (Oily and Carboniferous) of Predicted gene 4951 (Gm4951) identified from a forward genetic screen in mice. GM4951 deficient mice developed NAFLD on high fat diet (HFD) with no changes in body weight or glucose metabolism. Moreover, HFD caused a reduction in the level of Gm4951, which in turn promoted the development of NAFLD. Predominantly expressed in hepatocytes, GM4951 was verified as an interferon inducible GTPase. The NAFLD in Gm4951 knockout mice was associated with decreased lipid oxidation in the liver and no defect in hepatic lipid secretion. After lipid loading, hepatocyte GM4951 translocated to lipid droplets (LDs), bringing with it hydroxysteroid 17ß-dehydrogenase 13 (HSD17B13), which in the absence of GM4951 did not undergo this translocation. We identified a rare non-obese mouse model of NAFLD caused by GM4951 deficiency and define a critical role for GTPase-mediated translocation in hepatic lipid metabolism.

Next-Generation Diprovocims with Potent Human and Murine TLR1/TLR2 Agonist Activity That Activate the Innate and Adaptive Immune Response.

The diprovocims, a new class of toll-like receptor (TLR) agonists, bear no similarity to prior TLR agonists, act through a well-defined mechanism (TLR1/TLR2 agonist), exhibit exquisite structure-activity relationships, and display in vivo adjuvant activity. They possess potent and efficacious agonist activity toward human TLR1/TLR2 but modest agonism toward the murine receptor. A manner by which diprovocims can be functionalized without impacting hTLR1/TLR2 activity is detailed, permitting future linkage to antigenic, targeting, or delivery moieties. Improvements in both potency and its low efficacy in the murine system were also achieved, permitting more effective use in animal models while maintaining the hTLR1/TLR2 activity. The prototypical member diprovocim-X exhibits the excellent potency/efficacy of diprovocim-1 in human cells, displays substantially improved potency/efficacy in mouse macrophages, and serves as an adjuvant in mice when coadministered with a nonimmunogenic antigen, indicating stimulation of the adaptive as well as innate immune response.

Modulation of autoimmune diabetes by N-ethyl-N-nitrosourea- induced mutations in non-obese diabetic mice.

Genetic association studies of type 1 diabetes (T1D) in humans, and in congenic non-obese diabetic (NOD) mice harboring DNA segments from T1D-resistant mice, face the challenge of assigning causation to specific gene variants among many within loci that affect disease risk. Here, we created random germline mutations in NOD/NckH mice and used automated meiotic mapping to identify mutations modifying T1D incidence and age of onset. In contrast with association studies in humans or congenic NOD mice, we analyzed a relatively small number of genetic changes in each pedigree, permitting implication of specific mutations as causative. Among 844 mice from 14 pedigrees bearing 594 coding/splicing changes, we identified seven mutations that accelerated T1D development, and five that delayed or suppressed T1D. Eleven mutations affected genes not previously known to influence T1D (Xpnpep1, Herc1, Srrm2, Rapgef1, Ppl, Zfp583, Aldh1l1, Col6a1, Ccdc13, Cd200r1, Atrnl1). A suppressor mutation in Coro1a validated the screen. Mutagenesis coupled with automated meiotic mapping can detect genes in which allelic variation influences T1D susceptibility in NOD mice. Variation of some of the orthologous/paralogous genes may influence T1D susceptibility in humans.

RNPS1 inhibits excessive tumor necrosis factor/tumor necrosis factor receptor signaling to support hematopoiesis in mice.

Null mutations of spliceosome components or cofactors are homozygous lethal in eukaryotes, but viable hypomorphic mutations provide an opportunity to understand the physiological impact of individual splicing proteins. We describe a viable missense allele (F181I) of Rnps1 encoding an essential regulator of splicing and nonsense-mediated decay (NMD), identified in a mouse genetic screen for altered immune cell development. Homozygous mice displayed a stem cell­intrinsic defect in hematopoiesis of all lineages due to excessive apoptosis induced by tumor necrosis factor (TNF)­dependent death signaling. Numerous transcript splice variants containing retained introns and skipped exons were detected at elevated frequencies in Rnps1F181I/F181I splenic CD8+ T cells and hematopoietic stem cells (HSCs), but NMD appeared normal. Strikingly, Tnf knockout rescued all hematopoietic cells to normal or near-normal levels in Rnps1F181I/F181I mice and dramatically reduced intron retention in Rnps1F181I/F181I CD8+ T cells and HSCs. Thus, RNPS1 is necessary for accurate splicing, without which disinhibited TNF signaling triggers hematopoietic cell death.

De novo germline mutation in the dual specificity phosphatase 10 gene accelerates autoimmune diabetes.

Insulin-dependent or type 1 diabetes (T1D) is a polygenic autoimmune disease. In humans, more than 60 loci carrying common variants that confer disease susceptibility have been identified by genome-wide association studies, with a low individual risk contribution for most variants excepting those of the major histocompatibility complex (MHC) region (40 to 50% of risk); hence the importance of missing heritability due in part to rare variants. Nonobese diabetic (NOD) mice recapitulate major features of the human disease including genetic aspects with a key role for the MHC haplotype and a series of Idd loci. Here we mapped in NOD mice rare variants arising from genetic drift and significantly impacting disease risk. To that aim we established by selective breeding two sublines of NOD mice from our inbred NOD/Nck colony exhibiting a significant difference in T1D incidence. Whole-genome sequencing of high (H)- and low (L)-incidence sublines (NOD/NckH and NOD/NckL) revealed a limited number of subline-specific variants. Treating age of diabetes onset as a quantitative trait in automated meiotic mapping (AMM), enhanced susceptibility in NOD/NckH mice was unambiguously attributed to a recessive missense mutation of Dusp10, which encodes a dual specificity phosphatase. The causative effect of the mutation was verified by targeting Dusp10 with CRISPR-Cas9 in NOD/NckL mice, a manipulation that significantly increased disease incidence. The Dusp10 mutation resulted in islet cell down-regulation of type I interferon signature genes, which may exert protective effects against autoimmune aggression. De novo mutations akin to rare human susceptibility variants can alter the T1D phenotype.

Sulfatides are endogenous ligands for the TLR4-MD-2 complex.

Many endogenous molecules, mostly proteins, purportedly activate the Toll-like receptor 4 (TLR4)-myeloid differentiation factor-2 (MD-2) complex, the innate immune receptor for lipopolysaccharide (LPS) derived from gram-negative bacteria. However, there is no structural evidence supporting direct TLR4-MD-2 activation by endogenous ligands. Sulfatides (3-O-sulfogalactosylceramides) are natural, abundant sulfated glycolipids that have variously been shown to initiate or suppress inflammatory responses. We show here that short fatty acid (FA) chain sulfatides directly activate mouse TLR4-MD-2 independent of CD14, trigger MyD88- and TRIF-dependent signaling, and stimulate tumor necrosis factor α (TNFα) and type I interferon (IFN) production in mouse macrophages. In contrast to the agonist activity toward the mouse receptor, the tested sulfatides antagonize TLR4-MD-2 activation by LPS in human macrophage-like cells. The agonistic and antagonistic activities of sulfatides require the presence of the sulfate group and are inversely related to the FA chain length. The crystal structure of mouse TLR4-MD-2 in complex with C16-sulfatide revealed that three C16-sulfatide molecules bound to the MD-2 hydrophobic pocket and induced an active dimer conformation of the receptor complex similar to that induced by LPS or lipid A. The three C16-sulfatide molecules partially mimicked the detailed interactions of lipid A to achieve receptor activation. Our results suggest that sulfatides may mediate sterile inflammation or suppress LPS-stimulated inflammation, and that additional endogenous negatively charged lipids with up to six lipid chains of limited length might also bind to TLR4-MD-2 and activate or inhibit this complex.

SLFN2 protection of tRNAs from stress-induced cleavage is essential for T cell-mediated immunity.

Reactive oxygen species (ROS) increase in activated T cells because of metabolic activity induced to support T cell proliferation and differentiation. We show that these ROS trigger an oxidative stress response that leads to translation repression. This response is countered by Schlafen 2 (SLFN2), which directly binds transfer RNAs (tRNAs) to protect them from cleavage by the ribonuclease angiogenin. T cell-specific SLFN2 deficiency results in the accumulation of tRNA fragments, which inhibit translation and promote stress-granule formation. Interleukin-2 receptor ß (IL-2Rß) and IL-2Rγ fail to be translationally up-regulated after T cell receptor stimulation, rendering SLFN2-deficient T cells insensitive to interleukin-2's mitogenic effects. SLFN2 confers resistance against the ROS-mediated translation-inhibitory effects of oxidative stress normally induced by T cell activation, permitting the robust protein synthesis necessary for T cell expansion and immunity.

Germline Saturation Mutagenesis Induces Skeletal Phenotypes in Mice.

Proper embryonic and postnatal skeletal development require coordination of myriad complex molecular mechanisms. Disruption of these processes, through genetic mutation, contributes to variation in skeletal development. We developed a high-throughput N-ethyl-N-nitrosourea (ENU)-induced saturation mutagenesis skeletal screening approach in mice to identify genes required for proper skeletal development. Here, we report initial results from live-animal X-ray and dual-energy X-ray absorptiometry (DXA) imaging of 27,607 G3 mice from 806 pedigrees, testing the effects of 32,198 coding/splicing mutations in 13,020 genes. A total of 39.7% of all autosomal genes were severely damaged or destroyed by mutations tested twice or more in the homozygous state. Results from our study demonstrate the feasibility of in vivo mutagenesis to identify mouse models of skeletal disease. Furthermore, our study demonstrates how ENU mutagenesis provides opportunities to create and characterize putative hypomorphic mutations in developmentally essential genes. Finally, we present a viable mouse model and case report of recessive skeletal disease caused by mutations in FAM20B. Results from this study, including engineered mouse models, are made publicly available via the online Mutagenetix database. © 2021 American Society for Bone and Mineral Research (ASBMR).

N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization.

Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1-/- mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.