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Enikanolaiye A*, Ruston J, Zeng R*, Taylor C, Schrock M, Buchovecky CM*, Shendure J, Acar E, Justice, MJ. 2020. Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology, Genome Research, 30(4):540-552. doi: 10.1101/gr.258400.119. PMID: 32317254.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

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Justice MJ, Hmeljak J, Sankaran VJ, Socolovsky M, Zon L. (2020) From blood development to disease: a paradigm for clinical translation. Dis Model Mech 13(1) dmm043661. doi: 10.1242/dmm.043661. PMID: 31836582

Translating basic research to the clinic is a primary aim of Disease Models & Mechanisms, and the recent successes in hematopoiesis research provide a blueprint of how fundamental biological research can provide solutions to important clinical problems. These advances were the main motivation for choosing hematopoiesis disorders as the focus of our inaugural meeting, ‘Blood Disorders: Models, Mechanisms and Therapies’, which was held in early October 2019. This Editorial discusses the reasons for and the challenges of interdisciplinary research in hematopoiesis, provides examples of how research in model systems is a key translational step towards effective treatments for blood disorders and summarizes what the community believes are the key exciting developments and challenges in this field.

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Qiu B, Ruston J, Granzier H, Justice MJ, Dowling JJ. 2019. Failure to identify modifiers of NEBULIN-related nemaline myopathy in two pre-clinical models of the disease. Biol Open 8(9). pii: bio044867. doi: 10.1242/bio.044867.

Nemaline myopathy is a rare neuromuscular disorder that affects 1 in 50,000 live births, with prevalence as high as 1 in 20,000 in certain populations. 13 genes have been linked to nemaline myopathy (NM), all of which are associated with the thin filament of the muscle sarcomere. Of the 13 associated genes, mutations in NEBULIN (NEB) accounts for up to 50% of all cases. Currently, the disease is incompletely understood and there are no available therapeutics for patients. To address this urgent need for effective treatments for patients affected by NM, we conducted a large scale chemical screen in a zebrafish model of NEB-related NM and an N-ethyl-N-nitrosourea (ENU)-based genetic screen in a mouse model of NEB exon 55 deletion, the most common NEBmutation in NM patients. Neither screen was able to identify a candidate for therapy development, highlighting the need to transition from conventional chemical therapeutics to gene-based therapies for the treatment of NM.

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Tracey, LJ*, Justice MJ. (2019). Off to a bad start: Cancer Initiation by Pluripotency Regulator PRDM14. Invited Review. Trends in Genetics. 35(7): 489-500. doi: 10.1016/j.tig.2019.04.004. PMID: 31130394

Despite advances in chemotherapies that improve cancer survival, most patients who relapse succumb to the disease due to the presence of cancer stem cells (CSCs), which are highly chemoresistant. The pluripotency factor PR domain 14 (PRDM14) has a key role in initiating many types of cancer. Normally, PRDM14 uses epigenetic mechanisms to establish and maintain the pluripotency of embryonic cells, and its role in cancer is similar. This important link between cancer and induced pluripotency is a key revelation for how CSCs may form: pluripotency genes, such as PRDM14, can expand stem-like cells as they promote ongoing DNA damage. PRDM14 and its protein-binding partners, the ETO/CBFA2T family, are ideal candidates for eliminating CSCs from relevant cancers, preventing relapse and improving long-term survival.

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Vashi N*, Justice MJ. (2019). Treating Rett syndrome: from mouse models to human therapies. Review. Mammalian Genome doi: 10.1007/s00335-019-09793-5

Rare diseases are very difficult to study mechanistically and to develop therapies for because of the scarcity of patients. Here, the rare neuro-metabolic disorder Rett syndrome (RTT) is discussed as a prototype for precision medicine, demonstrating how mouse models have led to an understanding of the development of symptoms. RTT is caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). Mecp2-mutant mice are being used in preclinical studies that target the MECP2 gene directly, or its downstream pathways. Importantly, this work may improve the health of RTT patients. Clinical presentation may vary widely among individuals based on their mutation, but also because of the degree of X chromosome inactivation and the presence of modifier genes. Because it is a complex disorder involving many organ systems, it is likely that recovery of RTT patients will involve a combination of treatments. Precision medicine is warranted to provide the best efficacy to individually treat RTT patients.

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Enikanolaiye A*, Justice MJ. (2019). Model systems inform rare disease diagnosis, therapeutic discovery and pre-clinical efficacy. Royal Society: Emerging Topics in Life Sciences. Rare Diseases: New Therapeutic Avenues 3 (1): 1 - 10

Model systems have played a large role in understanding human diseases and are instrumental in taking basic research findings to the clinic; however, for rare diseases, model systems play an even larger role. Here, we outline how model organisms are crucial for confirming causal associations, understanding functional mechanisms and developing therapies for disease. As diseases that have been studied extensively through genetics and molecular biology, cystic fibrosis and Rett syndrome are portrayed as primary examples of how genetic diagnosis, model organism development and therapies have led to improved patient health. Considering which model to use, yeast, worms, flies, fish, mice or larger animals requires a careful evaluation of experimental genetic tools and gene pathway conservation. Recent advances in genome editing will aid in confirming diagnoses and developing model systems for rare disease. Genetic or chemical screening for disease suppression may reveal functional pathway members and provide candidate entry points for developing therapies. Model organisms may also be used in drug discovery and as preclinical models as a prelude to testing treatments in patient populations. Now, model organisms will increasingly be used as platforms for understanding variation in rare disease severity and onset, thereby informing therapeutic intervention.

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Hmeljak J, Justice MJ. (2019). From gene to treatment: supporting rare disease translational research through model systems. Dis Model Mech 12(2) pii: dmm039271. doi: 10.1242/dmm.039271.

PR domain-containing 14 (Prdm14) is a pluripotency regulator central to embryonic stem cell identity and primordial germ cell specification. Genomic regions containing PRDM14 are often amplified leading to misexpression in human cancer. Prdm14 expression in mouse hematopoietic stem cells (HSC) leads to progenitor cell expansion prior to the development of T-cell acute lymphoblastic leukemia (T-ALL), consistent with PRDM14’s role in cancer initiation. Here, we demonstrate mechanistic insight into PRDM14-driven leukemias in vivo. Mass spectrometry revealed novel PRDM14-protein interactions including histone H1, RNA-binding proteins, and the master hematopoietic regulator CBFA2T3. In mouse leukemic cells, CBFA2T3 and PRDM14 associate independently of the related ETO family member CBFA2T2, PRDM14’s primary protein partner in pluripotent cells. CBFA2T3 plays crucial roles in HSC self-renewal and lineage commitment, and participates in oncogenic translocations in acute myeloid leukemia. These results suggest a model whereby PRDM14 recruits CBFA2T3 to DNA, leading to gene misregulation causing progenitor cell expansion and lineage perturbations preceding T-ALL development. Strikingly, Prdm14-induced T-ALL does not occur in mice deficient for Cbfa2t3, demonstrating that Cbfa2t3 is required for leukemogenesis. Moreover, T-ALL develops in Cbfa2t3 heterozygotes with a significantly longer latency, suggesting that PRDM14-associated T-ALL is sensitive to Cbfa2t3levels. Our study highlights how an oncogenic protein uses a native protein in progenitor cells to initiate leukemia, providing insight into PRDM14-driven oncogenesis in other cell types. IMPLICATIONS: The pluripotency regulator PRDM14 requires the master hematopoietic regulator CBFA2T3 to initiate leukemia in progenitor cells, demonstrating an oncogenic role for CBFA2T3 and providing an avenue for targeting cancer-initiating cells.

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Tracey, LJ*, Brooke-Bisschop, T, Jansen, P, Campos, EI, Vermeulen, M, and Justice, MJ. (2019) The pluripotency regulator PRDM14 requires hematopoietic regulator CBFA2T3 to initiate leukemia in mice. Molecular Cancer Research 17(7): 489-500, doi: 10.1158/1541-7786.MCR-18-1327.

PR domain-containing 14 (Prdm14) is a pluripotency regulator central to embryonic stem cell identity and primordial germ cell specification. Genomic regions containing PRDM14 are often amplified leading to misexpression in human cancer. Prdm14 expression in mouse hematopoietic stem cells (HSC) leads to progenitor cell expansion prior to the development of T-cell acute lymphoblastic leukemia (T-ALL), consistent with PRDM14’s role in cancer initiation. Here, we demonstrate mechanistic insight into PRDM14-driven leukemias in vivo. Mass spectrometry revealed novel PRDM14-protein interactions including histone H1, RNA-binding proteins, and the master hematopoietic regulator CBFA2T3. In mouse leukemic cells, CBFA2T3 and PRDM14 associate independently of the related ETO family member CBFA2T2, PRDM14’s primary protein partner in pluripotent cells. CBFA2T3 plays crucial roles in HSC self-renewal and lineage commitment, and participates in oncogenic translocations in acute myeloid leukemia. These results suggest a model whereby PRDM14 recruits CBFA2T3 to DNA, leading to gene misregulation causing progenitor cell expansion and lineage perturbations preceding T-ALL development. Strikingly, Prdm14-induced T-ALL does not occur in mice deficient for Cbfa2t3, demonstrating that Cbfa2t3 is required for leukemogenesis. Moreover, T-ALL develops in Cbfa2t3 heterozygotes with a significantly longer latency, suggesting that PRDM14-associated T-ALL is sensitive to Cbfa2t3levels. Our study highlights how an oncogenic protein uses a native protein in progenitor cells to initiate leukemia, providing insight into PRDM14-driven oncogenesis in other cell types. IMPLICATIONS: The pluripotency regulator PRDM14 requires the master hematopoietic regulator CBFA2T3 to initiate leukemia in progenitor cells, demonstrating an oncogenic role for CBFA2T3 and providing an avenue for targeting cancer-initiating cells.

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Moore BA, Leonard BC, Sebbag L, Edwards SG, Cooper A, Imai DM, Straiton E, Santos L, Reilly C, Griffey SM, Bower L, Clary D, Mason J, Roux MJ, Meziane H, Herault Y; International Mouse Phenotyping Consortium, McKerlie C, Flenniken AM, Nutter LMJ, Berberovic Z, Owen C, Newbigging S, Adissu H, Eskandarian M, Hsu CW, Kalaga S, Udensi U, Asomugha C, Bohat R, Gallegos JJ, Seavitt JR, Heaney JD, Beaudet AL, Dickinson ME, Justice MJ, … , Moshiri A. Identification of genes required for eye development by high-throughput screening of mouse knockouts. Commun Biol. 2018 Dec 21;1:236. doi: 10.1038/s42003-018-0226-0.

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.

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Albrecht, NE, Alevy, J, Jiang, D, Burger, CA, Liu, BI, Li, F, Wang, J, Kim, S-Y, Hsu, C-W, Kalaga, S, Udensi, U, Asomugha, C, Bohat, R, Gaspero, A, Justice, MJ, Westenskow, PD, Yamamoto, S, Seavitt, JR, Beaudet, AL, Dickinson, ME, Samuel, MA. (2018). Rapid and integrative discovery of retina regulatory molecules. Cell Reports. 24(9): 2506-2519.

Retinal function relies on precisely organized neurons and synapses and a properly patterned vasculature to support them. Alterations in these features can result in vision loss. However, our understanding of retinal organization pathways remains incomplete because of a lack of methods to rapidly identify neuron and vasculature regulators in mammals. Here we developed a pipeline for the identification of neural and synaptic integrity genes by high-throughput retinal screening (INSiGHT) that analyzes candidate expression, vascular patterning, cellular organization, and synaptic arrangement. Using this system, we examined 102 mutant mouse lines and identified 16 unique retinal regulatory genes. Fifteen of these candidates are identified as novel retina regulators, and many (9 of 16) are associated with human neural diseases. These results expand the genetic landscape involved in retinal circuit organization and provide a road map for continued discovery of mammalian retinal regulators and disease-causing alleles.

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Kyle SM, Vashi N, Justice MJ. 2018. Rett syndrome: a neurological disorder with metabolic components. Open Biol. 2018 Feb;8(2). pii: 170216. doi: 10.1098/rsob.170216. Review.

Rett syndrome (RTT) is a neurological disorder caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2), a ubiquitously expressed transcriptional regulator. Despite remarkable scientific progress since its discovery, the mechanism by which MECP2 mutations cause RTT symptoms is largely unknown. Consequently, treatment options for patients are currently limited and centred on symptom relief. Thought to be an entirely neurological disorder, RTT research has focused on the role of MECP2 in the central nervous system. However, the variety of phenotypes identified in Mecp2 mutant mouse models and RTT patients implicate important roles for MeCP2 in peripheral systems. Here, we review the history of RTT, highlighting breakthroughs in the field that have led us to present day. We explore the current evidence supporting metabolic dysfunction as a component of RTT, presenting recent studies that have revealed perturbed lipid metabolism in the brain and peripheral tissues of mouse models and patients. Such findings may have an impact on the quality of life of RTT patients as both dietary and drug intervention can alter lipid metabolism. Ultimately, we conclude that a thorough knowledge of MeCP2’s varied functional targets in the brain and body will be required to treat this complex syndrome.

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Karp NA, Mason J, Beaudet AL, Benjamini Y, Bower L, Braun RE, Brown SDM, Chesler EJ, Dickinson ME, Flenniken AM, Fuchs H, Angelis MH, Gao X, Guo S, Greenaway S, Heller R, Herault Y, Justice MJ, Kurbatova N, Lelliott CJ, Lloyd KCK, Mallon AM, Mank JE, Masuya H, McKerlie C, Meehan TF, Mott RF, Murray SA, Parkinson H, Ramirez-Solis R, Santos L, Seavitt JR, Smedley D, Sorg T, Speak AO, Steel KP, Svenson KL; International Mouse Phenotyping Consortium, Wakana S, West D, Wells S, Westerberg H, Yaacoby S, White JK. 2017. Prevalence of sexual dimorphism in mammalian phenotypic traits. Nat Commun. 2017 Jun 26;8:15475. doi: 10.1038/ncomms15475.

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans.

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Nguyen AT, Prado MA, Schmidt PJ, Sendamarai AK, Wilson-Grady JT, Min M, Campagna DR, Tian G, Shi Y, Dederer V, Kawan M, Kuehnle N, Paulo JA, Yao Y, Weiss MJ, Justice MJ, Gygi SP, Fleming MD, Finley D. 2017. UBE2O remodels the proteome during terminal erythroid differentiation. Science. 2017 Aug 4;357(6350). pii: eaan0218. doi: 10.1126/science.aan0218.

During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o-/- mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.

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Bowl MR, Simon MM, Ingham NJ, Greenaway S, Santos L, Cater H, Taylor S, Mason J, Kurbatova N, Pearson S, Bower LR, Clary DA, Meziane H, Reilly P, Minowa O, Kelsey L; International Mouse Phenotyping Consortium, Tocchini-Valentini GP, Gao X, Bradley A, Skarnes WC, Moore M, Beaudet AL, Justice MJ, …, Brown SDM. 2017. A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction. Nat Commun. 2017 Oct 12;8(1):886. doi: 10.1038/s41467-017-00595-4.

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.

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Meehan TF, Conte N, West DB, Jacobsen JO, Mason J, Warren J, …, Justice MJ, Murray SA, Svenson KL, Braun RE, White JK, Bradley A, Flicek P, Wells S, Skarnes WC, Adams DJ, Parkinson H, Mallon AM, Brown SDM, Smedley D. 2017. Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium. Nat Genet. 49(8):1231-1238

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

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Dharmadhikari, AV, Sun, JJ, Gogolewski, K, Carofino, BL, Ustiyan, V, Hill, M, Majewski, T, Szafranski, P, Justice, MJ, Ray, RS, Dickinson, ME, Kaliniche nko, VV, Gambin, A and Stankiewicz, P. 2016. Lethal lung hypoplasia and vascular defects in mice with conditional Foxf1 overexpression. Biol Open Sep 16. pii: bio.019208

FOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with the neonatally lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in any human disease conditions. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking-in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 embryonic day (E)15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage-sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV.

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Kyle, SM, Saha, PK, Brown, HM, Chan, LC, and Justice, MJ. 2016. MeCP2 co-ordinates liver lipid metabolism with the NCoR1/HDAC3 corepressor complex. Human Molecular Genetics, 25(14): 3029-3041.

Rett syndrome (RTT; OMIM 312750), a progressive neurological disorder, is caused by mutations in methyl-CpG-binding protein 2 (MECP2; OMIM 300005), a ubiquitously expressed factor. A genetic suppressor screen designed to identify therapeutic targets surprisingly revealed that downregulation of the cholesterol biosynthesis pathway improves neurological phenotypes in Mecp2 mutant mice. Here, we show that MeCP2 plays a direct role in regulating lipid metabolism. Mecp2 deletion in mice results in a host of severe metabolic defects caused by lipid accumulation, including insulin resistance, fatty liver, perturbed energy utilization, and adipose inflammation by macrophage infiltration. We show that MeCP2 regulates lipid homeostasis by anchoring the repressor complex containing NCoR1 and HDAC3 to its lipogenesis targets in hepatocytes. Consistently, we find that liver targeted deletion of Mecp2 causes fatty liver disease and dyslipidemia similar to HDAC3 liver-specific deletion. These findings position MeCP2 as a novel component in metabolic homeostasis. Rett syndrome patients also show signs of peripheral dyslipidemia; thus, together these data suggest that RTT should be classified as a neurological disorder with systemic metabolic components. We previously showed that treatment of Mecp2 mice with statin drugs alleviated motor symptoms and improved health and longevity. Lipid metabolism is a highly treatable target; therefore, our results shed light on new metabolic pathways for treatment of Rett syndrome.

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Dickinson, M.E. and the International Mouse Phenotyping Consortium (of 83 authors, MJ Justice is one of the senior authors). 2016. High-throughput discovery of novel developmental phenotypes. Nature 537(7621): 508-514.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

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Carofino, B.L., Ayanga, B., Tracey, L, Brooke-Bisschop, T., and Justice, M.J. 2016. PRDM14 promotes RAG-dependent Notch1 driver mutations in mouse T-ALL. Biology Open 5(5): 645-653

PRDM14 is an epigenetic regulator known for maintaining embryonic stem cell identity and resetting potency in primordial germ cells. However, hematopoietic expression of Prdm14 at supraphysiological levels results in fully penetrant and rapid-onset T-cell acute lymphoblastic leukemia (T-ALL) in the mouse. Here, we show that PRDM14-induced T-ALLs are driven by NOTCH1, a frequently mutated driver of human T-ALL. Notch1 is activated in this murine model via RAG-dependent promoter deletions and subsequent production of truncated, ligand-independent protein from downstream regions of the Notch1 locus. These T-ALLs also have focal changes in H3K4me3 deposition at the Notch1 locus and global increases in both H3K4me1 and H3K4me3. Using a PRDM14-FLAG mouse model, we show that PRDM14 binds within an intron of Notch1 prior to leukemia development. Our data support the idea that PRDM14 binding promotes a chromatin state that allows access of the RAG recombinase complex to cryptic RAG signal sequences embedded at the Notch1 locus. Indeed, breeding into a RAG recombination-deficient background abrogates T-ALL development and prevents Notch1 deletions, while allowing for transient hematopoietic stem cell (HSC)-like pre-leukemia cell expansion. Together, our data suggest that PRDM14 expands a progenitor cell population while promoting a permissive epigenetic state for the creation of driver mutations (here, in Notch1), enabling cancer development through the misappropriation of endogenous cellular DNA recombination machinery.

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Carofino, BL and Justice, MJ. 2015. Tissue-specific regulation of oncogene expression using Cre-inducible ROSA26 knock-in transgenic mice, Current Protocols in Mouse Biology 5(2): 187 - 204.

Cre-inducible mouse models are often utilized for the spatial and temporal expression of oncogenes. With the wide number of Cre recombinase lines available, inducible transgenesis represents a tractable approach to achieve discrete oncogene expression. Here, we describe a protocol for targeting Cre-inducible genes to the ubiquitously expressed ROSA26 locus. Gene targeting provides several advantages over standard transgenic techniques, including a known site of integration and previously characterized pattern of expression. Historically, an inherent instability of ROSA26 targeting vectors has hampered the efficiency of developing ROSA26 knock-in lines. In this protocol, we provide individual steps for utilizing Gateway recombination for cloning as well as detailed instructions for screening targeted ES cell clones. By following this protocol, one can achieve germline transmission of a ROSA26 knock-in line within several months.

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Sen, P, Dharmadhikari, AV, Majewski, T, Mohammad, M, Kalin, TV, Zabielska, J, Ren, X, Bray, M, Brown, H, Welty, S, Thevananther, S, Langston, C, Szafranski, P, Justice, MJ, Kalinichenko, VV, Gambin, A, Belmont, J, and Stankiewicz, P 2014. Comparative analyses of lung transcriptomes in patients with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins and in Foxf1 heterozygous knockout mice. PLoS One 9(4): 394390.

Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a developmental disorder of the lungs, primarily affecting their vasculature. FOXF1 haploinsufficiency due to heterozygous genomic deletions and point mutations have been reported in most patients with ACDMPV. The majority of mice with heterozygous loss-of-function of Foxf1 exhibit neonatal lethality with evidence of pulmonary hemorrhage in some of them. By comparing transcriptomes of human ACDMPV lungs with control lungs using expression arrays, we found that several genes and pathways involved in lung development, angiogenesis, and in pulmonary hypertension development, were deregulated. Similar transcriptional changes were found in lungs of the postnatal day 0.5 Foxf1+/- mice when compared to their wildtype littermate controls; 14 genes, COL15A1, COL18A1, COL6A2, ESM1, FSCN1, GRINA, IGFBP3, IL1B, MALL, NOS3, RASL11B, MATN2, PRKCDBP, and SIRPA, were found common to both ACDMPV and Foxf1 heterozygous lungs. Our results advance knowledge toward understanding of the molecular mechanism of ACDMPV, lung development, and its vasculature pathology. These data may also be useful for understanding etiologies of other lung disorders, e.g. pulmonary hypertension, bronchopulmonary dysplasia, or cancer.

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Allache, R, Lachance, S, Guyot, MC, De Marco, P, Merello, E, Justice, MJ, Capra, V and Kibar, Z 2014. Novel mutations in Lrp6 orthologues in mouse and human neural tube defects affect a highly dosage-sensitive Wnt non canonical planar cell polarity pathway. Human Molecular Genetics 23(7): 1687-1699. PMID: 24203697.

Wnt signaling has been classified as canonical Wnt/β-catenin-dependent or non-canonical planar cell polarity (PCP) pathway. Misregulation of either pathway is linked mainly to cancer or neural tube defects (NTDs), respectively. Both pathways seem to antagonize each other, and recent studies have implicated a number of molecular switches that activate one pathway while simultaneously inhibiting the other thereby partially mediating this antagonism. The lipoprotein receptor-related protein Lrp6 is crucial for the activation of the Wnt/β-catenin pathway, but its function in Wnt/PCP signaling remains largely unknown. In this study, we investigate the role of Lrp6 as a molecular switch between both Wnt pathways in a novel ENU mouse mutant of Lrp6 (Skax26(m1Jus)) and in human NTDs. We demonstrate that Skax26(m1Jus) represents a hypermorphic allele of Lrp6 with increased Wnt canonical and abolished PCP-induced JNK activities. We also show that Lrp6(Skax26-Jus) genetically interacts with a PCP mutant (Vangl2(Lp)) where double heterozygotes showed an increased frequency of NTDs and defects in cochlear hair cells’ polarity. Importantly, our study also demonstrates the association of rare and novel missense mutations in LRP6 that is an inhibitor rather than an activator of the PCP pathway with human NTDs. We show that three LRP6 mutations in NTDs led to a reduced Wnt canonical activity and enhanced PCP signaling. Our data confirm an inhibitory role of Lrp6 in PCP signaling in neurulation and indicate the importance of a tightly regulated and highly dosage-sensitive antagonism between both Wnt pathways in this process.

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Justice, MJ, Buchovecky, CM, Kyle, SM and Djukic, A 2014. A role for metabolism in Rett Syndrome pathogenesis: new clinical findings and potential treatment targets. Rare Diseases 1, e27265.

Rett syndrome (RTT), an X-linked neurological disorder caused by mutations in MECP2, may have a metabolic component. We reported a genetic suppressor screen in a Mecp2-null mouse model to identify pathways for therapeutic improvement of RTT symptoms. Of note, one suppressor mutation implied that cholesterol homeostasis was perturbed in Mecp2 null mice; indeed, cholesterol synthesis was elevated in the brain and body system. Remarkably, the genetic effect of downregulating the cholesterol pathway could be mimicked chemically by statin drugs, improving motor symptoms, and increasing longevity in the mouse. Our work linked cholesterol metabolism to RTT pathology for the first time. Both neurological and systemic effects of perturbed cholesterol homeostasis overlap with many RTT symptoms. Here we show in patients that peripheral cholesterol, triglycerides, and/or LDLs may be elevated early in RTT disease onset, providing a biomarker for patients that could be aided by therapeutic interventions that modulate lipid metabolism.

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Buchovecky, CM, Hill, MG, Borkey, JM, Kyle, SM, and Justice, MJ 2013. Evaluation of Rett syndrome symptom improvement by metabolic modulators in Mecp2-mutant mice. Current Protocols in Mouse Biology, 3: 187-204.

Mouse models recapitulate many symptoms of Rett Syndrome, an X-linked disorder caused by mutations in methyl-CpG-binding protein 2 (MECP2). The study of Mecp2-null male mice has provided insight into pathogenesis of the disorder-most recently, dysregulation of cholesterol and lipid metabolism. Perisymptomatic treatment with statin drugs successfully mitigates the effects of this metabolic syndrome, increases longevity, and improves motor function. Described here is a metabolic drug screening protocol and timeline for symptom evaluation in Mecp2-mutant mice. Specifically, mice are treated twice weekly with a compound of interest alongside subjective health assessments, bi-weekly body composition measurements, and blood chemistries. Throughout treatment, behavioral phenotyping tests are carried out at specific time points. This protocol is highly adaptable to other neurological diseases; however, the time for completion depends on the specific mutant model under study. The protocol highlights the use of techniques described in several different Current Protocols in Mouse Biology articles to carry out testing in a preclinical model. Curr. Protoc. Mouse Biol. 3:187-204 © 2013 by John Wiley & Sons, Inc.

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Probst, FJ, Corrigan, RR, Gaudio, D, Salinger, AP, Lorenzo, I, Gao, S, Chiu, I, Xia, A, Oghalai, JS, and Justice, MJ 2013. A Point Mutation in the Gene for Asparagine-Linked Glycosylation 10B (Alg10b) Causes Nonsyndromic Hearing Impairment in Mice. PLoS One 8 (11):e80408.

The study of mouse hearing impairment mutants has led to the identification of a number of human hearing impairment genes and has greatly furthered our understanding of the physiology of hearing. The novel mouse mutant neurological/sensory 5 (nse5) demonstrates a significantly reduced or absent startle response to sound and is therefore a potential murine model of human hearing impairment. Genetic analysis of 500 intercross progeny localized the mutant locus to a 524 kilobase (kb) interval on mouse chromosome 15. A missense mutation in a highly-conserved amino acid was found in the asparagine-linked glycosylation 10B gene (Alg10b), which is within the critical interval for the nse5 mutation. A 20.4 kb transgene containing a wildtype copy of the Alg10b gene rescued the mutant phenotype in nse5/nse5 homozygous animals, confirming that the mutation in Alg10b is responsible for the nse5/nse5 mutant phenotype. Homozygous nse5/nse5 mutants had abnormal auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and cochlear microphonics (CMs). Endocochlear potentials (EPs), on the other hand, were normal. ABRs and DPOAEs also confirmed the rescue of the mutant nse5/nse5 phenotype by the wildtype Alg10b transgene. These results suggested a defect in the outer hair cells of mutant animals, which was confirmed by histologic analysis. This is the first report of mutation in a gene involved in the asparagine (N)-linked glycosylation pathway causing nonsyndromic hearing impairment, and it suggests that the hearing apparatus, and the outer hair cells in particular, are exquisitely sensitive to perturbations of the N-linked glycosylation pathway.

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Carofino, BL, Ayanga, B, and Justice, MJ 2013. A mouse model for inducible overexpression of the pluripotency factor Prdm14 results in rapid-onset highly penetrant T-cell acute lymphoblastic leukemia (T-ALL), Disease Models and Mechanisms 6(6): 1494-1506. PMID: 24046360

PRDM14 functions in embryonic stem cell (ESC) maintenance to promote the expression of pluripotency-associated genes while suppressing differentiation genes. Expression of PRDM14 is tightly regulated and typically limited to ESCs and primordial germ cells; however, aberrant expression is associated with tumour initiation in a wide variety of human cancers, including breast cancer and leukemia. Here, we describe the generation of a Cre-recombinase-inducible mouse model for the spatial and temporal control of Prdm14 misexpression [ROSA26 floxed-stop Prdm14 (R26PR)]. When R26PR is mated to either of two Cre lines, Mx1-cre or MMTV-cre, mice develop early-onset T-cell acute lymphoblastic leukemia (T-ALL) with median overall survival of 41 and 64 days for R26PR;Mx1-cre and R26PR;MMTV-cre, respectively. T-ALL is characterized by the accumulation of immature single-positive CD8 cells and their widespread infiltration. Leukemia is preceded by a dramatic expansion of cells resembling hematopoietic stem cells and lymphoid-committed progenitors prior to disease onset, accompanied by a blockage in B-cell differentiation at the early pro-B stage. Rapid-onset PRDM14-induced T-ALL requires factors that are present in stem and progenitor cells: R26PR;dLck-cre animals, which express Prdm14 starting at the double-positive stage of thymocyte development, do not develop disease. PRDM14-induced leukemic cells contain high levels of activated NOTCH1 and downstream NOTCH1 targets, including MYC and HES1, and are sensitive to pharmacological inhibition of NOTCH1 with the γ-secretase inhibitor DAPT. Greater than 50% of human T-ALLs harbour activating mutations in NOTCH1; thus, our model carries clinically relevant molecular aberrations. The penetrance, short latency and involvement of the NOTCH1 pathway will make this hematopoietic R26PR mouse model ideal for future studies on disease initiation, relapse and novel therapeutic drug combinations. Furthermore, breeding R26PR to additional Cre lines will allow for the continued development of novel cancer models.

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Buchovecky, CM, Turley, SC, Brown, HM, Kyle, SM, McDonald, JG, Liu, B, Pieper, A, Huang, W, Katz, D, Russell, DW, Shendure, J, and Justice, MJ 2013. A suppressor screen in mouse Mecp2 implicates cholesterol metabolism in Rett syndrome, Nature Genetics45(9): 1013-1020. PMID:23892605.

Mutations in MECP2, encoding methyl CpG-binding protein 2, cause Rett syndrome, the most severe autism spectrum disorder. Re-expressing Mecp2 in symptomatic Mecp2-null mice markedly improves function and longevity, providing hope that therapeutic intervention is possible in humans. To identify pathways in disease pathology for therapeutic intervention, we carried out a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis suppressor screen in Mecp2-null mice and isolated five suppressors that ameliorate the symptoms of Mecp2 loss. We show that a stop codon mutation in Sqle, encoding squalene epoxidase, a rate-limiting enzyme in cholesterol biosynthesis, underlies suppression in one line. Subsequently, we also show that lipid metabolism is perturbed in the brains and livers of Mecp2-null male mice. Consistently, statin drugs improve systemic perturbations of lipid metabolism, alleviate motor symptoms and confer increased longevity in Mecp2 mutant mice. Our genetic screen therefore points to cholesterol homeostasis as a potential target for the treatment of patients with Rett syndrome.

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Simko, SJ, Voicu, H, Carofino, BL, and Justice, MJ 2012. Mouse lymphoblastic leukemias induced by aberrant Prdm14 expression demonstrate widespread copy number alterations also found in human ALL. Cancers 4: 1050-1066. PMID: 23487523

Aberrant expression and activation of oncogenes in somatic cells has been associated with cancer initiation. Required for reacquisition of pluripotency in the developing germ cell, PRDM14 initiates lymphoblastic leukemia when misexpressed in murine bone marrow. Activation of pluripotency in somatic cells can lead to aneuploidy and copy number alterations during iPS cell generation, and we hypothesized that PRDM14-induced lymphoblastic leukemias would demonstrate significant chromosomal damage. High-resolution oligo array comparative genomic hybridization demonstrated infrequent aneuploidy but frequent amplification and deletion, with amplifications occurring in a 5:1 ratio with deletions. Many deletions (i.e., Cdkn2a, Ebf1, Pax5, Ikzf1) involved B-cell development genes and tumour suppressor genes, recapitulating deletions occurring in human leukemia. Pathways opposing senescence were frequently deactivated via Cdkn2a deletion or Tbx2 amplification, with corollary gene expression. Additionally, gene expression studies of abnormal pre-leukemic B-precursors showed downregulation of genes involved in chromosomal stability (i.e.,Xrcc6) and failure to upregulate DNA repair pathways. We propose a model of leukemogenesis, triggered by pluripotency genes like Prdm14, which involves ongoing DNA damage and failure to activate non-homologous end-joining secondary to aberrant gene expression.

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Dettman, EJ, Simko, SJ, Ayanga, B, Carofino, B, Margolin, J, Morse, HC, and Justice, MJ 2011. Prdm14 initiates lymphoblastic leukemia after expanding a population of cells resembling common lymphoid progenitors. Oncogene30(25):2859-73 PMID:21339739

Understanding the heterogeneous genetic mechanisms of tumour initiation in lymphoid leukemias (LL) will lead to improvements in prognostic classification and treatment regimens. In previous studies of mouse leukemias, we showed that retroviral insertion at the ecotropic viral insertion site 32 locus leads to increased expression of Prdm14, a pluripotency gene implicated in the self-renewal capacity of embryonic stem cells and the early stages of breast cancer. Here, we show that PRDM14 is also overexpressed in ∼25% of human lymphoid neoplasms, with increased frequencies in T-cell acute LL and hyperdiploid precursor B-cell acute LL. To test if Prdm14 over expression could initiate leukemia, mice were transduced with bone marrow cells transfected with a Prdm14 expression vector. LLs developed in 96% of female mice and 42% of male mice. Before the onset of leukemia, differentiation of transduced cells was biased up to 1000-fold toward cells with features of common lymphoid progenitors (CLPs), and lymphoid differentiation showed a relative block at the pro-B stage. Microarray gene expression analysis of expanded CLP-like cells before the onset of leukemia demonstrated upregulation of genes involved in pluripotency, tumour initiation, early B-lineage commitment, Wnt/Ras signaling and the epithelial-to-mesenchymal transition. Among the dysregulated genes were imprinted genes and non-coding RNAs including Dlk1 and Meg3, which are also key pluripotency mediators. Heightened expression of the estrogen-dependent oncogene, Myb, in tumours suggests a basis for the increased frequency of cancer in female mice. These data provide the first direct evidence for the association of Prdm14 with cancer initiation in an in vivo mouse model and in human lymphoid malignancies, while suggesting mechanisms for Prdm14’s mode of action.

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Fairfield H, Gilbert GJ, Barter M, Corrigan RR, Curtain M, Ding Y, D'Ascenzo M, Gerhardt DJ, He C, Huang W, Richmond T, Rowe L, Probst FJ, Bergstrom DE, Murray SA, Bult C, Richardson J, Kile BT, Gut I, Hager J, Sigurdsson S, Mauceli E, Di Palma F, Lindblad-Toh K, Cunningham ML, Cox TC, Justice MJ, Spector MS, Lowe SW, Albert T, Donahue LR, Jeddeloh J, Shendure J, Reinholdt LG. 2011. Mutation discovery in mice by whole exome sequencing. Genome Biol. 2011 Sep 14;12(9):R86. PMID: 21917142

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.

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Zheng L, Michelson Y, Freger V, Avraham Z, Venken KJ, Bellen HJ, Justice MJ*, Wides R*. 2011. Drosophila Ten-m and filamin affect motor neuron growth cone guidance. PLoS One. 2011;6(8):e22956. PMID: 21857973 *Joint corresponding authors.

The Drosophila Ten-m (also called Tenascin-major, or odd Oz (odz)) gene has been associated with a pair-rule phenotype. We identified and characterized new alleles of Drosophila Ten-m to establish that this gene is not responsible for segmentation defects but rather causes defects in motor neuron axon routing. In Ten-m mutants the inter-segmental nerve (ISN) often crosses segment boundaries and fasciculates with the ISN in the adjacent segment. Ten-m is expressed in the central nervous system and epidermal stripes during the stages when the growth cones of the neurons that form the ISN navigate to their targets. Over-expression of Ten-m in epidermal cells also leads to ISN misrouting. We also found that Filamin, an actin binding protein, physically interacts with the Ten-m protein. Mutations in cheerio, which encodes Filamin, cause defects in motor neuron axon routing like those of Ten-m. During embryonic development, the expression of Filamin and Ten-m partially overlap in ectodermal cells. These results suggest that Ten-m and Filamin in epidermal cells might together influence growth cone progression.

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