Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci

Westrick, Randal J.; Tomberg, Kärt; Siebert, Amy E; Zhu, Guojing; Winn, Mary E.; Dobies, Sarah L.; Manning, Sara L.; Brake, Marisa A; Cleuren, Audrey C. A.; Hobbs, Linzi M; Mishack, Lena M; Johnston, Alexander; Kotnik, Emilee N.; Siemieniak, David; Xu, Jishu; Li, Jun Z.; Saunders, Thomas L.; Ginsburg, David

doi:10.1073/pnas.1705762114

Cited by 14 publications

(21 citation statements)

References 62 publications

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“…The suppressor screen reported here represents the largest screen yet carried out in the mouse, and, by using massively parallel sequencing and association analysis, the screen identifies the largest number of candidate genes. Applications of modifier screens in the mouse are powerful (Carpinelli et al 2004;Matera et al 2008;Westrick et al 2017). Even so, the mutations are identified based on their ability to suppress or enhance a mutant phenotype.…”

Section: Discussionmentioning

confidence: 99%

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

et al. 2020

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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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Section: Discussionmentioning

confidence: 99%

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

et al. 2020

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“…Generation of vector and target-specific guide RNA (sgRNA) was designed as described (Westrick et al 2017). The three RBM48-specific sgRNAs (Table S2) Integrated DNA Technologies (IDT, Coralville, IL), is listed in Table S2 and described in Figure S1.…”

Section: Design and Generation Of Rbm48-targeting Crispr/cas9 Construmentioning

confidence: 99%

Genetic Analysis of Human RNA Binding Motif Protein 48 (RBM48) Reveals an Essential Role in U12-Type Intron Splicing

Siebert

Corll

Gronevelt

et al. 2020

Preprint

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U12-type (minor) introns are found in most multicellular eukaryotes, and constitute ~0.5% of all introns in species with a minor spliceosome required for their splicing. However, the biological relevance of U12-type introns is not well understood. It is known that mutations resulting in aberrant U12-type intron splicing cause developmental defects in both plants and animals. We recently reported that maize RNA Binding Motif Protein 48 (RBM48) is an essential splicing factor for U12-type introns. Maize rbm48 mutants display aberrant genome-wide U12-type intron splicing. This leads to severe defects in endosperm development, resulting in non-viable seeds. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to establish the evolutionary conservation of RBM48 dependent U12-type intron splicing between maize and humans. Comparative RNA-seq analysis performed on RBM48 deficient human cell lines and maize endosperm defined a subset of orthologous minor U12-type containing genes (MIGs) displaying aberrant splicing of U12-type introns in both species. Mutations in the majority of these MIGs have been reported to cause developmental defects in both plants and animals. Thus, a comparison of RNA-seq data between distantly related species containing mutations in RBM48 identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects. Our results elucidate deeply conserved post-transcriptional processing mechanisms that are required for normal growth and development of eukaryotes with a minor spliceosome.

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“…In contrast, the phenotype-based genetic screening can be a powerful method to rapidly reveal new gene targets of therapeutic potential, as evidenced by those genetic suppressor screens in lower animal models (7)(8)(9)(10)(11)(12). However, conducting genetic suppressor screen can be extremely difficult in vertebrates because of the challenge of colony management efforts (13)(14)(15). To overcome this bottleneck, we turned our attention to the experimentally accessible zebrafish model, and have successfully established an insertional mutagenesis screen-based platform for discovering genetic modifiers of cardiomyopathies (16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

Retinoid X receptor alpha is a spatiotemporally-specific therapeutic target for doxorubicin-induced cardiomyopathy in adult zebrafish

Ding

Zhang

et al. 2018

Preprint

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While the genetic suppressor screen is efficient in suggesting therapeutic genes, this strategy has yet to be successful for cardiomyopathies in vertebrates. To develop such a strategy, we recently established a mutagenesis screen platform in zebrafish for systematic discovery of genetic modifiers of doxorubicin-induced cardiomyopathy (DIC). Here, we further revealed both molecular and cellular insights of the first salutary modifier emerged from the screen, i.e. gene-breaking transposon (GBT) 0419 that affects the retinoid X receptor alpha a (rxraa) gene.First, by rescuing the mutation in tissue-specific manner with multiple Cre-loxP systems, we demonstrated that the endothelial, but not myocardial or epicardial, function of rxraa is primary to this cardioprotective effects. Next, we showed that the rxraa-associated salutary effects on DIC were conferred partially by the activation of retinoid acid (RA) signaling. Finally, we identified isotretinoin and bexarotene, 2 US Food and Drug Administration-approved RXRA agonists that are effective in treating adult zebrafish DIC when administered during the early, but not the late, phase of DIC progression. Collectively, we provided the first in vivo genetic evidence in supporting RXRA as the therapeutic target for DIC, and uncovered a previously unrecognized spatiotemporally-restricted mechanism for this gene-based therapeutic strategy. Our study also justified that searching salutary modifiers via zebrafish mutagenesis screen can be effective in discovering new therapeutic targets for cardiomyopathies.

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Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci

Cited by 14 publications

References 62 publications

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

Genetic Analysis of Human RNA Binding Motif Protein 48 (RBM48) Reveals an Essential Role in U12-Type Intron Splicing

Retinoid X receptor alpha is a spatiotemporally-specific therapeutic target for doxorubicin-induced cardiomyopathy in adult zebrafish

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