Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression

West, David B.; Engelhard, Eric K.; Adkisson, Michael; Nava, Alessia; Kirov, Julia V.; Cipollone, Andreanna; Willis, Brandon; Rapp, Jared; Jong, Pieter J. de; Lloyd, Kevin C K

doi:10.1371/journal.pgen.1005691

Cited by 30 publications

(42 citation statements)

References 72 publications

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“…Insertion of foreign DNA fragments into the genome may have unintended effects on the expression and function of neighboring genes. In a recent study, West et al (15) have used RNA-seq to analyze in a systematic manner the transcriptional impact of targeted mouse mutations on local changes in gene expression. They measured the expression levels of genes within +/− 500 kb of the targeted gene in tissues from 44 homozygous mutants generated by the International Knockout Mouse Consortium (16) compared with C57BL/6N inbred control mice.…”

Section: Resultsmentioning

confidence: 99%

“…The targeting strategies insert a bacterial beta-galactosidase reporter ( lacZ gene) downstream of the target gene endogenous promoter and a neomycin resistance selection cassette, similar to the strategy used to produce Mapk6 lacZ mutant mice. The authors found that one or more neighboring genes is down-regulated or up-regulated in up to 59% of the mouse mutants, depending on the targeting approach (15). Various mechanisms have been proposed to explain this local transcriptional dysregulation, including disruption of regulatory and insulating elements with cis-acting consequences and effects of the exogenous neo r promoter on expression of 3’ genes.…”

Section: Resultsmentioning

confidence: 99%

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Reevaluation of the Role of ERK3 in Perinatal Survival and Post-Natal Growth Using New Genetically-Engineered Mouse Models

Soulez¹,

Saba-El-Leil²,

Turgeon

et al. 2018

Preprint

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38The physiological functions of the atypical MAP kinase ERK3 remain poorly characterized. 39Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus 40 (Mapk6 lacZ ) showed that inactivation of ERK3 in Mapk6 lacZ mice leads to perinatal lethality 41 associated with intrauterine growth restriction, defective lung maturation, and neuromuscular 42 anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel 43 mouse models expressing a catalytically-inactive (Mapk6 KD ) or conditional (Mapk6  ) allele of 44 ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive 45 the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant 46 mice reached adulthood, were fertile and showed no apparent health problem. However, analysis 47 56 57 Extracellular signal-regulated kinase 3 (ERK3) and ERK4 are atypical members of the mitogen-58 activated protein (MAP) kinase family (1). Despite their identification more than 25 years ago, 59 very little is known about their physiological functions. The ERK3 gene (MAPK6 in human) is 60 expressed ubiquitously in adult mammalian tissues, while ERK4 gene (MAPK4) shows a 61 restricted expression profile being detected mainly in brain tissue (2-4) 62 (www.gtexportal.org/home). During mouse embryogenesis, ERK3 mRNA and protein levels 63 peak around embryonic day 11, after the onset of organogenesis, and then decline thereafter (5). 64Expression of ERK3 is also up-regulated during in vitro differentiation of cell line models along 65 the neuronal or muscle lineage (2, 6). These early observations have suggested a potential role 66 for ERK3 signaling in cell differentiation and tissue development. 67 68 Analogous to classical MAP kinases, ERK3 is activated by phosphorylation of the activation 69 loop, which stimulates its intrinsic catalytic activity and affinity for the substrate MK5 (7). 70Activation loop phosphorylation is mediated by Group I p21-activated kinases (8, 9), and is 71 reversed by the action of the MAP kinase phosphatase DUSP2 (10). Of note, it has been 72 previously proposed that ERK3 enzymatic activity is dispensable for MK5 activation and that 73 ERK3 may exert a scaffolding function (11). However, other experiments using coupled in vitro 74 kinase assays did not suppport this hypothesis (7). A more recent study reported that 75 overexpression of ERK3 induces rounding of MDA-MB-231 breast cancer cells by an unknown 76 mechanism independent of its kinase activity (12). The physiological importance of catalytic and 77 non-catalytic functions of ERK3 remains an open question. 78 * Mixed C57Bl/6J-129/Sv genetic background

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Reevaluation of the Role of ERK3 in Perinatal Survival and Post-Natal Growth Using New Genetically-Engineered Mouse Models

Soulez¹,

Saba-El-Leil²,

Turgeon

et al. 2018

Preprint

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“…The conditional allele reported here removes exon 3 of Erk3 and leaves only a single FRT and a single loxP sites in intron 2. The insertion of a lacZ-neomycin-resistance-cassette often altered transcription of genes in the flanking DNA around the targeted gene, and accordingly the phenotype of the mutation (26-29). This may be due to the loss or disruption of intragenic regulatory elements, the constitutive promoter driving the neomycin gene, removal of insulating DNA in the targeted alleles, and local silencing due to disruption of normal chromatin organization by the exogenous construct (26).…”

Section: Discussionmentioning

confidence: 99%

“…The insertion of a lacZ-neomycin-resistance-cassette often altered transcription of genes in the flanking DNA around the targeted gene, and accordingly the phenotype of the mutation (26-29). This may be due to the loss or disruption of intragenic regulatory elements, the constitutive promoter driving the neomycin gene, removal of insulating DNA in the targeted alleles, and local silencing due to disruption of normal chromatin organization by the exogenous construct (26). The detailed transcriptome analysis of 29 targeted alleles in mice revealed that down-regulated genes flanking the targets were rather equally distributed 5’ and 3’ of the target and the median distance from the target was 34kb (26).…”

Section: Discussionmentioning

confidence: 99%

“…This may be due to the loss or disruption of intragenic regulatory elements, the constitutive promoter driving the neomycin gene, removal of insulating DNA in the targeted alleles, and local silencing due to disruption of normal chromatin organization by the exogenous construct (26). The detailed transcriptome analysis of 29 targeted alleles in mice revealed that down-regulated genes flanking the targets were rather equally distributed 5’ and 3’ of the target and the median distance from the target was 34kb (26). In this regard, it is interesting that on chromosome 9 the gene for an essential subunit of RNA polymerase II, leo1, is 40 kb 3’ to the Erk3 gene.…”

Section: Discussionmentioning

confidence: 99%

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Germline deletion reveals a non-essential role of the atypical MAPK6/ERK3

Ronkina

Schuster-Gossler

Hansmann

et al. 2018

Preprint

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words) 24MAPK6/ERK3 is an atypical member of the MAPKs. An essential role has been suggested by the 25 perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to 26 pulmonary disfunction and by defects in function, activation and positive selection of T cells. To 27 study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon3 28 flanked by LoxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female 29 germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is 30 detected, providing first evidence for the existence of the ERK3/MK5 signaling complex in vivo. 31In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and 32 fertile, do not display pulmonary hypoplasia, acute respiratory failure, abnormal T cell 33 development, reduction of thymocyte numbers or altered T cells selection. Hence, ERK3 is 34 dispensable for pulmonary and T-cell functions. The perinatal lethality, lung and T-cell defects of 35 the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-36 neomycin-resistance-cassette. The knockout mouse of the closely related atypical MAPK 37 ERK4/MAPK4 is also normal suggesting redundant functions of both protein kinases. 38 Introduction (630 words) 39 40 Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules 41 where the downstream effector kinases regulate cell proliferation, differentiation and cell death 42 by phosphorylation of protein substrates. MAPK are also regulators in many physiological 43 processes including development and immune response. Multiple MAPKs were described in 44 mammalian cells, which can be divided in five groups, the 'classical' mitogen-responsive MAPKs 45 (ERK1 and ERK2), the stress-activated JNKs (JNK1-3) and p38 MAPK (a,b,g,d), the big MAPK ERK5, 46 and the atypical MAPKs ERK3, ERK4 and ERK7 (1). MAPKs activity is classically regulated by dual 47 5) ERK3_1-target-seq.-FW: TGGACAGAGCACTGGAAG, 6) ERK3-loxP-RC: 131 CTTAAGACAGGAGTGTGGATC. 5+6 primer combination amplifies 943 bp of WT, 1055 bp of 132 exon3 floxed or 336 bp of exon3 deleted fragment. 133 PCR reactions were separated on 2% agarose gels and images acquired using INTAS Gel 134 documentation system. 135 136 137

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The pro‐apoptotic Bax gene modifies susceptibility to craniofacial dysmorphology following gastrulation‐stage alcohol exposure

Fish

Mendoza-Romero

Love

et al. 2022

Birth Defects Research

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Background During early development, alcohol exposure causes apoptotic cell death in discrete regions of the embryo which are associated with distinctive patterns of later‐life abnormalities. In gastrulation, which occurs during the third week of human pregnancy, alcohol targets the ectoderm, the precursor of the eyes, face, and brain. This midline tissue loss leads to the craniofacial dysmorphologies, such as microphthalmia and a smooth philtrum, which define fetal alcohol syndrome (FAS). An important regulator of alcohol‐induced cell death is the pro‐apoptotic protein Bax. The current study determines if mice lacking the Bax gene are less susceptible to the pathogenic effects of gastrulation‐stage alcohol exposure. Methods Male and female Bax+/− mice mated to produce embryos with full (−/−) or partial (+/−) Bax deletions, or Bax+/+ wild‐type controls. On Gestational Day 7 (GD 7), embryos received two alcohol (2.9 g/kg, 4 hr apart), or control exposures. A subset of embryos was collected 12 hr later and examined for the presence of apoptotic cell death, while others were examined on GD 17 for the presence of FAS‐like facial features. Results Full Bax deletion reduced embryonic apoptotic cell death and the incidence of fetal eye and face malformations, indicating that Bax normally facilitates the development of alcohol‐induced defects. An RNA‐seq analysis of GD 7 Bax+/+ and Bax−/− embryos revealed 63 differentially expressed genes, some of which may interact with the Bax deletion to further protect against apoptosis. Conclusions Overall, these experiments identify that Bax is a primary teratogenic mechanism of gastrulation‐stage alcohol exposure.

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Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression

Cited by 30 publications

References 72 publications

Reevaluation of the Role of ERK3 in Perinatal Survival and Post-Natal Growth Using New Genetically-Engineered Mouse Models

Reevaluation of the Role of ERK3 in Perinatal Survival and Post-Natal Growth Using New Genetically-Engineered Mouse Models

Germline deletion reveals a non-essential role of the atypical MAPK6/ERK3

The pro‐apoptotic Bax gene modifies susceptibility to craniofacial dysmorphology following gastrulation‐stage alcohol exposure

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