High resolution mapping and positional cloning of ENU-induced mutations in the Rw region of mouse chromosome 5
- Yung-Hao Ching1,
- Robert J Munroe1,
- Jennifer L Moran2,
- Anna K Barker1,
- Evan Mauceli2,
- Tim Fennell2,
- Frederica diPalma2,
- Kerstin Lindblad-Toh2, 4,
- Lindsay M Abcunas1,
- Joyanna F Gilmour3,
- Tanya P Harris1,
- Susan L Kloet1,
- Yunhai Luo1,
- John L McElwee1,
- Weipeng Mu1,
- Hyo K Park1,
- David L Rogal1,
- Kerry J Schimenti1,
- Lishuang Shen1,
- Mami Shindo1,
- James Y Shou1,
- Erin K Stenson1,
- Patrick J Stover3 and
- John C Schimenti1Email author
© Ching et al; licensee BioMed Central Ltd. 2010
Received: 1 October 2010
Accepted: 30 November 2010
Published: 30 November 2010
Forward genetic screens in mice provide an unbiased means to identify genes and other functional genetic elements in the genome. Previously, a large scale ENU mutagenesis screen was conducted to query the functional content of a ~50 Mb region of the mouse genome on proximal Chr 5. The majority of phenotypic mutants recovered were embryonic lethals.
We report the high resolution genetic mapping, complementation analyses, and positional cloning of mutations in the target region. The collection of identified alleles include several with known or presumed functions for which no mutant models have been reported (Tbc1d14, Nol14, Tyms, Cad, Fbxl5, Haus3), and mutations in genes we or others previously reported (Tapt1, Rest, Ugdh, Paxip1, Hmx1, Otoe, Nsun7). We also confirmed the causative nature of a homeotic mutation with a targeted allele, mapped a lethal mutation to a large gene desert, and localized a spermiogenesis mutation to a region in which no annotated genes have coding mutations. The mutation in Tbc1d14 provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis.
This collection of alleles contributes to the goal of assigning biological functions to all known genes, as well as identifying novel functional elements that would be missed by reverse genetic approaches.
Since the human and mouse genomes were sequenced several years ago, it has been recognized that the next major challenge is to uncover the functional content of the genome. This was a motivation behind large scale projects such as ENCODE (Encyclopedia of DNA elements) and KOMP (Knockout Mouse Project; http://www.nih.gov/science/models/mouse/knockout/komp.html). The former used molecular strategies to identify features of human chromatin , and the next iteration (modENCODE) is geared to identifying functional genomic elements in vivo using non-vertebrate models http://www.genome.gov/26524507. KOMP uses a reverse genetic approach to identify the functions of all known and annotated mouse protein coding genes, by generating germline or conditional null alleles in embryonic stem (ES) cells.
Forward genetic mutagenesis in mice is most commonly conducted via whole animal mutagenesis with the point mutagen N-ethyl-N-nitrosourea (ENU). This approach has succeeded in identifying new genes that play important roles in biological processes and in modeling disease [3–8]. Two key advantages over reverse genetics are that: 1) ENU can cause non-null alleles, enabling the dissection of protein function and yielding variants that are more relevant to disease-causing mutations in humans; and 2) as a non-biased approach, genetic elements or genes can be identified that would never have been implicated ab initio to have specific activities or roles.
In multiple organisms, point mutagens have been employed in two basic strategies: random and region-directed. The former has the benefit of scanning the whole genome, whereas the latter allows one to focus resources on a microcosm of the genome. Several years ago, we initiated a larger scale regional screen of proximal mouse Chr 5 spanned by an inversion called rump-white (Rw). This recessive lethal balancer facilitated the isolation and maintenance of mutations recovered in the screen. Predominantly lethal mutations were recovered (a total of 37) . Here, we report on the mapping and cloning of much of this collection. Several mutations were identified in genes for which no models previously existed, providing new biological insights and models of interest. Intriguingly, we also mapped one mutation to a gene desert, in which the nearest protein-coding gene is over 1 megabase away.
Results and Discussion
The rough locations of the Rw region ENU-induced mutations were reported by Wilson et al , with most of them being localized to intervals that were too large to allow effective candidate gene selection. Additionally, since many of the mutations were located in common large intervals, allelism amongst them was possible. To map the mutations at higher resolution, we continued and expanded upon two strategies initiated by Wilson et al: deletion mapping and recombination mapping .
Positional Cloning of Mutations Summary
Mutations causes homeotic-like skeletal transformations and perinatal lethality.
Vasculogenesis; DNA repair; epigenetic regulation
missense in TBC1 domain
M4-J57 (1.6 Mb)
TBC1 domain family; putative RAB GTPase activation protein.
SA site, exon 3 nonsense, exon 3
J36-M268 600 kb
Nucleolar protein homolog; ribosome biosynthesis.
T > A (ASN > LYS)
J63-M176 (1.1 Mb)
C893G (P > R)
RE1-silencing transcription factor
carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase.
missense LEU > PRO in exon 4.
M388-J88 (1.8 Mb)
Subunit of augmin (HAUS) complex that regulates centrosome and spindle integrity.
missense MET > LYS
Part of E3 ubiquitin ligase complex that regulates iron homeostasis.
UDP-glucose dehyrogenase; GAG biosynthesis; gastrulation.
Required for normal sperm motility and male fertility.
Otoferlin; inner hair cell neurotransmission
Complementation analyses of the mutant collection
Positional Cloning of Mutations
Once mutations were mapped to intervals under 5 cM, or to regions containing a small number of annotated genes, we initiated searches for causative mutations. The strategies varied during the course of the project as technology advanced. In most cases, annotated genes were prioritized for mutational analysis by considering the following: 1) phenotypes of described knockouts; 2) whether the genes had orthologs in other species with mutant phenotype information; and 3) gene expression patterns from microarrays or EST library origins. Primer pairs were then designed to amplify exonic sequences from the prioritized candidate genes, using genomic DNA of mutant heterozygotes (or homozygous embryonic material in some cases) as templates. PCR products were analyzed by denaturing HPLC (to detect mismatches as heteroduplexes) or direct sequencing. This led to the identification of several mutations summarized below. The causative nature of the identified mutations is relatively confident in cases where two alleles were identified, or when the mutated gene has a similar knockout phenotype. Otherwise, follow-on confirmation of causality will be required by methods such as non-complementation with a targeted or gene-trap allele (as in the case of Tapt1; see below).
The following paragraphs in this section describe positional cloning of new alleles, or relevant additional information on previously identified alleles. Table 1 also overviews all alleles identified in this project.
This perinatal lethal mutation was identified previously as affecting a novel gene, Tapt1 (Transmembrane anterior posterior transformation 1) . Homozygous mutants exhibit homeotic transformation of the skeleton. To confirm that the point mutation is causative for the phenotype, we performed a complementation test between L5Jcs1 and a mice carrying a deletion allele of Tapt1 (Tapt1 KO ; see Methods). Intercrosses between Tapt1 KO /Rw and L5Jcs1/Rw animals produced litters (N = 31 pups) in which only Rw offspring (21) survived to wean age. The remaining animals (10) died shortly after birth, before the Rw phenotype could be determined. Given that Rw/Rw causes embryonic lethality shortly after gastrulation, the expected number of non-Rw animals for those surviving to wean age is 7, and the lack thereof is significant (Chi square = 10.5; P = .0012). This confirms that the mutation in Tapt1 is causative for the L5Jcs1 lethal phenotype, and indicates that Tapt1 L5Jcs1 is a null or severe hypomorph.
L5Jcs5 was mapped to a 1.6 Mb region containing ~17 genes whose knockout phenotypes are not known. Sequencing of several of these genes (not all to completion) revealed only 1 mutation in Tbc1d14. This gene encodes a protein containing a TBC (Tre-2/Bub2/Cdc16) domain characteristic of RAB-GAPs, proteins that activate GTPase activity of RAB proteins [12, 13]. RABs are guanine nucleotide binding proteins that mediate membrane-associated protein transport. The mutation causes a SER > GLY change in amino acid 433 (NP_001106833; or 413 for NP_598671), which resides in the TBC1 domain. This amino acid is conserved in mammals (dog; human), zebrafish and chickens. There have been no studies regarding the function of TBC1D14 in cells or animals, however the crystal structure of the human protein has been reported .
Interestingly, none of the 39 mouse genes annotated as a RAB-GAP (Gene Ontology term: Rab GTPase activator activity; GO:0005097) has been associated with a mutant phenotype of embryonic lethality. Thus, if indeed the Tbc1d14 mutation proves to causative for the L5Jcs5 phenotype, this would be the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis.
L5Jcs11 is an early postimplantation mutant overtly defective in gastrulation . It was mapped genetically to a ~1.1 Mb interval by a combination of deletion mapping (Dpp6 df4J complemented L5Jcs11, defining the proximal end as being distal to D5Jcs63) and recombination mapping (defining the distal end of the critical region). Sequencing of candidate genes in the region identified a T > A transversion in the third nucleotide of the codon encoding amino acid 106 of TYMS. This causes a predicted asparagine to lysine change. The structure and function of thymidylate synthase has been characterized extensively in various eukaryotes. The protein is a symmetric dimer, with each subunit ranging from 30 to 35 kDa depending on the organism. However, in protozoa and plants, TYMS and DHFR are produced on the same polypeptide (reviewed in ).
The main role of TYMS is to convert deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) using a 5,10-methylenetetrahydrofolate cofactor. dTMP is essential for the synthesis of DNA but not RNA, and therefore it is found at especially high levels in cells undergoing rapid cell division. Consequently, anti-TYMS drugs are used in cancer treatment, as are other chemicals that inhibit this pathway (such as methotrexate, a DHFR inhibitor) . TYMS inhibitor drugs primarily target the dUMP substrate or the folate binding site on TYMS.
Since ASN106 is not specified as being a residue essential for TYMS function , we sought additional evidence that the mutation is responsible for the drastic phenotype of L5Jcs11. TYMS autoregulates its levels by binding its mRNA to decrease translation . Consistent with a decrease in functional TYMS in cells bearing this allele, Western blots revealed that L5Jcs11 heterozygotes have ~2.6 fold elevated TYMS protein in the liver relative to WT mice (Figure 3b). Mutant heterozygotes also exhibited 2.3 fold lower DHFR protein levels (Figure 3b). ASN106 is located close to the folate binding motif of TYMS, suggesting that the enzyme is defective in binding or effectively utilizing the major substrate N5, N10-Methylene H4 folate.
To further explore the effects of this mutation on mice, we performed inner cell mass (ICM) outgrowth assays. As shown in Figure 3c, although trophoblast cells from cultured blastocysts could grow, consistent with the ability to implant, mutant ICMs were unable to outgrow from hatched embryos. This indicates that the rapid cell division and DNA synthesis occurring in embryonic cells cannot be supported without normal TYMS function, consistent with failed postimplantation development of the embryo proper.
HAUS3 is a component of the multiprotein HAUS complex, homologous to the Drosophila Augmin complex. In mammalian cells, HAUS regulates centrosome and spindle integrity. Its disruption causes destabilization of kinetocore microtubules and centrosome disruption, and thus is critical for genome stability . Interestingly, HAUS3 was found to be mutated early in the development of a subset of lobular breast cancers . Though we haven't noted any obvious defects in heterozygous animals, this model might be useful for investigating the effects on breast cancer frequency or progression in tumor susceptible backgrounds. Homozygosity for L5Jcs31 causes pre- or peri-implantation lethality, indicating the essential nature of the augmin complex in mammalian cell growth or early development.
This mutation was identified as a midgestation recessive lethal showing neural tube defects . The allele was mapped by virtue of non-complementation with the Hdh df9J deletion. Sequencing of candidate genes in the region led to the identification of a T to A transition in exon 3 of Fbxl5, resulting in the replacement of methionine by lysine at amino acid position 127.
FBXL5 (F box and leucine-rich repeat protein 5) is part of an SCF (SKP1-cullin-F-box) ubiquitin ligase complex that plays a key role in regulating iron homeostasis [22, 23]. It serves as a sensor of iron and oxygen levels by tethering the iron regulatory proteins IRP1/2 to an E3 ligase complex for ubiquitination and proteasome degradation, or alternatively, increasing transferrin receptor transcription and inhibiting ferritin production under conditions of low intracellular iron. The mutation resides in the conserved iron-binding hemerythrin domain of FBXL5, changing a highly conserved (invariant among vertebrates) MET immediately following a key histidine residue that is involved in coordinating Fe . We postulate that this compromises or eliminates iron binding by the protein, thus disrupting its sensor function. Our findings that mutation of Fbxl5 causes embryonic lethality underscores the known critical nature of iron homeostasis during development (reviewed in ).
The L5Jcs13 embryonic lethal allele maps to a gene desert
Remarkably, the L5Jcs13 critical region (289 kb) lies within a much larger gene desert that contains only 1 RefSeq gene (Pcdh7) in 7.7 Mb. The nearest gene is G6pd2, located 1.3 Mb distally, which marks the beginning of a gene rich region. The current data raises the possibility that the L5Jcs13 mutation affects a long range regulatory sequence, as appears to be the case with an ENU-induced allele of the quaking gene that is located 40-640 kb upstream , or the callipyge point mutation in sheep that affects transcription of genes up to several hundred kilobases away [26–28]. Alternatively, the mutation could disrupt the local chromatin structure, or interaction of chromatin with the nuclear matrix, in a manner that affects the expression of other genes. Finally, it is possible that there is an uncharacterized non-coding RNA in the critical region that is mutated. There is one reported case of a mouse ENU-induced mutation in a microRNA gene causing progressive hearing loss .
Ste5Jcs3 maps to a region lacking mutated genes with mutations or implicated spermatogenesis roles
This spermiogenesis mutation was localized to a 1.6 Mb region (Figure 1) containing 4 annotated genes: Tbc1d19, Rbpj, Cckar and Stim2. We sequenced all of these (cDNA or exons from genomic DNA) and found no mutations. Additionally, the latter 3 have been mutated by gene targeting to produce phenotypes not consistent with specific male infertility. CCKAR-deficient mice are viable with no fertility defects [30, 31]. Mutation of Rbpj causes embryonic lethality. Stim2 null mice are slightly runted and die by 4-5 weeks of age . That study reported T cell defects but did not address germ cell development. Tbc1d19 appears to be widely expressed based on EST library representation (Unigene), and, like Tbc1d14 (mutated in L5Jcs5), encodes a likely Rab-Gap containing the TBC domain. Further sequencing analyses will be required to identify the mutation and determine if it affects a novel element, the expression of any of these genes, or the expression of genes located outside the genetically defined interval.
This report summarizes work on a region-specific saturation mutagenesis project that was initiated before the mouse genome was sequenced. We reasoned that such regional screens (using deletions or balancer inversions such as Rw) provided powerful advantages for phenotype characterization and gene identification, including simplified stock maintenance using a single visibly marked balancer chromosome, and the knowledge of a mutation's location. Coupled with nested deletions generated in the same region , it was possible to rapidly sublocalize mutations to regions upon which to concentrate recombination mapping and guide efficient complementation analyses. The sum of this work was the molecular identification of 16 mutant alleles representing 13 genes. For 10 of these genes, the mutants are/were the first reported in mice (Table 1), and 6 are presented here for the first time. The remaining mutations have been mapped to relatively small intervals.
Perhaps the most interesting mutation thus far is L5Jcs13, which is a recessive embryonic lethal allele mapping to a gene desert. Clearly, this region contains a sequence element of crucial importance to mouse development, and close scrutiny of this region for evidence of cryptic genes (using ab initio gene prediction programs), highly conserved elements or transcription units have not revealed any candidate elements that contain mutations (data not shown). In an era where all protein-coding genes will soon be knocked out in ES cells by the KOMP project, the identification of apparently novel, non-genic, essential genomic elements such as L5Jcs13 underscores the continuing value of unbiased forward genetic screens.
The drawback of forward genetic screens in mice has been the cost and time involved in identifying causative mutations. For genome-wide screens, genetic crosses must be conducted in order to localize mutations, followed by traditional positional cloning efforts guided by candidate gene prioritization. Region-specific screens ameliorate the mapping part, and in one study the approach was taken to directly resequence all exons (and their flanking sequences) in the target region to identify causative mutations . Potentially causative mutations were identified in 31 of the 41 mutant lines examined, and most of the potentially causative mutations were in non-coding regions within or near the transcription unit. The failure to identify the other 10 mutations amongst annotated genes, and the cases of L5Jcs13 and Ste5Jcs5, indicate that there is much we don't know about the functional content of the genome. Furthermore, we must caution that for the potentially causative mutations presented here and in other studies such as the aforementioned, functional validation must be obtained. This caveat also underscores the value of genetic mapping, which contributes to validation of a mutation identified by sequencing.
The advent of high-throughput DNA sequencing technologies can be transformative for forward genetics in mice. Here, we identified three mutations by Solexa/Illumina resequencing of exon-enriched DNA from critical regions. However, the rapidly declining cost of whole genome sequencing will soon negate the benefit of such sequence enrichment, or even genetic mapping in advance of sequencing (bearing in mind the issue of validation mentioned above). Additionally, whole genome sequencing has the important advantage of enabling the identification of non-coding regulatory elements.
Sequence capture and next generation sequencing
A custom Agilent oligonucleotide array was designed to sequence exons, UTRs and promoters of all RefSeq genes in non-recombinant intervals of several mutations (from this and unrelated projects). The array included all RefSeq genes between D5Mit388 and D5Mit268 (mm8; NCBI Build 36). Solution-based hybrid capture of L5Jcs14/+B6, L5Jcs31/+B6 and C57BL/6J genomic DNAs to transcribed RNA oligonucleotide baits was performed as described . Libraries made from captured DNA were sequenced using 51 or 76 bp paired-end reads with an Illumina Genome Analyzer. Candidate single nucleotide changes were identified by 1) aligning high quality sequence reads to the reference C57BL/6J mm8 genome sequence, 2) identification of single nucleotide polymorphisms (SNPs) between each sample against the reference, and 3) filtering for heterozygous SNPs unique to the mutant sample. In addition to Sanger sequencing, genotyping of L5Jcs14 SNPs with the Sequenom iPlex technology was performed to confirm the L5Jcs14 mutation.
For L5Jcs13, 70 primer pairs were developed to amplify the 289 kb critical region. The products averaged a length of 4.5 kb and overlapped approximately 400 bp on each end. Amplification was initially attempted on all products using Roche Expand Long Range Polymerase or iProof Polymerase (Bio-Rad). Out of the 70 primer pairs, 51 showed some level of successful amplification. Amplimers were treated with FastAP Alkaline Phosphatase plus Exonuclease I, pooled, then sequenced on an Illumina GA genome analyzer using chemistry that generated 43 nucleotide, single-end reads. The sequence was aligned to C57BL/6J ("B6") genomic sequence using Novoalign v2.07 and Stampy 1.0.9. Further comparison was done with the C3H/HeJ reference sequence from the Sanger Institute. Data was analyzed for single nucleotide polymorphisms (SNPs), insertions, and deletions using GATK v 1.4418 (The Genome Analysis Toolkit; http://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_Toolkit) and VarScan v 2.2.3 http://sourceforge.net/projects/varscan/. Candidate SNPs were compared to the NCBI SNP database dbSNP, and ultimately re-sequenced by standard methods to resolve whether a nucleotide difference was a SNP, a mutation, or a database or sequencing error.
Generation of Tapt1 mice
ES cells containing a deletion of the entire Tapt1 locus were obtained from the KOMP Repository (clone name Tapt1_AG12, produced by Regeneron, Inc.). The targeted cells were of strain C57BL/6N, and the official allele name is Tapt1 tm1(KOMP)Vic . Cells were microinjected into albino CD1 blastocysts to produce chimeras.
Western blot analysis
Snap-frozen liver samples were lysed in 10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM DTT, 1% Triton X-100, and Mammalian Protease Inhibitor Cocktail (Sigma). Tissue lysates were loaded onto 12% SDS-PAGE gels with 25 μg total protein/lane; protein concentrations were determined using the Lowry-Bensadoun method . Proteins were then transferred to an Immobilon-P PVDF Membrane (Millipore). The membrane was blocked overnight at 4°C in phosphate-buffered saline with 10% nonfat dry milk and 1% NP40. The membrane was incubated for two hours at room temperature in one of two primary antibodies: 1:1000 -anti-TYMS (Zymed) or 1:2000 anti-DHFR (Sigma). After four washes (phosphate buffered saline with 0.1% Tween-20) of 10 minutes each, the membranes were incubated for 1 hour in 1:10,000 HRP-conjugated goat anti-mouse antibody for TYMS or 1:20,000 HRP-conjugated goat anti-rabbit for DHFR (Pierce). After four washes of 10 minutes each, membranes were developed in SuperSignal West Pico Chemiluminescent Substrate (Pierce) and films exposed. As a loading control, membranes were also probed with 1:100,000 HRP-conjugated anti-beta actin (Abcam). Quantification of protein bands was done by digitizing the films, then analyzing the images with ImageJ software (NIH).
Blastocysts (E3.5) were plated onto gelatinized tissue culture dishes, cultured for 5 days in ES cell medium, and harvested for genotyping as described previously .
Mutation Mapping and screening
For recombination mapping, Rw/L5Jcs# animals were crossed to C5Cast/C5Cast (a strain in which the proximal portion of Chr 5 from Mus castaneous was rendered partially congenic in strain C3HeB/FeJ), and non-Rw F1 progeny (L5Jcs#/C5Cast) were intercrossed to produce F2's. Genomic DNA isolated from tails from F2 animals were genotyped with polymorphic markers within the Rw region to identify recombinants. The locations of the mutations were determined indirectly, in that we excluded intervals in which a mutation resides by virtue of being able to obtain homozygosity for the parental B6 allele in live offspring.
Markers not in the MGD database are:
D5Jcs640 GCCAGGTTAATACAAGCTCCA and TCTCCTTCTTCCTTCTCTTCTCTTC
D5Jcs85 GGGCTTTTAGACGAGCAGAG and TGGGTTCAGAACGAAGGTCT
D5Jcs24 AAACATGTCAGGGCCAGAAG and TGTGCTTCCATTCATTTATGC
Mutation screening by dHPLC was performed on a Transgenomic Wave machine, designed to detect heteroduplexes. The screening was generally performed on heterozygous DNAs. Otherwise, amplified DNAs were sequenced by standard Sanger protocols in an automated sequencer.
All experiments using mice were conducted with approval of Cornell's Institutional Animal Care and Use Committee, protocol # 2004-0038.
AKB, LMA, JYS, HKP, EKS, DLR and SLK were undergraduates at Cornell when they conducted the research.
This work was support by NIH grant HD35984 and a NHGRI medical sequencing grant to the Broad Institute. We thank Lauren Imbroglio, Stacy Gabriel, Ross Sawford, Jeremy Johnson and the Broad Institute's Genetic Analysis Platform and Genome Sequencing Platform for SNP genotyping, hybrid capture and Illumina sequencing.
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