Bayesian modeling of skewed X inactivation in genetically diverse mice identifies a novel Xce allele associated with copy number changes

Female mammals are functional mosaics of their parental X-linked gene expression due to X chromosome inactivation (XCI). This process inactivates one copy of the X chromosome in each cell during embryogenesis and that state is maintained clonally through mitosis. In mice, the choice of which parental X chromosome remains active is determined by the X chromosome controlling element (Xce), which has been mapped to a 176-kb candidate interval. A series of functional Xce alleles has been characterized or inferred for classical inbred strains based on biased, or skewed, inactivation of the parental X chromosomes in crosses between strains. To further explore the function structure basis and location of the Xce, we measured allele-specific expression of X-linked genes in a large population of F1 females generated from Collaborative Cross (CC) strains. Using published sequence data and applying a Bayesian “Pólya urn” model of XCI skew, we report two major findings. First, inter-individual variability in XCI suggests mouse epiblasts contain on average 20–30 cells contributing to brain. Second, CC founder strain NOD/ShiLtJ has a novel and unique functional allele, Xce^g, that is the weakest in the Xce allelic series. Despite phylogenetic analysis confirming that NOD/ShiLtJ carries a haplotype almost identical to the well-characterized C57BL/6J (Xce^b), we observed unexpected patterns of XCI skewing in females carrying the NOD/ShiLtJ haplotype within the Xce. Copy number variation is common at the Xce locus and we conclude that the observed allelic series is a product of independent and recurring duplications shared between weak Xce alleles.     

Nonrandom X Chromosome Inactivation Is Influenced by Multiple Regions on the Murine X Chromosome

During the development of female mammals, one of the two X chromosomes is inactivated, serving as a dosage-compensation mechanism to equalize the expression of X-linked genes in females and males. While the choice of which X chromosome to inactivate is normally random, X chromosome inactivation can be skewed in F1 hybrid mice, as determined by alleles at the X chromosome controlling element (Xce), a locus defined genetically by Cattanach over 40 years ago. Four Xce alleles have been defined in inbred mice in order of the tendency of the X chromosome to remain active: Xce^a < Xce^b < Xce^c < Xce^d. While the identity of the Xce locus remains unknown, previous efforts to map sequences responsible for the Xce effect in hybrid mice have localized the Xce to candidate regions that overlap the X chromosome inactivation center (Xic), which includes the Xist and Tsix genes. Here, we have intercrossed 129S1/SvImJ, which carries the Xce^a allele, and Mus musculus castaneus EiJ, which carries the Xce^c allele, to generate recombinant lines with single or double recombinant breakpoints near or within the Xce candidate region. In female progeny of 129S1/SvImJ females mated to recombinant males, we have measured the X chromosome inactivation ratio using allele-specific expression assays of genes on the X chromosome. We have identified regions, both proximal and distal to Xist/Tsix, that contribute to the choice of which X chromosome to inactivate, indicating that multiple elements on the X chromosome contribute to the Xce.     

Mapping the murine Xce locus with (CA)n repeats

The X Chromosome (Chr) controlling element locus (Xce) in the mouse has been shown to influence the X inactivation process. Xce maps to the central region of the X Chr, which also contains the Xist sequence, itself possibly implicated in the X inactivation process. Three microsatellite markers spanning the Xist locus have been isolated from an Xist containing YAC. All three microsatellite markers showed complete linkage with Xce in recombinants for the central span of the mouse X Chr between Ta and Mo ^blo and strong linkage disequilibrium with Xce in all but one of the inbred mouse strains tested. In the standard Xce ^b typing strain JU/Ct, the two microsatellites most closely flanking Xist fail to carry the allelic forms expected if Xist and Xce are synonymous. Alternative explanations for this finding are presented in the context of our search for understanding the relation between Xist and Xce.     

Genetic and parent-of-origin influences on X chromosome choice in Xce heterozygous mice

X chromosome inactivation is unique among dosage compensation mechanisms in that the two X chromosomes in females are treated differently within the same cell; one X chromosome is stably silenced while the other remains active. It is widely believed that, when X inactivation is initiated, each cell makes a random choice of which X chromosome will be silenced. In mice, only one genetic locus, the X-linked X controlling element (Xce), is known to influence this choice, because animals that are heterozygous at Xce have X-inactivation patterns that differ markedly from a mean of 0.50. To document other genetic and epigenetic influences on choice, we have performed a population-based study of the effect of Xce genotype on X-inactivation patterns. In B6CAST F₁ females (Xce^b/Xce^c), the X-inactivation pattern followed a symmetric distribution with a mean of 0.29 (SD = 0.08). Surprisingly, however, in a population of Xce^b/Xce^c heterozygous B6CAST F₂ females, we observed significant differences in both the mean (p = 0.004) and variance (p = 0.004) of the X-inactivation patterns. This finding is incompatible with a single-locus model and suggests that additional genetic factors also influence X chromosome choice. We show that both parent-of-origin and naturally occurring genetic variation at autosomal loci contribute to these differences. Taken together, these data reveal further genetic complexity in this epigenetic control pathway.     

Nucleotide variation in wild and inbred mice

The house mouse is a well-established model organism, particularly for studying the genetics of complex traits. However, most studies of mice use classical inbred strains, whose genomes derive from multiple species. Relatively little is known about the distribution of genetic variation among these species or how variation among strains relates to variation in the wild. We sequenced intronic regions of five X-linked loci in large samples of wild Mus domesticus and M. musculus, and we found low levels of nucleotide diversity in both species. We compared these data to published data from short portions of six X-linked and 18 autosomal loci in wild mice. We estimate that M. domesticus and M. musculus diverged <500,000 years ago. Consistent with this recent divergence, some gene genealogies were reciprocally monophyletic between these species, while others were paraphyletic or polyphyletic. In general, the X chromosome was more differentiated than the autosomes. We resequenced classical inbred strains for all 29 loci and found that inbred strains contain only a small amount of the genetic variation seen in wild mice. Notably, the X chromosome contains proportionately less variation among inbred strains than do the autosomes. Moreover, variation among inbred strains derives from differences between species as well as from differences within species, and these proportions differ in different genomic regions. Wild mice thus provide a reservoir of additional genetic variation that may be useful for mapping studies. Together these results suggest that wild mice will be a valuable complement to laboratory strains for studying the genetics of complex traits.     

Characterization of X Chromosome Inactivation Using Integrated Analysis of Whole-Exome and mRNA Sequencing

In females, X chromosome inactivation (XCI) is an epigenetic, gene dosage compensatory mechanism by inactivation of one copy of X in cells. Random XCI of one of the parental chromosomes results in an approximately equal proportion of cells expressing alleles from either the maternally or paternally inherited active X, and is defined by the XCI ratio. Skewed XCI ratio is suggestive of non-random inactivation, which can play an important role in X-linked genetic conditions. Current methods rely on indirect, semi-quantitative DNA methylation-based assay to estimate XCI ratio. Here we report a direct approach to estimate XCI ratio by integrated, family-trio based whole-exome and mRNA sequencing using phase-by-transmission of alleles coupled with allele-specific expression analysis. We applied this method to in silico data and to a clinical patient with mild cognitive impairment but no clear diagnosis or understanding molecular mechanism underlying the phenotype. Simulation showed that phased and unphased heterozygous allele expression can be used to estimate XCI ratio. Segregation analysis of the patient''s exome uncovered a de novo, interstitial, 1.7 Mb deletion on Xp22.31 that originated on the paternally inherited X and previously been associated with heterogeneous, neurological phenotype. Phased, allelic expression data suggested an 83∶20 moderately skewed XCI that favored the expression of the maternally inherited, cytogenetically normal X and suggested that the deleterious affect of the de novo event on the paternal copy may be offset by skewed XCI that favors expression of the wild-type X. This study shows the utility of integrated sequencing approach in XCI ratio estimation.     

Nucleoside phosphorylase 2 (Np-2) of mice

Isozyme patterns of nucleoside phosphorylase (NP) in 16 inbred strains, two recombinant inbred, one congenic, and three species of wild mice were studied. Evidence is provided for a genetic locus, Np-2, encoding an electrophoretic variant which is expressed exclusively in erythrocytes of certain inbred strains. This finding establishes the occurrence of genetic polymorphism of NP among inbred strains of mice. In addition, the Npla allele previously reported only in inbred strains has been observed in one of the species of wild mice (Mus musculus castaneus) studied.     

Genetic dissection of testis weight in mice: quantitative trait locus analysis using F2 intercrosses between strains with extreme testis weight, and association study using Y-consomic strains

In the present study, dissection of genetic bases of testis weight in mice was performed. Autosomes and the X chromosome were searched using traditional quantitative trait locus (QTL) scans, and the Y chromosome was searched by association studies of Y-consomic strains. QTL analysis was performed in ??DDD?×???CBA F₂ mice; the inbred mouse DDD has the heaviest testes, whereas the inbred mouse CBA has the lightest testes. Two significant testis weight QTLs were identified on chromosomes 1 and X. A DDD allele was associated with increased and decreased testis weight at the locus on chromosomes 1 and X, respectively. In the reciprocal cross ??CBA?×???DDD F₂ mice, QTL on chromosome 1, and not on chromosome X, had a significant effect on testis weight. The DDD allele at the X-linked locus could not sustain testis weight in combination with the Y chromosome of the CBA strain. The Y chromosome per se had a significant effect on testis weight, i.e., DH-Chr Y^DDD had significantly heavier testes than DH-Chr Y^CBA. On the basis of the results of Y-chromosome-wide association studies using 17 Y-consomic strains, variations in Uty, Usp9y, and Sry were significantly associated with testis weight. Thus, testis weight is a complex quantitative phenotype controlled by multiple genes on autosomes and sex chromosomes and their interactions.     

Clonal and atypical Toxoplasma strain differences in virulence vary with mouse sub-species

The severe virulence of Toxoplasma gondii in classical laboratory inbred mouse strains contradicts the hypothesis that house mice (Mus musculus) are the most important intermediate hosts for its transmission and evolution because death of the mouse before parasite transmission equals death of the parasite. However, the classical laboratory inbred mouse strains (Mus musculus domesticus), commonly used to test Toxoplasma strain differences in virulence, do not capture the genetic diversity within Mus musculus. Thus, it is possible that Toxoplasma strains that are severely virulent in laboratory inbred mice are avirulent in some other mouse sub-species. Here, we present insight into the responses of individual mouse strains, representing strains of the genetically divergent Mus musculus musculus, Mus musculus castaneus and Mus musculus domesticus, to infection with individual clonal and atypical Toxoplasma strains. We observed that, unlike M. m. domesticus, M. m. musculus and M. m. castaneus are resistant to the clonal Toxoplasma strains. For M. m. musculus, we show that this is due to a locus on chromosome 11 that includes the genes that encode the interferon gamma (IFNG)-inducible immunity-related GTPases (Irgs) that can kill the parasite by localising and subsequently vesiculating the parasitophorous vacuole membrane. However, despite the localization of known effector Irgs to the Toxoplasma parasitophorous vacuole membrane, we observed that some atypical Toxoplasma strains are virulent in all the mouse strains tested. The virulence of these atypical strains in M. m. musculus could not be attributed to individual rhoptry protein 5 (ROP5) alleles, a secreted parasite pseudokinase that antagonises the canonical effector Irgs and is indispensable for parasite virulence in laboratory inbred mice (M. m. domesticus). We conclude that murine resistance to Toxoplasma is modulated by complex interactions between host and parasite genotypes and may be independent of known effector Irgs on murine chromosome 11.     

TheD17Tu5 locus in thet complex: implications for the origin oft haplotypes and inbred strains

The mouse × Chinese hamster cell line R4 4-1 contains only one mouse chromosome, the bulk of which corresponds toMus musculus chromosomes 17 and 18 (MMU17 and MMU18, respectively). A genomic library was prepared from the R4 4-1 DNA, and a mouse clone was isolated from the library, which—with the help of somatic cell hybrids-could be mapped to the MMU17. A locus defined by a 2.7-kb longBam HI probe from this clone was designatedD17Tu5 (Tu for Tübingen). The locus proved to be polymorphic among inbred strains and wild mice. By testing of recombinant inbred strains and partialt haplotypes, theD17Tu5 locus could be mapped to a position between theD17Leh66E andD17Rp17 loci within thet complex. Two alleles were found at this locus,D17Tu5 ^a andD17Tu5 ^b , defined byTaq I restriction fragment length polymorphism. Both alleles are present among inbred strains and wild mice of the speciesM. domesticus. All completet haplotypes tested carry theD17Tu5 ^a allele and all tested wild mice of the speciesM. musculus, with the exception of those bearingt haplotypes, carry theD17Tu5 ^b allele. Additional alleles are found in some populations of wild mice and in other species of the genusMus. The distribution of the two alleles among the inbred strains correlates well with their known or postulated genealogy. Their distribution between the two species ofMus and among the mice withT haplotypes suggests a relatively recent origin of thet haplotypes.     

Sperm Morphology in Two House Mouse Subspecies: Do Wild-Derived Strains and Wild Mice Tell the Same Story?

Being subject to intense post-copulatory selection, sperm size is a principal determining component of male fitness. Although previous studies have presented comparative sperm size data at higher taxonomic levels, information on the evolution of sperm size within species is generally lacking. Here, we studied two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus, which undergo incipient speciation. We measured four sperm dimensions from cauda epididymis smears of 28 wild-caught mice of both subspecies. As inbred mouse strains are frequently used as proxies for exploring evolutionary processes, we further studied four wild-derived inbred strains from each subspecies. The subspecies differed significantly in terms of sperm head length and midpiece length, and these differences were consistent for wild mice and wild-derived strains pooled over genomes. When the inbred strains were analyzed individually, however, their strain-specific values were in some cases significantly shifted from subspecies-specific values derived from wild mice. We conclude that: (1) the size of sperm components differ in the two house mouse subspecies studied, and that (2) wild-derived strains reflect this natural polymorphism, serving as a potential tool to identify the genetic variation driving these evolutionary processes. Nevertheless, we suggest that more strains should be used in future experiments to account for natural variation and to avoid confounding results due to reduced variability and/or founder effect in the individual strains.     

Wild Mice As Bountiful Resources of Novel Genetic Variants for Quantitative Traits

Most traits of biological importance, including traits for human complex diseases (e.g., obesity and diabetes), are continuously distributed. These complex or quantitative traits are controlled by multiple genetic loci called QTLs (quantitative trait loci), environments and their interactions. The laboratory mouse has long been used as a pilot animal model for understanding the genetic architecture of quantitative traits. Next-generation sequencing analyses and genome-wide SNP (single nucleotide polymorphism) analyses of mouse genomes have revealed that classical inbred strains commonly used throughout the world are derived from a few fancy mice with limited and non-randomly distributed genetic diversity that occurs in nature and also indicated that their genomes are predominantly Mus musculus domesticus in origin. Many QTLs for a huge variety of traits have so far been discovered from a very limited gene pool of classical inbred strains. However, wild M. musculus mice consisting of five subspecies widely inhabit areas all over the world, and hence a number of novel QTLs may still lie undiscovered in gene pools of the wild mice. Some of the QTLs are expected to improve our understanding of human complex diseases. Using wild M. musculus subspecies in Asia as examples, this review illustrates that wild mice are untapped natural resources for valuable QTL discovery.     

Familial Skewed X Chromosome Inactivation in Adrenoleukodystrophy Manifesting Heterozygotes from a Chinese Pedigree

Background

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder caused by mutations in the ABCD1 gene. Approximately 20% of X-ALD female carriers may develop neurological symptoms. Skewed X chromosome inactivation (XCI) has been proposed to influence the manifestation of symptoms in X-ALD carriers, but data remain conflicting so far. We identified a three generation kindred, with five heterozygous females, including two manifesting carriers. XCI pattern and the ABCD1 allele expression were assessed in order to determine if symptoms in X-ALD carriers could be related to skewed XCI and whether skewing within this family is more consistent with genetically influenced or completely random XCI.

Results

We found a high frequency of skewing in this family. Four of five females had skewed XCI, including two manifesting carriers favoring the mutant allele, one asymptomatic carrier favoring the normal allele, and one female who was not an X-ALD carrier. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, XIST promoter mutations, were not consistent with our results.

Conclusions

Our data support that skewed XCI in favor of the mutant ABCD1 allele would be associated with the manifestation of heterozygous symptoms. Furthermore, XCI skewing in this family is genetically influenced. However, the underlying mechanism remains to be substantiated by further experiments.     

Analysis of wild-derived mice for Fv-1 and Fv-2 murine leukemia virus restriction loci: a novel wild mouse Fv-1 allele responsible for lack of host range restriction.

Wild-derived mice originally obtained from Asia, Africa, North America, and Europe were typed for in vitro sensitivity to ecotropic murine leukemia viruses and for susceptibility to Friend virus-induced disease. Cell cultures established from some wild mouse populations were generally less sensitive to exogenous virus than were cell cultures from laboratory mice. Wild mice also differed from inbred strains in their in vitro sensitivity to the host range subgroups defined by restriction at the Fv-1 locus. None of the wild mice showed the Fv-1n or Fv-1b restriction patterns characteristic of most inbred strains, several mice resembled the few inbred strains carrying Fv-1nr, and most differed from laboratory mice in that they did not restrict either N- or B-tropic murine leukemia viruses. Analysis of genetic crosses of Mus spretus and Mus musculus praetextus demonstrated that the nonrestrictive phenotype is controlled by a novel allele at the Fv-1 locus, designated Fv-10. The wild mice were also tested for sensitivity to Friend virus complex-induced erythroblastosis to type for Fv-2. Only M. spretus was resistant to virus-induced splenomegaly and did not restrict replication of Friend virus helper murine leukemia virus. Genetic studies confirmed that this mouse carries the resistance allele at Fv-2.     

Common inbred strains of the laboratory mouse that are susceptible to infection by mouse xenotropic gammaretroviruses and the human-derived retrovirus XMRV

Laboratory mouse strains carry endogenous copies of the xenotropic mouse leukemia viruses (X-MLVs), named for their inability to infect cells of the laboratory mouse. This resistance to exogenous infection is due to a nonpermissive variant of the XPR1 gammaretrovirus receptor, a resistance that also limits in vivo expression of germ line X-MLV proviruses capable of producing infectious virus. Because laboratory mice vary widely in their proviral contents and in their virus expression patterns, we screened inbred strains for sequence and functional variants of the XPR1 receptor. We also typed inbred strains and wild mouse species for an endogenous provirus, Bxv1, that is capable of producing infectious X-MLV and that also contributes to the generation of pathogenic recombinant MLVs. We identified the active Bxv1 provirus in many common inbred strains and in some Japanese Mus molossinus mice but in none of the other wild mouse species that carry X-MLVs. Our screening for Xpr1 variants identified the permissive Xpr1(sxv) allele in 7 strains of laboratory mice, including a Bxv1-positive strain, F/St, which is characterized by lifelong X-MLV viremia. Cells from three strains carrying Xpr1(sxv), namely, SWR, SJL, and SIM.R, were shown to be infectable by X-MLV and XMRV; these strains carry different alleles at Fv1 and vary in their sensitivities to specific X/P-MLV isolates and XMRV. Several strains with Xpr1(sxv) lack the active Bxv1 provirus or other endogenous X-MLVs and may provide a useful model system to evaluate the in vivo spread of these gammaretroviruses and their disease potential in their natural host.     

Genetic variation of milk proteins in mice

A survey for qualitative and quantitative variation in milk proteins from 58 inbred strains of mice revealed two electrophoretic variants. One is in a whey acidic protein of milk of YBR mice and the other is in a curd protein of the Asian house mouse, Mus musculus castaneus. The whey acidic protein variant is shown to be under the control of a single Mendelian autosomal gene with alleles expressed in a codominant manner. This gene is designated Wap, is not identical to Eg, is not X linked, and is either unlinked or loosely linked to the coat color genes a and b.     

The telocentric tandem repeat at the p-arm is not conserved in Mus musculus subspecies

Mouse chromosomes, with the exception of the Y chromosome, are telocentric. The telomere at the p-arm is separated from the centromere by the tL1 sequence and TLC tandem repeats. A previous report showed that the TLC array was also conserved in other strains of the subgenus Mus. These results suggest that the TLC arrays promote the stable evolutionary maintenance of a telocentric karyotype in the subgenus Mus. In this study, we investigated the degree of conservation of TLC arrays among a variety of wild-derived inbred strains, all of which are descendants of wild mice captured in several areas of the world. Genomic PCR analysis indicates that the sequential order of telomere-tL1 is highly conserved in all strains, whereas tL1-TLC is not. Next, Southern blot analysis of DNAs isolated from a panel of mouse subspecies showed both Mus musculus domesticus and Mus musculus castaneus subspecies possess TLC arrays. Unexpectedly, this repeat appears to be lost in almost all Mus musculus musculus and Mus musculus molossinus subspecies, which show a clear geographic divide. These results indicate that either other unknown sequences were replaced by the TLC repeat or almost all M. m. musculus and M. m. molossinus subspecies do not have any sequence between the telomere and minor satellites. Our observation suggests that the TLC array might be evolutionarily unstable and not essential for murine chromosomal conformation. This is the first example of the subspecies-specific large genome alterations in mice.