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Hybrid sterility (HS) is an early postzygotic reproductive isolation mechanism observed in all sexually reproducing species. Infertility of hybrids prevents gene flow between incipient species and leads to speciation. While Drosophila studies have focused almost exclusively on the genic control of HS, two other model species, Mus musculus and budding yeast, provided the first experimental evidence of hybrid sterility governed by the nongenic effects of DNA sequence divergence. Here, we propose that the nongenic effect of increasing DNA divergence between closely related species may impair mutual recognition of homologous chromosomes and disrupt their synapsis. Unsynapsed or mispaired homologs can induce early meiotic arrest, or their random segregation can cause aneuploidy of spermatids and sperm cells. Impaired recognition of homologs may thus act as a universal chromosomal checkpoint contributing to the complexity of genetic control of HS. Chromosomal HS controlled by the Prdm9 gene in mice and HS driven by the mismatch repair machinery in yeast are currently the most advanced examples of chromosomal homology search-based HS. More focus on the cellular and molecular phenotypes of meiosis will be needed to further validate the role of homolog recognition in hybrid sterility and speciation.

Supplementary Figures 1-7. Supplementary Figure 1: Assembly of the PWD genome and alignment to the C57BL/6 genome. Supplementary Figure 2: RNA-seq read assignment. Supplementary Figure 3: MeDIP-seq quality controls. Supplementary Figure 4: Histology and distribution of tumors in B6 x PWD consomic mice. Supplementary Figure 5: Transcriptome analyses identify modifier candidate genes on other chromosomes than chromosome 5. Supplementary Figure 6: Strain-specific DNA methylation on chromosome 5 is correlated to CpG availability. Supplementary Figure 7: Assessment of cell types in the intestinal crypt.

Genetic predisposition affects the penetrance of tumor-initiating mutations, such as APC mutations that stabilize β-catenin and cause intestinal tumors in mice and humans. However, the mechanisms involved in genetically predisposed penetrance are not well understood. Here, we analyzed tumor multiplicity and gene expression in tumor-prone ApcMin/+ mice on highly variant C57BL/6J (B6) and PWD/Ph (PWD) genetic backgrounds. (B6 × PWD) F1 APCMin offspring mice were largely free of intestinal adenoma, and several chromosome substitution (consomic) strains carrying single PWD chromosomes on the B6 genetic background displayed reduced adenoma numbers. Multiple dosage-dependent modifier loci on PWD chromosome 5 each contributed to tumor suppression. Activation of β-catenin–driven and stem cell–specific gene expression in the presence of ApcMin or following APC loss remained moderate in intestines carrying PWD chromosome 5, suggesting that PWD variants restrict adenoma initiation by controlling stem cell homeostasis. Gene expression of modifier candidates and DNA methylation on chromosome 5 were predominantly cis controlled and largely reflected parental patterns, providing a genetic basis for inheritance of tumor susceptibility. Human SNP variants of several modifier candidates were depleted in colorectal cancer genomes, suggesting that similar mechanisms may also affect the penetrance of cancer driver mutations in humans. Overall, our analysis highlights the strong impact that multiple genetic variants acting in networks can exert on tumor development.Significance:These findings in mice show that, in addition to accidental mutations, cancer risk is determined by networks of individual gene variants.

Supplementary tables 1-7. Supplementary Table 1: GSEA signatures and references (Wnt target genes are taken from the HALLMARK Signature). Supplementary Table 2: 58 differentially expressed genes on chromosome 5, B6 vs C5 APC min normal intestine. Supplementary Table 3: 116 differentially expressed genes not located on chromosome 5, B6 vs C5 APC min normal intestine. Supplementary table 4: Differentially expressed genes on chromosome 5 regulated in cis, trans, or both. Supplementary table 5: Candidate modifier genes differentially expressed between B6 or PWD chromosome 5. Supplementary Table 6: Polymorphisms in human orthologs of mouse genes located on chromosome 5, polymorphic between B6 and PWD, and expressed in the intestine. Supplementary Table 7: PWD polymorphisms on chr5 in genes that are expressed in the intestine and/or in adenoma.

PRDM9-mediated reproductive isolation was first described in offspring ofMus musculus musculusstrain PWD/Ph andMus musculus domesticusstrain C57BL/6J. Male F1-hybrids do not complete chromosome synapsis and arrest meiosis at Prophase I. Currently, all data supports an oligogenic control of hybrid sterility based on incompatibilities between PRDM9 and hybrid-sterility locusHstx2inMus musculushybrids. Erosion of PRDM9 binding sites was proposed to result in asymmetric binding on diverged homologs of intersubspecific F1hybrids. Numerous alleles ofPrdm9have been characterized for different subspecies ofMus musculus, but only a few were analyzed for their impact on hybrid sterility. We analyzedPrdm9diversity in natural wild mouse populations from Europe, Asia, and the Middle East and identified several novelPrdm9alleles. We established that a singlePrdm9allele is associated witht-haplotype Chromosome 17 in all three subspecies ofMus musculusand characterized the phylogenetic relationships of novelPrdm9alleles with established sterility alleles. Novel wildPrdm9alleles produced F1-hybrid male offspring that were either fertile or showedPrdm9-dependent reduction of fertility and high levels of asynapsis. Fertility or sterility phenotypes segregated purely with thePrdm9genotype, although theMus musculus musculusbackground varied. Our data substantiate that hybrid sterility is under oligogenic control withPrdm9as the leading player but is consistent with a nonbinary regulation of hybrid sterility and gradual fertility decline when homologs diverge.

Genetic predisposition affects the penetrance of tumor-initiating mutations, such as APC mutations that stabilize β-catenin and cause intestinal tumors in mice and humans. However, the mechanisms involved in genetically predisposed penetrance are not well understood. Here, we analyzed tumor multiplicity and gene expression in tumor-prone ApcMin/+ mice on highly variant C57BL/6J (B6) and PWD/Ph (PWD) genetic backgrounds. (B6 × PWD) F1 APCMin offspring mice were largely free of intestinal adenoma, and several chromosome substitution (consomic) strains carrying single PWD chromosomes on the B6 genetic background displayed reduced adenoma numbers. Multiple dosage-dependent modifier loci on PWD chromosome 5 each contributed to tumor suppression. Activation of β-catenin–driven and stem cell–specific gene expression in the presence of ApcMin or following APC loss remained moderate in intestines carrying PWD chromosome 5, suggesting that PWD variants restrict adenoma initiation by controlling stem cell homeostasis. Gene expression of modifier candidates and DNA methylation on chromosome 5 were predominantly cis controlled and largely reflected parental patterns, providing a genetic basis for inheritance of tumor susceptibility. Human SNP variants of several modifier candidates were depleted in colorectal cancer genomes, suggesting that similar mechanisms may also affect the penetrance of cancer driver mutations in humans. Overall, our analysis highlights the strong impact that multiple genetic variants acting in networks can exert on tumor development. Significance: These findings in mice show that, in addition to accidental mutations, cancer risk is determined by networks of individual gene variants.

Hybrid sterility is a critical step in the evolution of reproductive barriers between diverging taxa during the process of speciation. Recent studies of young subspecies of the house mouse revealed a multigenic nature and frequent polymorphism of hybrid sterility genes as well as the recurrent engagement of the meiosis-specific gene PR domain-containing 9 (Prdm9) and X-linked loci. Prdm9-controlled hybrid sterility is essentially chromosomal in nature, conditioned by the sequence divergence between subspecies. Depending on the Prdm9 interallelic interactions and the X-linked Hstx2 locus, the same homologs either regularly recombine and synapse, or show impaired DNA DSB repair, asynapsis, and early meiotic arrest. Thus, Prdm9-dependent hybrid sterility points to incompatibilities affecting meiotic recombination as a possible mechanism of reproductive isolation between (sub)species.

The classical definition posits hybrid sterility as a phenomenon when two parental taxa each of which is fertile produce a hybrid that is sterile. The first hybrid sterility gene in vertebrates, Prdm9, coding for a histone methyltransferase, was identified in crosses between two laboratory mouse strains derived from Mus mus musculus and M. m. domesticus subspecies. The unique function of PRDM9 protein in the initiation of meiotic recombination led to the discovery of the basic molecular mechanism of hybrid sterility in laboratory crosses. However, the role of this protein as a component of reproductive barrier outside the laboratory model remained unclear. Here, we show that the Prdm9 allelic incompatibilities represent the primary cause of reduced fertility in intersubspecific hybrids between M. m. musculus and M. m. domesticus including 16 musculus and domesticus wild-derived strains. Disruption of fertility phenotypes correlated with the rate of failure of synapsis between homologous chromosomes in meiosis I and with early meiotic arrest. All phenotypes were restored to normal when the domesticus Prdm9dom2 allele was substituted with the Prdm9dom2H humanized variant. To conclude, our data show for the first time the male infertility of wild-derived musculus and domesticus subspecies F1 hybrids controlled by Prdm9 as the major hybrid sterility gene. The impairment of fertility surrogates, testes weight and sperm count, correlated with increasing difficulties of meiotic synapsis of homologous chromosomes and with meiotic arrest, which we suppose reflect the increasing asymmetry of PRDM9-dependent DNA double-strand breaks.

During meiosis, the recombination-initiating DNA double-strand breaks (DSBs) are repaired by crossovers or noncrossovers (gene conversions). While crossovers are easily detectable, noncrossover identification is hampered by the small size of their converted tracts and the necessity of sequence polymorphism. We report identification and characterization of a mouse chromosome-wide set of noncrossovers by next-generation sequencing of 10 mouse intersubspecific chromosome substitution strains. Based on 94 identified noncrossovers, we determined the mean length of a conversion tract to be 32 bp. The spatial chromosome-wide distribution of noncrossovers and crossovers significantly differed, although both sets overlapped the known hotspots of PRDM9-directed histone methylation and DNA DSBs, thus supporting their origin in the standard DSB repair pathway. A significant deficit of noncrossovers descending from asymmetric DSBs proved their proposed adverse effect on meiotic recombination and pointed to sister chromatids as an alternative template for their repair. The finding has implications for the molecular mechanism of hybrid sterility in mice from crosses between closely related Mus musculus musculus and Mus musculus domesticus subspecies.

Genetic predisposition affects the penetrance of tumor-initiating mutations, such as APC mutations that stabilize β-catenin and cause intestinal tumors in mice and humans. However, the mechanisms involved in genetically predisposed penetrance are not well understood. Here, we analyzed tumor multiplicity and gene expression in tumor-prone

During meiosis, the recombination-initiating DNA double-strand breaks (DSBs) are repaired by crossovers or noncrossovers (gene conversions). While crossovers are easily detectable, the noncrossover identification is hampered by the small size of their converted tracts and the necessity of sequence polymorphism to detect them. We report identification and characterization of a mouse chromosome-wide set of noncrossovers by NGS sequencing of 10 mouse chromosome substitution strains. Based on 94 identified noncrossovers we determined the mean length of a conversion tract to be 32 basepairs. The spatial chromosome-wide distribution of noncrossovers and crossovers significantly differed, though both sets overlapped the known hotspots of PRDM9-directed histone methylation and DNA DSBs, thus proving their origin in the standard DSB repair pathway. A significant deficit of noncrossovers descending from asymmetric DSBs pointed to sister chromatids as an alternative template for their repair. The finding has implications for the molecular mechanism of hybrid sterility in mice.

F1 hybrids between mouse inbred strains PWD and C57BL/6 represent the most thoroughly genetically defined model of hybrid sterility in vertebrates. Hybrid male sterility can be fully reconstituted from three components of this model, namely thePrdm9hybrid sterility gene, intersubspecific homeology ofMus musculus musculusandMus musculus domesticusautosomes, and the X-linkedHstx2locus.Hstx2modulates the extent ofPrdm9-dependent meiotic arrest and harbors two additional genetic factors responsible for intersubspecific introgression-induced oligospermia (Hstx1) and reduced global meiotic recombination rate (Meir1). To facilitate positional cloning and to overcome the recombination suppression within the 4.3 Mb genomicDob interval encompassing theHstx2locus we designedHstx2-CRISPR and SPO11/Cas9 transgenes aimed to induce DNA double-strand breaks specifically within theHstx2locus. The resulting recombinant reduced theHstx2locus to 2.70 Mb (Chr X:66.51-69.21 Mb). The newly definedHstx2still operates as the major X-linked factor of the F1 hybrid sterility, controls meiotic chromosome synapsis, and modifies meiotic recombination rate. Despite extensive further crosses, the 2.70 MbHstx2interval behaved as a recombination cold spot with reduced PRDM9-mediated H3K4 hotspots and absence of DMC1-defined DNA DSB hotspots. To search for structural anomalies as a possible cause of recombination suppression we used optical mapping of theHstx2interval and observed high incidence of subspecies-specific structural variants along the X chromosome, with a striking copy number polymorphism of the microRNAMir465cluster. Finally, we analyzed the role of one of theHstx2candidate genes, the Fmr1 neighbor (Fmr1nb) gene in male fertility.Article summaryEarly meiotic arrest of mouse intersubspecific hybrids depends on the interaction between thePrdm9gene and Hybrid sterility X2 (Hstx2) locus on chromosome X. Lustyk et al. conducted high-resolution genetic and physical mapping of theHstx2locus, reduced it to 2.7 Mb interval within a constitutive recombination cold spot and found that the newly definedHstx2still operates as the X-linked hybrid sterility factor, controls meiotic chromosome synapsis, and modifies recombination rate. Optical mapping of theHstx2genomic region excluded inversion as a cause of recombination suppression and revealed a striking copy number polymorphism of the microRNAMir465cluster.

Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution because of reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers, leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects co-occur with a genome-wide delay in assembling DSB-promoting proteins on autosome axes and loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins in wild type. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated DSB-promoting proteins.

PR domain containing 9 (Prdm9) is specifying hotspots of meiotic recombination but in hybrids between two mouse subspecies Prdm9 controls failure of meiotic chromosome synapsis and hybrid male sterility. We have previously reported that Prdm9-controlled asynapsis and meiotic arrest are conditioned by the inter-subspecific heterozygosity of the hybrid genome and we presumed that the insufficient number of properly repaired PRDM9-dependent DNA double-strand breaks (DSBs) causes asynapsis of chromosomes and meiotic arrest (Gregorova et al., 2018). We now extend the evidence for the lack of properly processed DSBs by improving meiotic chromosome synapsis with exogenous DSBs. A single injection of chemotherapeutic drug cisplatin increased frequency of RPA and DMC1 foci at the zygotene stage of sterile hybrids, enhanced homolog recognition and increased the proportion of spermatocytes with fully synapsed homologs at pachytene. The results bring a new evidence for a DSB-dependent mechanism of synapsis failure and infertility of intersubspecific hybrids.

The infertility of hybrids between closely related (sub)species is one of the early reproductive isolation mechanisms leading to speciation. Since the genetic control of hybrid sterility (HS) is complex, only a few HS genes have been identified so far, including Prdm9, the only known vertebrate HS gene. Prdm9 causes infertility in male hybrids between PWD/Ph and C57BL/6J mouse strains derived from Mus m. musculus and Mus m. domesticus subspecies. However, within species Prdm9 determines the sites of programmed DNA double-strand breaks (DSBs) and meiotic recombination hotspots. In sterile hybrids, different PRDM9 variants do not bind both homologous chromosomes equally, suggesting that the asymmetry or lack of symmetry of binding sites could explain the failure of meiotic chromosomes to synapse. To investigate the relation between asynapsis and meiotic arrest, we prepared intersubspecific hybrids with stretches of consubspecific homology on several pairs of autosomes and analyzed their ability to form synaptonemal complexes. Eight examined autosomes with more than 27 Mb of consubspecific homology fully restored their synapsis and we predicted that a minimum of two symmetric DSBs per chromosome are necessary for successful meiosis. Moreover, targeted suppression of asynapsis in four chromosomes most sensitive to asynapsis partially rescued spermatogenesis, thus pointing to chromosomal quality of hybrid sterility. We hypothesize that reduced ability of meiotic chromosomes to synapse, determined by impaired recombination between evolutionary diverged homologous chromosomes could function as an ancient mechanism of hybrid sterility occurring in various sexually reproducing species.

Interspecific hybridization between closely related mammalian species, including various species of the genus Mus, is commonly associated with abnormal growth of the placenta and hybrid foetuses, a phenomenon known as hybrid placental dysplasia (HPD). The role of HPD in speciation is anticipated but still poorly understood. Here, we studied placental and foetal growth in F1 crosses between four inbred mouse strains derived from two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus. These subspecies are in the early stage of speciation and still hybridize in nature. In accordance with the maternal-foetal genomic conflict hypothesis, we found different parental influences on placental and foetal development, with placental weight most affected by the father's body weight and foetal weight by the mother's body weight. After removing the effects of parents' body weight, we did not find any significant differences in foetal or placental weights between intra-subspecific and inter-subspecific F1 crosses. Nevertheless, we found that the variability in placental weight in inter-subspecific crosses is linked to the X chromosome, similarly as for HPD in interspecific mouse crosses. Our results suggest that maternal-foetal genomic conflict occurs in the house mouse system, but has not yet diverged sufficiently to cause abnormalities in placental and foetal growth in inter-subspecific crosses. HPD is thus unlikely to contribute to speciation in the house mouse system. However, we cannot rule out that it might have contributed to other speciation events in the genus Mus, where differences in the levels of polyandry exist between the species.

Hybrid sterility is one of the reproductive isolation mechanisms leading to speciation. Prdm9, the only known vertebrate hybrid-sterility gene, causes failure of meiotic chromosome synapsis and infertility in male hybrids that are the offspring of two mouse subspecies. Within species, Prdm9 determines the sites of programmed DNA double-strand breaks (DSBs) and meiotic recombination hotspots. To investigate the relation between Prdm9-controlled meiotic arrest and asynapsis, we inserted random stretches of consubspecific homology on several autosomal pairs in sterile hybrids, and analyzed their ability to form synaptonemal complexes and to rescue male fertility. Twenty-seven or more megabases of consubspecific (belonging to the same subspecies) homology fully restored synapsis in a given autosomal pair, and we predicted that two or more DSBs within symmetric hotspots per chromosome are necessary for successful meiosis. We hypothesize that impaired recombination between evolutionarily diverged chromosomes could function as one of the mechanisms of hybrid sterility occurring in various sexually reproducing species., eLife digest It has been known for centuries that hybrids between closely related species are often infertile. This hybrid sterility was an enigma for Charles Darwin, who understood that it influenced how new species formed but could not fit it with his theory of evolution by natural selection. Sex cells – in mammals, the egg or sperm cells – form by a process called meiosis. During meiosis, chromosomes formed of DNA inherited from the mother pair up with the equivalent chromosomes inherited from the father and exchange sections of their DNA. This process is called synapsis and homologous recombination. A gene called Prdm9 determines where the DNA will break and recombine. Prdm9 plays a major role in determining whether the male hybrid offspring of two laboratory strains of mice (which come from different subspecies) are sterile. In sterile hybrids, the two versions of Prdm9 interact in ways that disturb the DNA repair process. However, these interactions are not enough on their own to cause hybrid males to be sterile. The currently prevailing view is that interactions between a large number of other – currently unidentified – genes also contribute to sterility. But could there be other processes involved that do not involve gene interactions? To investigate, Gregorova, Gergelits et al. utilized strains of hybrid mice where a pair of chromosomes of one subspecies was substituted by the corresponding pair from the other subspecies. This generated hybrids with stretches of DNA that came entirely from a single subspecies. Having such a stretch that lasted for 27 million or more DNA base pairs fully restored synapsis in a given pair of chromosomes during meiosis. Hybrid sterility was reversed when synapsis was restored in the four chromosomes that were most strongly affected by synapsis not occurring. The results presented by Gregorova, Gergelits et al. provide a direct link between Prdm9-controlled chromosome synapsis and the interruption of meiosis. Hybrid sterility occurs in all sexually reproducing organisms, as does chromosome pairing during meiosis. Thus Prdm9 could control a particular case of a more universal mechanism that enables new species to form.

Hybrid sterility genes prevent gene flow between related taxa, making the speciation process irreversible. To understand the genetic control and molecular mechanisms of hybrid sterility in mammals, hybrid sterility genes were mapped in crosses of wild-derived inbred strains originating from house mouse subspecies Mus musculus domesticus, Mus musculus musculus , and Mus musculus molossinus and from more distant mouse species Mus spretus and Mus macedonicus . Hybrid sterility genes in mice obey the same rules (large X-effect and Haldane’s rule) as observed in Drosophila and other species. The first hybrid sterility gene in vertebrates identified at the sequence level is M. m. domesticus hybrid sterility 1 ( Hst1 ) locus that was recognized as “PR domain containing 9, Prdm9 ,” encoding meiosis-specific H3K4 tri-methyltransferase.

Laboratory mice are valuable in biomedical research in part because of the extraordinary diversity of genetic resources that are available for studies of complex genetic traits and as models for human biology and disease. Chromosome substitution strains (CSSs) are important in this resource portfolio because of their demonstrated use for gene discovery, genetic and epigenetic studies, functional characterizations, and systems analysis. CSSs are made by replacing a single chromosome in a host strain with the corresponding chromosome from a donor strain. A complete CSS panel involves a total of 22 engineered inbred strains, one for each of the 19 autosomes, one each for the X and Y chromosomes, and one for mitochondria. A genome survey simply involves comparing each phenotype for each of the CSSs with the phenotypes of the host strain. The CSS panels that are available for laboratory mice have been used to dissect a remarkable variety of phenotypes and to characterize an impressive array of disease models. These surveys have revealed considerable phenotypic diversity even among closely related progenitor strains, evidence for strong epistasis and for heritable epigenetic changes. Perhaps most importantly, and presumably because of their unique genetic constitution, CSSs, and congenic strains derived from them, the genetic variants underlying quantitative trait loci (QTLs) are readily identified and functionally characterized. Together these studies show that CSSs are important resource for laboratory mice.

Hybrid sterility as a postzygotic reproductive isolation mechanism has been studied for over 80 years, yet the first identifications of hybrid sterility genes in Drosophila and mouse are quite recent. To study the genetic architecture of F(1) hybrid sterility between young subspecies of house mouse Mus m. domesticus and M. m. musculus, we conducted QTL analysis of a backcross between inbred strains representing these two subspecies and probed the role of individual chromosomes in hybrid sterility using the intersubspecific chromosome substitution strains. We provide direct evidence that the asymmetry in male infertility between reciprocal crosses is conferred by the middle region of M. m. musculus Chr X, thus excluding other potential candidates such as Y, imprinted genes, and mitochondrial DNA. QTL analysis identified strong hybrid sterility loci on Chr 17 and Chr X and predicted a set of interchangeable autosomal loci, a subset of which is sufficient to activate the Dobzhansky-Muller incompatibility of the strong loci. Overall, our results indicate the oligogenic nature of F(1) hybrid sterility, which should be amenable to reconstruction by proper combination of chromosome substitution strains. Such a prefabricated model system should help to uncover the gene networks and molecular mechanisms underlying hybrid sterility.

Adult BMD, an important risk factor for fracture, is the result of genetic and environmental interactions. A quantitative trait locus (QTL) for the phenotype of volumetric BMD (vBMD), named Bmd8, was found on mid-distal chromosome (Chr) 6 in mice. This region is homologous to human Chr 3p25. The B6.C3H-6T (6T) congenic mouse was previously created to study this QTL. Using block haplotyping of the 6T congenic region, expression analysis in the mouse, and examination of nonsynonymous SNPs, peroxisome proliferator activated receptor gamma (Pparg) was determined to be the most likely candidate gene for the Bmd8 QTL of the 630 genes located in the congenic region. Furthermore, in the C3H/HeJ (C3H) strain, which is the donor strain for the 6T congenic, several polymorphisms were found in the Pparg gene. On challenge with a high-fat diet, we found that the 6T mouse has a lower areal BMD (aBMD) and volume fraction of trabecular bone (BV/TV%) of the distal femur compared with B6 mice. Interactions between SNPs in the PPARG gene and dietary fat for the phenotype of BMD were examined in the Framingham Offspring Cohort. This analysis showed that there was a similar interaction of the PPARG gene and diet (fat intake) on aBMD in both men and women. These findings suggest that dietary fat has a significant influence on BMD that is dependent on the alleles present for the PPARG gene.

Extensive linkage disequilibrium among classical laboratory strains represents an obstacle in the high-resolution haplotype mapping of mouse quantitative trait loci (QTL). To determine the potential of wild-derived mouse strains for fine QTL mapping, we constructed a haplotype map of a 250-kb region of the t-complex on chromosome 17 containing the Hybrid sterility 1 (Hst1) gene. We resequenced 33 loci from up to 80 chromosomes of five mouse (sub)species. Trans-species single-nucleotide polymorphisms (SNPs) were rare between Mus m. musculus (Mmmu) and Mus m. domesticus (Mmd). The haplotypes in Mmmu and Mmd differed and therefore strains from these subspecies should not be combined for haplotype-associated mapping. The haplotypes of t-chromosomes differed from all non-t Mmmu and Mmd haplotypes. Half of the SNPs and SN indels but only one of seven longer rearrangements found in classical laboratory strains were useful for haplotype mapping in the wild-derived M. m. domesticus. The largest Mmd haplotype block contained three genes of a highly conserved synteny. The lengths of the haplotype blocks deduced from 36 domesticus chromosomes were in tens of kilobases, suggesting that the wild-derived Mmd strains are suitable for fine interval-specific mapping.

Consomic (chromosome substitution) strains (CSs) represent the most recent addition to the mouse genetic resources aimed to geneticaly analyze complex trait loci (QTLs). In this study, we report the development of a set of 28 mouse intersubspecific CSs. In each CS, we replaced a single chromosome of the C57BL/6J (B6) inbred strain (mostly Mus m. domesticus) with its homolog from the PWD/Ph inbred strain of the Mus m. musculus subspecies. These two progenitor subspecies diverged less than 1 million years ago and accumulated a large number of genetic differences that constitute a rich resource of genetic variation for QTL analyses. Altogether, the 18 consomic, nine subconsomic, and one conplastic strain covered all 19 autosomes, X and Y sex chromosomes, and mitochondrial DNA. Most CSs had significantly lower reproductive fitness compared with the progenitor strains. CSs homosomic for chromosomes 10 and 11, and the C57BL/6J-Chr X males, failed to reproduce and were substituted by less affected subconsomics carrying either a proximal, central, or distal part of the respective chromosome. A genome-wide scan of 965 DNA markers revealed 99.87% purity of the B6 genetic background. Thirty-three nonsynonymous substitutions were uncovered in the protein-coding regions of the mitochondrial DNA of the B6.PWD-mt conplastic strain. A pilot-phenotyping experiment project revealed a high number of variations among B6.PWD consomics.

Three orthologous genes encoding programmed cell death 2 (PDCD2), TATA-binding protein (TBP), and proteasomal subunit C5 (PSMB1) proteins have been shown previously to be nonrandomly distributed in both mammalian and invertebrate genomes. Here we analyze a conserved synteny of the PDCD2, TBP, and PSMB1 orthologs in four nonmammalian vertebrates. Homologous genes of the chicken, zebrafish, fugu, and Tetraodon nigroviridis were identified. A chicken cosmid harboring the orthologs of these three genes was completely sequenced. The fish genes were analyzed in silico. In all seven vertebrates thus far investigated, the PDCD2 and TBP genes are located tail-to-tail. In six tested species but the zebrafish, the PSMB1 gene mapped head-to-head or in the close vicinity to the TBP, but even in the zebrafish, all three genes were syntenic. In contrast, a three times reused synteny breakpoint in the 5'-region from PDCD2 was detected. A comparative analysis revealed the distribution of putative matrix-attached regions (MARs), which may affect the synteny conservation.

The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact potygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

All of the mouse models of human trisomy 21 syndrome that have been studied so far are based on segmental trisomies, encompassing, to a varying extent, distal chromosome 16. Their comparison with one or more unrelated and non-overlapping segmental trisomies may help to distinguish the effects of specific triplicated genes from the phenotypes caused by less specific developmental instability mechanisms. In this paper, the Ts43H segmental trisomy of mouse chromosome 17 is presented as such an alternative model. The trisomy stretches over 32.5 Mb of proximal chromosome 17 and includes 486 genes. The triplicated interval carries seven blocks of synteny with five human chromosomes. The block syntenic to human chromosome 21 contains 20 genes.

Autosomal trisomies and monosomies bring serious threats to embryonic development through transcriptional disarray caused primarily by the dosage effect of the aneuploid part of the genome. The present study compared the effect of a mouse-viable 30-Mb segmental trisomy on the genome-wide transcriptional profile of somatic (liver) cells and male germ cells. Although the 1.6-fold change in expression of triplicated genes reflected the gene dosage in liver cells, the extra copy genes were compensated in early pachytene spermatocytes, showing 1.18-fold increase. Although more pronounced, the dosage compensation of trisomic genes was concordant with the incidence of HORMAD2 protein and histone gammaH2AX markers of unsynapsed chromatin. A possible explanation for this includes insufficient sensitivity to detect the meiotic silencing of unsynapsed chromatin markers in the 30-Mb region of the chromosome or an earlier silencing effect of another epigenetic factor. Taken together, our results indicate that the meiotic silencing of unsynapsed chromatin is the major, but most likely not the only, factor driving the dosage compensation of triplicated genes in primary spermatocytes.

Autosomal trisomies and monosomies bring serious threats to embryonic development through transcriptional disarray caused primarily by the dosage effect of the aneuploid part of the genome. The present study compared the effect of a mouse-viable 30-Mb segmental trisomy on the genome-wide transcriptional profile of somatic (liver) cells and male germ cells. Although the 1.6-fold change in expression of triplicated genes reflected the gene dosage in liver cells, the extra copy genes were compensated in early pachytene spermatocytes, showing 1.18-fold increase. Although more pronounced, the dosage compensation of trisomic genes was concordant with the incidence of HORMAD2 protein and histone gammaH2AX markers of unsynapsed chromatin. A possible explanation for this includes insufficient sensitivity to detect the meiotic silencing of unsynapsed chromatin markers in the 30-Mb region of the chromosome or an earlier silencing effect of another epigenetic factor. Taken together, our results indicate that the meiotic silencing of unsynapsed chromatin is the major, but most likely not the only, factor driving the dosage compensation of triplicated genes in primary spermatocytes.

This issue of Mammalian Genome contains a short narrative report for each of the 21 mouse chromosomes produced by the mouse Chromosome Committees. Committees are constituted from individuals whose research focuses on specific chromosomal regions or who are interested in contributing their insight and knowledge to developing a consensus map of a particular chromosome. These committees meet regularly at the annual International Mouse Genome Conference and are responsible for maintaining the public electronic files summarizing the status of their chromosome. Complete Chromosome Committee Report files can be viewed or downloaded from the Mouse Genome Database (MGD) via the World Wide Web at URL address

Speciation genes restrict gene flow between the incipient species and related taxa. Three decades ago, we mapped a mammalian speciation gene, hybrid sterility 1 ( Hst1 ), in the intersubspecific hybrids of house mouse. Here, we identify this gene as Prdm9 , encoding a histone H3 lysine 4 trimethyltransferase. We rescued infertility in male hybrids with bacterial artificial chromosomes carrying Prdm9 from a strain with the “fertility” Hst1 f allele. Sterile hybrids display down-regulated microrchidia 2B ( Morc2b ) and fail to compartmentalize γH2AX into the pachynema sex (XY) body. These defects, seen also in Prdm9 -null mutants, are rescued by the Prdm9 transgene. Identification of a vertebrate hybrid sterility gene reveals a role for epigenetics in speciation and opens a window to a hybrid sterility gene network.

The recognition of mammalian genes encoded within a mouse yeast artificial chromosome (YAC) by the yeast transcription and RNA processing machinery was investigated. Transcripts from five genes known to be encoded by the YAC were all found in the total yeast RNA. Of 12 mouse introns assayed, six were correctly spliced by the yeast. However, an abnormal transcription of mouse DNA was also observed. Three genes of three tested were transcribed both from their sense and antisense strands and all tested microsatellite, inter-repetitive and anonymous mouse loci were detected in the YAC clone RNA. An RNA transcript from a well defined intergenic region of two head-to-head oriented mouse genes was detected by RT-PCR and by RNase protection assay. These results indicate the presence of multiple yeast-specific transcription sites in the mouse DNA. 3' RACE experiments demonstrated the inability of the yeast to use the mouse polyadenylation signals. Thus, a method for isolation of mammalian exons based on a YAC clone RNA is likely to produce a high background, because the enrichment with mammalian exons in the YAC RNA is low. Nevertheless, YAC clones can serve as in vivo test tubes to study the conservation of RNA processing sequences.

Transcriptional regulation of the MHC class I genes leading to their developmental and tissue specific expression is still poorly understood in spite of the recovery of a large variety of cis -controlling sequences and trans -acting factors pertaining to the 5′ enhancer and the downstream regulatory element. Here we produced a series transgenic lines of mice with a genomic subclone of the H2-K b gene consisting of 367 bp of the 5′ upstream region, the coding region and 1.5 kb of the 3′ downstream region and carrying all hitherto known regulatory sequences. The comparison of nine transgenic lines carrying the same H2-K b transgene made it possible to ask whether the cis -information present in the transgene was sufficient for the tissue- and developmental-specific expression and its copy number dependence. We found the proper developmental onset of expression of the transgene at day 13 p.c. and correct tissue specific mRNA levels in adult mice. While in lymphoid tissues and in lung the number of transgene copies still correlated with RNA levels, the copy number dependence was completely lost in liver, kidney and embryonic tissues. Comparison with previously published H2-K b transgenes indicates that the H2-K b locus-controlling region is composed of more than one element.

Experimental animal models are essential to obtain basic knowledge of the underlying biological mechanisms in human diseases. Here, we review major contributions to biomedical research and discoveries that were obtained in the mouse model by using forward genetics approaches and that provided key insights into the biology of human diseases and paved the way for the development of novel therapeutic approaches.

A continuous blood volume monitoring (CBVM) device (Inline Diagnostics, Riverdale, USA) was used to study response to prescribed ultrafiltration during haemodialysis (HD) in 66 stabilised HD patients. Fifty percent of patients showed the expected linear decrease in BV right from the beginning of HD (group 1), 32% exhibited no decrease at all (group 2), while eighteen percent formed the transient group 3 which showed a plateau of varying length after which a decrease occurred. The correct setting of dry weight was verified through evaluation of the ratio of extracellular fluid volume to total body water (VEC/TBW) in 26 patients by means of whole body multifrequency impedometry MFI (Xitron Tech., San Diego, USA) and through measurement of the Vena Cava Inferior diameter (VCID) pre and post HD (in 6 and 5 patients from groups 1 and 3 and from group 2, respectively). The mean VEC/TBW in groups 1 and 3 was 0.56 pre and 0.51 post HD as compared to 0.583 and 0.551 in group 2. VCID decreased on average by 14.1% in groups 1 and 3 but remained stable in group 2. Both findings thus confirmed inadequately high estimation of dry weight. Since CBVM is extremely easy to perform it can be used as a method of choice in detecting inadequately high prescribed dry weight. The status of the cardiovascular system must always be considered before final judgement is made.

According to the Dobzhansky-Muller model, hybrid sterility is a consequence of the independent evolution of related taxa resulting in incompatible genomic interactions of their hybrids. The model implies that the incompatibilities evolve randomly, unless a particular gene or nongenic sequence diverges much faster than the rest of the genome. Here we propose that asynapsis of heterospecific chromosomes in meiotic prophase provides a recurrently evolving trigger for the meiotic arrest of interspecific F1 hybrids. We observed extensive asynapsis of chromosomes and disturbance of the sex body in >95% of pachynemas of Mus m. musculus × Mus m. domesticus sterile F1 males. Asynapsis was not preceded by a failure of double-strand break induction, and the rate of meiotic crossing over was not affected in synapsed chromosomes. DNA double-strand break repair was delayed or failed in unsynapsed autosomes, and misexpression of chromosome X and chromosome Y genes was detected in single pachynemas and by genome-wide expression profiling. Oocytes of F1 hybrid females showed the same kind of synaptic problems but with the incidence reduced to half. Most of the oocytes with pachytene asynapsis were eliminated before birth. We propose the heterospecific pairing of homologous chromosomes as a preexisting condition of asynapsis in interspecific hybrids. The asynapsis may represent a universal mechanistic basis of F1 hybrid sterility manifested by pachytene arrest. It is tempting to speculate that a fast-evolving subset of the noncoding genomic sequence important for chromosome pairing and synapsis may be the culprit.

Hybrid sterility as a postzygotic reproductive isolation mechanism has been studied for over 80 years, yet the first identifications of hybrid sterility genes in Drosophila and mouse are quite recent. To study the genetic architecture of F(1) hybrid sterility between young subspecies of house mouse Mus m. domesticus and M. m. musculus, we conducted QTL analysis of a backcross between inbred strains representing these two subspecies and probed the role of individual chromosomes in hybrid sterility using the intersubspecific chromosome substitution strains. We provide direct evidence that the asymmetry in male infertility between reciprocal crosses is conferred by the middle region of M. m. musculus Chr X, thus excluding other potential candidates such as Y, imprinted genes, and mitochondrial DNA. QTL analysis identified strong hybrid sterility loci on Chr 17 and Chr X and predicted a set of interchangeable autosomal loci, a subset of which is sufficient to activate the Dobzhansky-Muller incompatibility of the strong loci. Overall, our results indicate the oligogenic nature of F(1) hybrid sterility, which should be amenable to reconstruction by proper combination of chromosome substitution strains. Such a prefabricated model system should help to uncover the gene networks and molecular mechanisms underlying hybrid sterility.

Hybrid sterility 1 (Hst1) is the major gene responsible for sterility of male hybrids between Mus musculus and certain laboratory strains. Thus, Hst1 is of importance in studying both postreproductive isolation of closely related species and male fertility. It has been mapped to mouse chromosome 17 in the region corresponding to the third inversion of the t haplotypes. The aim of the present study was to construct a physical map of the Hst1 region as the first step in an effort to clone the gene. Three yeast artificial chromosome (YAC) libraries (Princeton, Whitehead, and ICRF) were screened with polymerase chain reaction (PCR) oligonucleotide primers and DNA probes specific for Joel previously mapped into the region of the third inversion. The isolated YAC clones were restriction mapped and arranged into contigs. Sixteen YAC clones were arranged into a single contig encompassing a region approximately 2000 kb long based on restriction mapping of highly overlapping but independently derived YAC clones. Five new loci in the region of the third inversion were mapped and the order and approximate physical distances of 12 loci established in this contig. The Hst1 gene maps approximately 0.2 cM proximal to the D17Ph1 locus encompassed by the YAC contig. Since the contig extends at least 1200 kb proximal to D17Ph1 , it should contain the Hst1 gene.

We compared the organization of satellite DNA (stDNA) and its chromosomal allocation in Mus domesticus and in Mus musculus. The two stDNAs show similar restriction fragment profiles after digestion (probed with M. domesticus stDNA) with some endonucleases of which restriction sequences are present in the 230-240 bp repetitive unit of the M. domesticus stDNA. In contrast, EcoRI digestion reveals that M. musculus stDNA lacks most of the GAATTC restriction sites, particularly at the level of the half-monomer. The chromosome distribution of stDNA (revealed by an M. domesticus stDNA probe) shows different patterns in the M. domesticus and M. musculus karyotypes, with about 60% of M. domesticus stDNA retained in the M. musculus genome. It is particularly noteworthy that the pericentromeric regions of M. musculus chromosomes 1 and X are totally devoid of M. domesticus stDNA sequences. In both groups, the differences in energy transfer between the stDNA-bound fluorochromes Hoechst 33258 and propidium iodide suggest that AT-rich repeated sequences have a much more clustered array in the M. domesticus stDNA, as if they are organized in tandem repeats longer than those of M. musculus. Considering the data as a whole, it seems likely that the evolutionary paths of the two stDNAs diverged after the generation of the ancestral 230-240 bp stDNA repetitive unit through the amplification, in the M. domesticus genome, of a family repeat which included the EcoRI GAATTC restriction sequence.

Approximately 30% of patients with hypertension have hepatic steatosis, and it has recently been proposed that fatty liver be considered a feature of the metabolic syndrome. Obesity, diet, and level of physical activity are likely factors modulating risk for hepatic steatosis, however genetic factors could also influence susceptibility or resistance to fatty liver in hypertensive or normotensive subjects. In genetic studies in spontaneously hypertensive rats (SHRs) and Brown Norway (BN) rats, we discovered that a variant form of sterol regulatory element binding transcription factor 1 (S rebf1 gene, SREBP-1 protein) underlies a quantitative trait locus (QTL) influencing hepatic cholesterol levels in response to a high cholesterol diet. Compared with the BN allele of Srebf1 , the SHR allele of Srebf1 includes variants in the promoter and coding regions that are linked to hepatic deficiency of SREBP-1 mRNA and protein, reduced expression of the SREBP-1 target gene stearoyl-CoA desaturase 1, reduced promoter activity for SREBP-1c, and relative protection from dietary induced accumulation of liver cholesterol. Genetic correction of reduced SREBP-1 activity by derivation of congenic and transgenic strains of SHR increased hepatic cholesterol levels, thereby confirming Srebf1 as a QTL influencing hepatic lipid metabolism in the rat. The Srebf1 variant regulating hepatic cholesterol did not appear to affect blood pressure. These findings (1) are consistent with the results of association studies indicating that common polymorphisms affecting SREBP-1 may influence cholesterol synthesis in humans and (2) indicate that variation in Srebf1 may influence risk for hepatic steatosis.