Evolutionary strata on the mouse X chromosome correspond to strata on the human X chromosome

Lahn and Page previously observed that genes on the human X chromosome were physically arranged along the chromosome in "strata," roughly ordered by degree of divergence from related genes on the Y chromosome. They hypothesized that this ordering results from a historical series of suppressions of recombination along the mammalian Y chromosome, thereby allowing formerly recombining X and Y chromosomal genes to diverge independently. Here predictions of this hypothesis are confirmed in a nonprimate mammalian order, Rodentia, through an analysis of eight gene pairs from the X and Y chromosomes of the house mouse, Mus musculus. The mouse X chromosome has been rearranged relative to the human X, so strata were not found in the same physical order on the mouse X. However, based on synonymous evolutionary distances, X-linked genes in M. musculus fall into the same strata as orthologous genes in humans, as predicted. The boundary between strata 2 and 3 is statistically significant, but the boundary between strata 1 and 2 is not significant in mice. An analysis of smaller fragments of Smcy, Smcx, Zfy, and Zfx from seven species of Mus confirmed that the strata in Mus musculus were representative of the genus Mus.     

Breaking the species barrier: derivation of germline-competent embryonic stem cells from Mus spretus x C57BL/6 hybrids

Embryonic stem (ES) cells, which can differentiate into almost all types of cells, have been derived from the house mouse Mus musculus, rat, rabbit, humans, and other species. Transmission of the genotype to the offspring of chimeras has been achieved only with M. musculus ES cells, limiting targeted mutagenesis using ES cells to this species. Mus spretus, which exhibits many genetic polymorphisms with M. musculus, displays dominant resistance to cancer and inflammation, making derived inbred strains very useful in positional cloning and interspecies mapping. We show here for the first time the derivation of ES cells from hybrid blastocysts, obtained by the mating of two different species, namely Mus musculus and Mus spretus, and their use for the generation of chimeric mice that transmit the Mus spretus genotype and phenotype to the offspring. These hybrid ES cells allow the genetic manipulation of Mus spretus, as an alternative to Mus musculus.     

Random X inactivation in the mule and horse placenta

In eutherian mammals, dosage compensation of X-linked genes is achieved by X chromosome inactivation. X inactivation is random in embryonic and adult tissues, but imprinted X inactivation (paternal X silencing) has been identified in the extra-embryonic membranes of the mouse, rat, and cow. Few other species have been studied for this trait, and the data from studies of the human placenta have been discordant or inconclusive. Here, we quantify X inactivation using RNA sequencing of placental tissue from reciprocal hybrids of horse and donkey (mule and hinny). In placental tissue from the equid hybrids and the horse parent, the allelic expression pattern was consistent with random X inactivation, and imprinted X inactivation can clearly be excluded. We characterized horse and donkey XIST gene and demonstrated that XIST allelic expression in female hybrid placental and fetal tissues is negatively correlated with the other X-linked genes chromosome-wide, which is consistent with the XIST-mediated mechanism of X inactivation discovered previously in mice. As the most structurally and morphologically diverse organ in mammals, the placenta also appears to show diverse mechanisms for dosage compensation that may result in differences in conceptus development across species.     

Dosage compensation of the mammalian X chromosome influences the phenotypic variability of X-linked dominant male-lethal disorders

In mammals females inactivate one of the two X chromosomes during early development to achieve an equal gene dosage between sexes. This process, named X chromosome inactivation (XCI), usually occurs randomly. However, in a few instances, non-random XCI may take place, thus modulating the phenotype observed in female patients carrying mutations in X-linked genes. Different aspects related to dosage compensation contribute to explain the influences of XCI on the phenotypic variability observed in female patients. The study of two X-linked dominant male-lethal disorders, such as the microphthalmia with linear skin lesions (MLS) syndrome and the oral-facial-digital type I (OFDI) syndrome, offers the opportunity to discuss this intriguing topic. In addition, recent data on the characterisation of a murine model for OFDI provide the opportunity to discuss how differences in the XCI between Homo sapiens and Mus musculus can justify the discrepancies between the phenotypes observed in OFDI patients and the corresponding murine model.     

Isolation,X location and activity of the marsupial homologue of <Emphasis Type="Italic">SLC16A2</Emphasis>, an <Emphasis Type="Italic">XIST</Emphasis>-flanking gene in eutherian mammals

X chromosome inactivation (XCI) achieves dosage compensation between males and females for most X-linked genes in eutherian mammals. It is a whole-chromosome effect under the control of the XIST locus, although some genes escape inactivation. Marsupial XCI differs from the eutherian process, implying fundamental changes in the XCI mechanism during the evolution of the two lineages. There is no direct evidence for the existence of a marsupial XIST homologue. XCI has been studied for only a handful of genes in any marsupial, and none in the model kangaroo Macropus eugenii (the tammar wallaby). We have therefore studied the sequence, location and activity of a gene SLC16A2 (solute carrier, family 16, class A, member 2) that flanks XIST on the human and mouse X chromosomes. A BAC clone containing the marsupial SLC16A2 was mapped to the end of the long arm of the tammar X chromosome and used in RNA FISH experiments to determine whether one or both loci are transcribed in female cells. In male and female cells, only a single signal was found, indicating that the marsupial SLC16A2 gene is silenced on the inactivated X.     

X chromosome inactivation and X-linked mental retardation

The expression of X-linked genes in females heterozygous for X-linked defects can be modulated by epigenetic control mechanisms that constitute the X chromosome inactivation pathway. At least four different effects have been found to influence, in females, the phenotypic expression of genes responsible for X-linked mental retardation (XLMR). First, non-random X inactivation, due either to stochastic or genetic factors, can result in tissues in which one cell type (for example, that in which the X chromosome carrying a mutant XLMR gene is active) dominates, instead of the normal mosaic cell population expected as a result of random X inactivation. Second, skewed inactivation of the normal X in individuals carrying a deletion of part of the X chromosome has been documented in a number of mentally retarded females. Third, functional disomy of X-linked genes that are expressed inappropriately due to the absence of X inactivation has been found in mentally retarded females with structurally abnormal X chromosomes that do not contain the X inactivation center. And fourth, dose-dependent overexpression of X-linked genes that normally “escape” X inactivation may account for the mental and developmental delay associated with increasing numbers of otherwise inactive X chromosomes in individuals with X chromosome aneuploidy. © 1996 Wiley-Liss, Inc.     

Replication timing properties, of the humanHPRT locus on active, inactive and reactivated X chromosomes

X chromosome inactivation is associated with a highly asynchronous pattern of DNA replication at most X-linked loci in females. We studied the human HPRT locus, which is subject to X inactivation and expressed from only the active homolog, with the goal of comparing replication properties between the active and inactive homologs in this region using a fluorescence in situ hybridization approach. We found that in normal female lymphoblasts this locus is replicated in a highly asynchronous manner across a broad, discrete 500–600 kb zone with earliest replication appearing at the gene coding sequence. This general timing profile is maintained in normal male lymphoblasts, as well as in hamster x human hybrid cells containing the active human X chromosome. However, the inactive human X chromosome in the hamster cell background does not appear to function in a fully equivalent manner to the normal inactive X chromosome in female cells. Furthermore, reactivation of the inactive human X chromosome in a hamster x human hybrid system by 5-azacytidine treatment and HAT selection restores early replication at the HPRT gene itself, but does not change the overall domain behavior.     

Involvement of the X chromosome in non-Hodgkin lymphoma

Gain of an X chromosome is observed as a secondary, acquired karyotypic alteration in a significant proportion of malignant lymphomas. To determine the potential involvement of X-linked genes in neoplastic development, we have analyzed the inactivation status of the supernumerary X chromosome in lymphomas in both male and female patients. In males, neither methylation of FMR1 nor expression of XIST was detected, demonstrating that the duplicated chromosome was not subject to inactivation. In females, both expressed polymorphisms and polymorphisms associated with methylation differences between the active and inactive X chromosome were analyzed to determine whether the duplicated chromosome was active or inactive. To facilitate this analysis, allele-specific PCR primers were designed for detection of previously described polymorphisms in the IDSX and G6PD genes. The female lymphomas were shown to be clonal in origin, and duplication of either the active (5 cases) or inactive (4 cases) X chromosome was observed. Correlations between clinical status and the inactivation status of the X chromosome involved in the duplication were not observed in our relatively small sample, although 4/4 informative cases with a t(14;18) showed duplication of the active X chromosome. In the course of these studies, we detected hypermethylation of the androgen receptor (AR) locus in an extremely high proportion of both male (7/9) and female (9/10) samples. These results are discussed with respect to whether sex chromosome aneuploidies in tumors are involved in, or simply the result of, the neoplastic process. Genes Chromosomes Cancer 28:246-257, 2000.     

Nomenclature and overview of the mouse (Mus musculus and Mus sp.) immunoglobulin kappa (IGK) genes

'Nomenclature and overview of the mouse (Mus musculus and Mus sp.) immunoglobulin kappa (IGK) Genes', the 19th report of the 'IMGT Locus in Focus' section, provides the first complete list of all the mouse (M. musculus) IGK genes. The mouse (M. musculus) locus spans 3,200 kb. The total number of mouse (M. musculus) IGK genes per haploid genome is 164 (174 if the orphons are included). The functional genomic repertoire comprises 93 IGKV belonging to 18 subgroups, 5 IGKJ and 1 IGKC gene. IMGT gene names and definitions of the mouse (M. musculus) IGK genes on chromosome 6 and IGK orphons are provided with the gene functionality and the number of alleles, according to the concepts of IMGT-ONTOLOGY and to rules of the IMGT Scientific chart, with the accession numbers of the IMGT reference sequences. These tables and figures are available at the IMGT Marie-Paule page of IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France.     

Methylation of L1Hs promoters is lower on the inactive X, has a tendency of being higher on autosomes in smaller genomes and shows inter-individual variability at some loci

LINE-1 repeats account for ~17% of the human genome. Little is known about their individual methylation patterns, because their repetitive, almost identical sequences make them difficult to be individually targeted. Here, we used bisulfite conversion to study methylation at individual LINE-1 repeats. The loci studied included 39 X-linked loci and 5 autosomal loci. On the X chromosome in women, we found statistically significant less methylation at almost all L1Hs compared with men. Methylation at L1P and L1M did not correlate with the inactivation status of the host DNA, while the majority of L1Hs that were possible to be studied lie in inactivated regions. To investigate whether the male-female differences at L1Hs on the X are linked to the inactivation process itself rather than to a mere influence of gender, we analyzed six of the L1Hs loci on the X chromosome in Turners and Klinefelters which have female and male phenotype, respectively, but with reversed number of X chromosomes. We could confirm that all samples with two X chromosomes are hypomethylated at the L1Hs loci. Therefore, the inactive X is hypomethylated at L1Hs; the latter could play an exclusive role in the X chromosome inactivation process. At autosomal L1Hs, methylation levels showed a correlation tendency between methylation level and genome size, with higher methylation observed at most loci in individuals with one X chromosome and the lowest in XXY individuals. In summary, loci-specific LINE-1 methylation levels show considerable plasticity and depend on genomic position and constitution.     

Proliferation and growth factor expression in abnormally enlarged placentas of mouse interspecific hybrids.

It has been shown previously that abnormal placental growth occurs in crosses and backcrosses between different mouse (Mus) species. In such crosses, late gestation placentas may weigh between 13 and 848 mg compared with a mean placental weight of approximately 100 mg in late gestation M. musculus intraspecific crosses. A locus on the X-chromosome was shown to segregate with placental dysplasia. Thus in the (M. musculus x M. spretus)F1 x M. musculus backcross, placental hyperplasia cosegregates with a M. spretus derived X-chromosome. Here we have investigated whether increased cell proliferation and aberrant expression of two genes that are involved in placental growth control, Igf2 and Esx1, may cause, or contribute to placental hyperplasia. Increased bromodeoxyuridine labeling of nuclei, reflecting enhanced proliferation, was indeed observed in hyperplastic placentas when compared with normal littermate placentas. Also, increased expression of Igf2 was seen in giant cells and spongiotrophoblast. However, when M. musculus x M. spretus F1 females were backcrossed with males that were heterozygous for a targeted mutation of the Igf2 gene, placentas that carried a M. spretus derived X-chromosome and were negative for a functional Igf2 allele exhibited an intermediate placental phenotype. Furthermore, in early developmental stages of placental hyperplasia, we observed a decreased expression of the X-chromosomal Esx1 gene. This finding suggests that abnormal expression of both Igf2 and Esx1 contributes to abnormal placental development in mouse interspecific hybrids. However, Esx1 is not regulated by IGF2.     

Mouse consomic strains: exploiting genetic divergence between Mus m. musculus and Mus m. domesticus subspecies

Consomic (chromosome substitution) strains (CSs) represent the most recent addition to the mouse genetic resources aimed to genetically 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.     

Word frequency analysis reveals enrichment of dinucleotide repeats on the human X chromosome and [GATA]n in the X escape region

Most of the human genome encodes neither protein nor known functional RNA, yet available approaches to seek meaningful information in the "noncoding" sequence are limited. The unique biology of the X chromosome, one of which is silenced in mammalian females, can yield clues into sequence motifs involved in chromosome packaging and function. Although autosomal chromatin has some capacity for inactivation, evidence indicates that sequences enriched on the X chromosome render it fully competent for silencing, except in specific regions that escape inactivation. Here we have used a linguistic approach by analyzing the frequency and distribution of nine base-pair genomic "words" throughout the human genome. Results identify previously unknown sequence differences on the human X chromosome. Notably, the dinucleotide repeats [AT]n, [AC]n, and [AG]n are significantly enriched across the X chromosome compared with autosomes. Moreover, a striking enrichment (>10-fold) of [GATA]n is revealed throughout the 10-Mb segment at Xp22 that escapes inactivation, and is confirmed by fluorescence in situ hybridization. A similar enrichment is found in other eutherian genomes. Our findings clearly demonstrate sequence differences relevant to the novel biology and evolution of the X chromosome. Furthermore, they implicate simple sequence repeats, linked to gene regulation and unusual DNA structures, in the regulation and formation of facultative heterochromatin. Results suggest a new paradigm whereby a regional escape from X inactivation is due to the presence of elements that prevent heterochromatinization, rather than the lack of other elements that promote it.     

Diagnosis of Wiskott-Aldrich syndrome by analysis of the X chromosome inactivation patterns in maternal leucocyte populations using the hypervariable DXS255 locus.

The Wiskott-Aldrich syndrome (WAS) is characterized by severe recurrent infections, petachiae and chronic eczema. The syndrome involves differentiation disorders in several haematopoietic cell lineages usually manifested as T lymphocyte deficiency, dysgammaglobulinaemia and thrombocytopenia. The defect is inherited in an X-linked recessive mode. A 1-year-old boy presented with otitis, upper respiratory infections, eczema, a persistent granulocytopenia and a dysgammaglobulinaemia. In his family five males in two generations had been shown to have WAS, which entailed a significant risk for the patient to have WAS. As the WAS gene or gene product is not delineated, the symptoms of the patient presented a diagnostic dilemma. If the boy had inherited the disease, his mother should be a WAS carrier. Segregation analysis in the family using the closely linked restriction fragment length polymorphisms (RFLP) DXS7, DXS255 and DXS14 did not exclude her carriership, although the probability was low. As a result of the differentiation arrest, obligate female WAS carriers manifest a unilateral X chromosome inactivation pattern in several haematopoietic cell lineages. Methylation analysis of the X chromosomal DXS255 loci exposed random X chromosome inactivation patterns in the peripheral blood granulocytes, T lymphocytes and B lymphocytes of the patient's mother. These findings excluded her WAS carriership and therefore excluded the diagnosis of WAS in the patient. This was further substantiated in a 1-year follow up with recovery from the haematological and immunological symptoms. These results demonstrated that X inactivation analysis in maternal leucocytes is decisive in the exclusion of the diagnosis of WAS.     

Mapping segmental and sequence variations among laboratory mice using BAC array CGH

We used arrays of 2069 BACs (1303 nonredundant autosomal clones) to map sequence variation among Mus spretus (SPRET/Ei and SPRET/Glasgow) and Mus musculus (C3H/HeJ, BALB/cJ, 129/J, DBA/2J, NIH, FVB/N, and C57BL/6) strains. We identified 80 clones representing 74 autosomal loci of copy number variation (|log(2)ratio| >/= 0.4). These variant loci distinguish laboratory strains. By FISH mapping, we determined that 63 BACs mapped to a single site on C57BL/6J chromosomes, while 17 clones mapped to multiple chromosomes (n = 16) or multiple sites on one chromosome (n = 1). We also show that small ratio changes (Delta log(2)ratio approximately 0.1) distinguish homozygous and heterozygous regions of the genome in interspecific backcross mice, providing an efficient method for genotyping progeny of backcrosses.     

Molecular analysis of APRT deficiency in mouse P19 teratocarcinoma stem cell line

We have used four gene probes specific for mouse chromosome 8, including adenine phosphoribosyltransferase (aprt), to demonstrate that the P19 teratocarcinoma stem cell line contains two disinct chromosome 8 homologs. One represents the common laboratory mouse C3H (Mus musculus domesticus) homolog while the second homolog was presumably contributed by a feralMus musculus musculus animal. Six cell lines with APRT heterozygous deficiencies were isolated from P19 subclones. A molecular analysis of these heterozygotes demonstrated that three arose by deletion of theMus musculus musculus aprt allele and three arose byaprt gene inactivation. APRT homozygous deficient cell lines were isolated from both classes of heterozygote; most contained little or no detectable APRT activity. When the heterozygous deficiency was due to deletion of theMus musculus musculus aprt allele, the most frequent event yielding homozygous deficient cell lines was associated with loss of heterozygosity for all tested markers on theMus musculus domesticus homolog indicating chromosome los. In contrast, when the initial event resulting in APRT heterozygous deficiency was gene inactivation, homozygotes arose predominantly from gene deletion or a second inactivation event. These results suggest a potential relationship between the first- and second-step events resulting in APRT deficiencies.     

The mouse (Mus musculus) T cell receptor delta variable (TRDV), diversity (TRDD) and joining (TRDJ) genes

'The Mouse (Mus musculus) T Cell Receptor Delta Variable (TRDV), Diversity (TRDD) and Joining (TRDJ) Genes', the 15th report of the 'IMGT Locus in Focus' section, comprises 7 tables entitled: (1) 'Number of mouse (Mus musculus) germline TRDV genes at 14D1-D2 and potential repertoire'; (2) 'Mouse (Mus musculus) germline TRDV genes at 14D1-D2'; (3) 'Mouse (Mus musculus) TRDV allele table'; (4) 'Mouse (Mus musculus) germline TRDD genes and alleles'; (5) 'Mouse (Mus musculus) germline TRDJ genes'; (6) 'Mouse (Mus musculus) TRDJ allele table', and (7) 'Correspondence between the different mouse (Mus musculus) TRDV gene nomenclatures'. These tables are available at the IMGT Marie-Paule page from IMGT, the international ImMunoGeneTics database (http://imgt.cines. fr:8104) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, Montpellier, France.