Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome.

The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts, separated by a hinge region. Using Hi-C in edited mouse cells with allelic deletions or inversions within the hinge, here we show that the conserved Dxz4 locus is necessary to maintain this bipartite structure. Dxz4 orientation controls the distribution of contacts on the Xi, as shown by a massive reversal in long-range contacts after Dxz4 inversion. Despite an increase in CTCF binding and chromatin accessibility on the Xi in Dxz4-edited cells, only minor changes in TAD structure and gene expression were detected, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of its bank of CTCF motifs, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi.

Multiple fetal anomalies associated with subtle subtelomeric chromosomal rearrangements.

We report two cases of multiple fetal anomalies detected by prenatal ultrasound and associated with subtle subtelomeric chromosomal rearrangements. The first case presented at 25 weeks of gestation with an enlarged cisterna magna and ventriculomegaly. Karyotyping of amniocytes showed a subtle terminal abnormality of chromosome 6q. Thereafter, screening of all unique chromosomal subtelomeric regions using a panel of telomere-specific, fluorescence in situ hybridization (FISH) probes revealed an unbalanced reciprocal translocation between 6q and 17p [46,XX.ish der(6)t(6;17)(q25.3;p13)(TelVysion6q-;TelVysion17p+)]. The second case presented at 25 weeks of gestation with tetralogy of Fallot and at 34 weeks of gestation had additional ultrasound findings of an arachnoid cyst and intrauterine growth restriction. Postnatal karyotyping of peripheral blood was performed and appeared normal. However, a cryptic deletion of the subtelomeric region of the long arm of chromosome 14 was identified when the infant's blood sample was used as a control for an oncology FISH probe. Thereafter, screening of all unique chromosomal subtelomeric regions using a panel of telomere-specific FISH probes revealed an unbalanced reciprocal translocation of chromosomes 14q and 20p [46,XY.ish der(14)t(14;20)(q32.3;p13)(IGH-, D14S308-,TelVysion20p+)mat]. These two cases add to a growing number of reports of cryptic subtelomeric chromosomal rearrangements associated with congenital anomalies. This is the first report of multiple, simultaneous FISH screening of the subtelomeric regions in amniotic fluid and has demonstrated the technical feasibility of this technique in the prenatal period.

Escape from X inactivation.

Although the process of X inactivation in mammalian cells silences the majority of genes on the inactivated X chromosome, some genes escape this chromosome-wide silencing. Genes that escape X inactivation present a unique opportunity to study the process of silencing and the mechanisms that protect some genes from being turned off. In this review, we will discuss evolutionary aspects of escape from X inactivation, in relation to the divergence of the sex chromosomes. Molecular characteristics, expression, and epigenetic modifications of genes that escape will be presented, including their developmental regulation and the implications of chromatin domains along the X chromosome in modeling the escape process.

Autosomal telomere exchange results in the rapid amplification and dispersion of Csf2ra genes in wild-derived mice.

Common laboratory strains such as C57BL/6J carry a single Csf2ra gene that maps to the distal end of Chromosome (Chr) 19. Here we report that several species of wild mice contain multiple Csf2ra genes. Using interspecific backcross mapping and in situ hybridization, we demonstrate that one of these species, Mus spretus, carries four Csf2ra genes dispersed among the distal tips of Chrs 4, 10, 13, and 19. Our data further suggest that these additional Csf2ra genes are not generated by retrotransposition, but rather by nonhomologous subtelomeric exchanges that could be mediated in part by ribosomal genes located at the subtelomeric regions of Chrs 4, 13, and 19. Although we do not know whether these additional Csf2ra genes are functionally active, our studies suggest that subtelomeric exchange provides a potent means for rapid gene amplification in the mouse.

Ring X and other structural X chromosome abnormalities: X inactivation and phenotype.

Patients who carry a structural abnormality of the X chromosome are a fascinating group who have provided opportunities to evaluate genotype/phenotype correlation in relation to X chromosome content and inactivation. Turner syndrome (TS) is most commonly associated with a 45,X karyotype and presents with an array of phenotypes, the main ones being poor viability in utero, ovarian failure and infertility, short stature, lymphedema, and other congenital malformations but usually not mental retardation. In some TS patients the karyotype shows both a normal X and a structurally rearranged X chromosome. These structural abnormalities, which include deletions, duplications, inversions, translocations, and rings, are associated with chromosome breaks and significant imbalance of gene content of the X chromosome. However, such abnormalities are generally well tolerated because of the preferential inactivation of the abnormal X, which can restore, at least in part, a balanced genetic makeup. This beneficial effect of X inactivation results in a mild phenotype in most patients with structural abnormalities of the X, similar to that found in TS patients with a 45,X karyotype. However, in cases of ring X chromosomes and of X/autosome translocations the incidence of mental retardation and other congenital abnormalities can be significantly higher than in TS. These abnormal phenotypes can be ascribed to failed or partial X inactivation and/or incomplete selection in favor of cells with normal balance of gene expression. In this article, we present phenotype/genotype correlation in female patients with structural abnormalities of the X and address the role of X inactivation and cell selection in the phenotypic findings. Our review emphasizes a subset of rare patients with ring X chromosomes who have provided evidence of a direct role for X inactivation in determining phenotypes.

Expression and conservation of processed copies of the RBMX gene.

RBMX and RBMY are members of an ancient pair of genes located on the sex chromosomes that encode RNA-binding proteins involved in splicing. These genes have differentiated and evolved separately on the X and Y Chromosomes. RBMY has acquired a testis-specific function, whereas, as shown here, RBMX is ubiquitously expressed and is subject to X inactivation. We have also found that multiple processed copies of RBMX are present in the human genome. RBMX-like sequences (RBMXLs) located on human Chrs 1, 4, 6, 9 (9p13 and 9p24), 11, 20, and X lack introns and thus probably result from retroposition events. We found RBMXLs to be conserved in primates and great apes at corresponding chromosomal locations, indicating that they arose prior to the divergence of human. Some of the RBMXLs show insertions, deletions, and stop codons, which would probably result in nonfunctional proteins. The RBMXL on Chr 20 is deleted in some individuals. Two of the largely intact RBMXLs, located on Chrs 1 and 9p13, are expressed in different tissues and may encode novel proteins involved in splicing in a tissue-specific manner. The RBMXL located at 9p13 is specifically expressed in testis, and to a lesser extent in brain, and may therefore play a role in testis function. This autosomal, testis-specific copy of RBMX could potentially compensate for RBMX that is presumably inactivated in male germ cells, in a manner analogous to autosomal retroposed copies of other X-linked genes.

Expression and chromosomal mapping of the mouse smooth muscle calponin gene.

Smooth muscle calponin (Cnn1) is a multifunctional protein whose expression is tightly restricted to differentiated smooth muscle cell (SMC) lineages during embryonic and post-natal life. As such, Cnn1 represents an ideal locus from which to dissect out regulatory elements that control its expression and hence the mature SMC phenotype. Previous work has focused on the expression and chromosomal mapping of the rat and human Cnn1 orthologs. In this report, we describe a unique pattern of Cnn1 expression during the growth and differentiation of BC3H1 cells, a mouse cell line that has transcriptional characteristics of both smooth and skeletal muscle lineages. Actively growing BC3H1 cells exhibit Cnn1 mRNA expression, which is extinguished when these cells are induced to differentiate upon serum withdrawal. Replating differentiated BC3H1 cells restores steady-state Cnn1 mRNA levels. The down-regulation of Cnn1 mRNA during BC3H1 differentiation coincides with the induction of myogenin, a skeletal muscle transcription factor that is not present in SMC lineages. Results from cycloheximide and actinomycin D studies suggest the existence of a labile repressor protein(s) that destabilizes the pool of Cnn1 mRNA and/or silences transcription of the Cnn1 locus. Mapping of the mouse Cnn1 locus to Chr 9, which is homologous to human Cnn1 on 19p13.2 and rat Cnn1 on 8q, suggests no gross rearrangement of this locus in the BC3H1 cell line. These results are the first to show reversible expression of Cnn1 and demonstrate the utility of the BC3H1 muscle cell line as a model system for the further characterization of Cnn1 gene regulation.

The amnionless gene, essential for mouse gastrulation, encodes a visceral-endoderm-specific protein with an extracellular cysteine-rich domain.

Fate-mapping experiments in the mouse have revealed that the primitive streak can be divided into three functional regions: the proximal region gives rise to germ cells and the extra-embryonic mesoderm of the yolk sac; the distal region generates cardiac mesoderm and node-derived axial mesendoderm; and the middle streak region produces the paraxial, intermediate and lateral plate mesoderm of the trunk. To gain insight into the mechanisms that mediate the assembly of the primitive streak into these functional regions, we have cloned and functionally identified the gene disrupted in the amnionless (amn) mouse, which has a recessive, embryonic lethal mutation that interferes specifically with the formation and/or specification of the middle primitive streak region during gastrulation. Here we report that the gene Amn encodes a novel type I transmembrane protein that is expressed exclusively in the extra-embryonic visceral endoderm layer during gastrulation. The extracellular region of the Amn protein contains a cysteine-rich domain with similarity to bone morphogenetic protein (BMP)-binding cysteine-rich domains in chordin, its Drosophila melanogaster homolog (Short gastrulation) and procollagen IIA (ref. 3). Our findings indicate that Amn may direct the production of trunk mesoderm derived from the middle streak by acting in the underlying visceral endoderm to modulate a BMP signaling pathway.

Karyotyping.

Relevant portions of the new International System for Human Cytogenetic Nomenclature (ISCN 1995) have been reproduced in this appendix (with permission from Karger, the original publisher). The new rules supersede all previous rules and include guidelines for cancer cytogenetics as well as new recommendations for nomenclature when in situ hybridization techniques are used in the analysis of chromosomes.

ISCN standard idiograms.

Chromosome banding is used mainly to identify both normal and rearranged chromosomes, to define chromosome breakpoints, and to describe the specific location of DNA sequences on chromosomes. A nomenclature has been developed to standardize the identification of chromosomes and the naming of chromosome bands. The system currently in use is An International System for Human Cytogenetic Nomenclature, referred to as "ISCN 1995." It is the report of the standing committee on human cytogenetic nomenclature edited by Felix Mitelman. The report includes a chromosome band nomenclature, as well as standard idiograms, which are "diagrammatic representations of a karyotype, which may be based on measurements of the chromosomes" (ISCN 1995). The idiograms presented here, with the permission of S. Karger and Cytogenetics and Cell Genetics, are drawings of G-banded chromosomes with band numbers indicated. Heterochromatic regions, which contain classes of repetitive DNA and can show individual differences in size, are indicated by patterned areas.

Chromosome banding techniques.

Chromosome banding techniques produce a series of consistent landmarks along the length of metaphase chromosomes that allow for both recognition of individual chromosomes within a genome and identification of specific segments of individual chromosomes. These landmarks facilitate assessment of chromosome normalcy, identification of sites of chromosome breaks and alterations, and location of specific genes. This unit covers these basic banding techniques (Q-banding, G-banding, and R-banding), which produce virtually identical patterns of bands along the length of human chromosomes, although the bands and polymorphic regions highlighted may differ with each technique. These techniques highlight reproducible landmarks along the length of the chromosome and specialized staining techniques can be used to highlight particular regions of chromosomes, such as heterochromatic and repeated-sequence segments. These specialized techniques, nucleolar organizer region (NOR) staining, centromeric heterochromatin staining (C-banding), methylated satellite DNA staining (distamycin-DAPI banding), and replication banding are also presented in this unit.

A novel B lymphocyte-associated adaptor protein, Bam32, regulates antigen receptor signaling downstream of phosphatidylinositol 3-kinase.

We have identified and characterized a novel src homology 2 (SH2) and pleckstrin homology (PH) domain-containing adaptor protein, designated Bam32 (for B cell adaptor molecule of 32 kD). cDNAs encoding the human and mouse Bam32 coding sequences were isolated and the human bam32 gene was mapped to chromosome 4q25-q27. Bam32 is expressed by B lymphocytes, but not T lymphocytes or nonhematopoietic cells. Human germinal center B cells show increased Bam32 expression, and resting B cells rapidly upregulate expression of Bam32 after ligation of CD40, but not immunoglobulin M. Bam32 is tyrosine-phosphorylated upon B cell antigen receptor (BCR) ligation or pervanadate stimulation and associates with phospholipase Cgamma2. After BCR ligation, Bam32 is recruited to the plasma membrane through its PH domain. Membrane recruitment requires phosphatidylinositol 3-kinase (PI3K) activity and an intact PI(3,4, 5)P(3)-binding motif, suggesting that membrane association occurs through binding to 3-phosphoinositides. Expression of Bam32 in B cells leads to a dose-dependent inhibition of BCR-induced activation of nuclear factor of activated T cells (NF-AT), which is blocked by deletion of the PH domain or mutation of the PI(3,4,5)P(3)-binding motif. Thus, Bam32 represents a novel B cell-associated adaptor that regulates BCR signaling downstream of PI3K.

Common trisomy mosaicism diagnosed in amniocytes involving chromosomes 13, 18, 20 and 21: karyotype-phenotype correlations.

Karyotype-phenotype correlations of common trisomy mosaicism prenatally diagnosed via amniocentesis was reviewed in 305 new cases from a collaboration of North American cytogenetic laboratories. Abnormal outcome was noted in 10/25 (40%) cases of 47,+13/46, 17/31 (54%) cases of 47,+18/46, 10/152 (6.5%) cases of 47,+20/46, and in 49/97 (50%) cases of 47,+21/46 mosaicism. Risk of abnormal outcome in pregnancies with less than 50% trisomic cells and greater than 50% trisomic cells were: 26% (4/15) versus 60% (6/10) for 47,+13/46, 52% (11/21) versus 75% (6/8) for 47,+18/46, 4.5% (6/132) versus 20% (4/20) 47,+20/46, and 45% (27/60) versus 59% (22/37) for 47,+21/46. Phenotypically normal liveborns were observed with mean trisomic cell lines of 9.3% for 47,+13/46, 8.6% for 47,+18/46, 27% for 47, +20/46, and 17% for 47,+21/46. Cytogenetic confirmation rates were 46% (6/13 cases) for 47,+13/46 mosaicism, 66% (8/12 cases) for 47, +18/46, 10% (10/97 cases) for 47,+20/46, and 44% (24/54 cases) for 47,+21/46. There were higher confirmation rates in pregnancies with abnormal versus normal outcome: 50% versus 44% for 47,+13/46 mosaicism, 100% versus 33% for 47,+18/46, 66% versus 7% for 47, +20/46, and 55% versus 40% for 47,+21/46. Repeat amniocentesis is not helpful in predicting clinical outcome. It may be considered when there is insufficient number of cells or cultures to establish a diagnosis. Fetal blood sampling may have a role in mosaic trisomy 13, 18, and 21 as the risk for abnormal outcome increases with positive confirmation: 1/5 (20%) normal cases versus 5/8 (62%) abnormal cases. High resolution ultrasound examination(s) is recommended for clinical correlation and to facilitate genetic counselling.

The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen.

The mammalian Y chromosome encodes male-specific minor histocompatibility (H-Y) Ags that are recognized by female T cells in an MHC-restricted manner. Two human H-Y epitopes presented by HLA-A2 and HLA-B7, respectively, have been identified previously and both are derived from the SMCY gene. We previously isolated CD8+ CTL clones that recognized a male-specific minor histocompatibility Ag presented by HLA-B8. In contrast to the SMCY-encoded H-Y epitopes, the B8/H-Y Ag was not presented by fibroblasts from male donors, suggesting that it was encoded by a novel gene. We now report that the HLA-B8-restricted H-Y epitope is defined by the octameric peptide LPHNHTDL corresponding to aa residues 566-573 of the human UTY protein. Transcription of the UTY gene is detected in a wide range of human tissues, but presentation of the UTY-derived H-Y epitope to CTL by cultured human cells shows significant cell-type specificity. Identification of this CTL-defined H-Y epitope should facilitate analysis of its contribution to graft/host interactions following sex-mismatched organ and bone marrow transplantation.

The human acid ceramidase gene (ASAH): structure, chromosomal location, mutation analysis, and expression.

Acid ceramidase (AC) is the lysosomal enzyme that degrades ceramide into sphingosine and fatty acid. A deficiency in human AC activity leads to the lysosomal storage disorder, Farber disease (FD). The human AC gene (HGMW-approved symbol ASAH) was cloned and characterized, revealing an organization similar to that of the murine AC gene. The human gene spans about 30 kb in length and contains 14 exons ranging in size from 46 to 1201 bp. The exon/intron junctions were determined and found to follow the GT-AG rule. The putative promoter region had a GC content over 60%, lacked a TATA box, and contained several sequences matching transcription factor binding sites, including nine SP-1 sites, one AP-1 site, and three CACC boxes. The promoter activity of a 475-bp fragment from within this region was demonstrated by chloramphenicol acyltransferase assays. Northern blotting revealed variable expression of the human AC RNA; i.e., expression of the major 2.4-kb transcript was high in heart and kidney, followed by lung and placenta, but low in pancreas, liver, brain, and skeletal muscle. Two minor AC transcripts of 1.7 and 1.2 kb also were detected in heart and skeletal muscle. The human AC gene was mapped to the chromosomal region 8p21.3-p22 by in situ hybridization and FISH analyses, syntenic with the mouse chromosomal location. Finally, three new missense mutations, E138V, R254G, and P362R, were identified in the human AC gene from FD patients. Mutant AC cDNAs containing these point mutations were constructed and examined using the FLAG-tagged expression system. Although the levels of protein expression for these mutant ACs were about equivalent to that of the controls, their enzymatic activity was markedly reduced, confirming their authenticity.

Genetic pathway to recurrent chromosome translocations in murine lymphoma involves V(D)J recombinase.

Chromosome translocations involving antigen receptor loci are a genetic hallmark of non-Hodgkin's lymphomas in humans. Most commonly, these translocations result in juxtaposition of the immunoglobulin heavy-chain (IgH) locus with one of several cellular proto-oncogenes, leading to deregulated oncogene expression. The V(D)J recombinase, which mediates physiologic rearrangements of antigen receptor genes, may play a mechanistic role in some lymphoma translocations, although evidence is indirect. A high incidence of B-lineage lymphomas has been observed in mice with severe combined immunodeficiency (SCID) and p53-null mutations. We show that these tumors are characteristic of the pro-B-cell stage of development and that they harbor recurrent translocations involving chromosomes 12 and 15. Fluorescence in situ hybridization (FISH) shows retention of IgH sequences on the derivative chromosome 12, implying that breakpoints involve the IgH locus. Pro-B-cell lymphomas were suppressed in SCID p53(-/-) mice by a Rag-2-null mutation, demonstrating that DNA breaks generated during V(D)J recombination are required for oncogenic transformation, and suggesting that t(12;15) arise during attempted IgH rearrangement in pro-B cells. These studies indicate that the oncogenic potential inherent in antigen receptor diversification is controlled in vivo by efficient rejoining of DNA ends generated during V(D)J recombination and an intact cellular response to DNA damage.

Cloning and characterization of the mouse interleukin enhancer binding factor 3 (Ilf3) homolog in a screen for RNA binding proteins.

In a screen for RNA-binding proteins expressed during murine spermatogenesis, we have identified a cDNA that encodes a protein of 911 amino acids that contains two copies of the double-stranded RNA-binding motif and has 80% identity with human Interleukin Enhancer Binding Factor 3 (ILF3). Linkage and cytogenetic analyses localized the Ilf3 cDNA to a portion of mouse Chr 9, which shows conserved synteny with a region of human Chr 19 where the human ILF3 gene had been previously localized, supporting that we had cloned the murine homolog of ILF3. Northern analysis indicated the Ilf3 gene is ubiquitously expressed in mouse adult tissues with high levels of expression in the brain, thymus, testis, and ovary. Polyclonal antibodies detected multiple protein species in a subset of the tissues expressing Ilf3 RNA. Immunoreactive species are present at high levels in the thymus, testis, ovary, and the spleen to a lesser extent. The high degree of sequence similarity between the mouse ILF3 protein and other dsRNA binding motif-containing proteins suggests a role in RNA metabolism, while the differential expression indicates the mouse ILF3 protein predominantly functions in tissues containing developing lymphocyte and germ cells.