On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation.

Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using an on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in transcriptional activity whereas at the peak of gene expression, in early erythroblasts, transcription is relatively stable. Later during differentiation as α-globin expression declines, we again observe more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of active transcriptional compartments as gene expression is regulated during differentiation.

Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells.

The alpha- and beta-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous alpha- and beta-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the alpha- and beta-globin genes are localized in areas of the nucleus that are discrete from alpha-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of alpha- and beta-globin genes was markedly different. beta-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse lambda5, CD4, CD8alpha, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, alpha-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with alpha-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the alpha-globin locus, or in mouse erythroblasts and lymphocytes derived from human alpha-globin transgenic mice. These data show that the normal regulated expression of alpha- and beta-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.

Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects.

We have examined the phenotypic effects of 21 independent deletions from the fully sequenced and annotated 356 kb telomeric region of the short arm of chromosome 16 (16p13.3). Fifteen genes contained within this region have been highly conserved throughout evolution and encode proteins involved in important housekeeping functions, synthesis of haemoglobin, signalling pathways and critical developmental pathways. Although a priori many of these genes would be considered candidates for critical haploinsufficient genes, none of the deletions within the 356 kb interval cause any discernible phenotype other than alpha thalassaemia whether inherited via the maternal or paternal line. These findings contrast with previous observations on patients with larger (> 1 Mb) deletions from the 16p telomere and therefore address the mechanisms by which monosomy gives rise to human genetic disease.

Sequence, structure and pathology of the fully annotated terminal 2 Mb of the short arm of human chromosome 16.

We have sequenced 1949 kb from the terminal Giemsa light band of human chromosome 16p, enabling us to fully annotate the region extending from the telomeric repeats to the previously published tuberous sclerosis disease 2 (TSC2) and polycystic kidney disease 1 (PKD1) genes. This region can be subdivided into two GC-rich, Alu-rich domains and one GC-rich, Alu-poor domain. The entire region is extremely gene rich, containing 100 confirmed genes and 20 predicted genes. Many of the genes encode widely expressed proteins orchestrating basic cellular processes (e.g. DNA recombination, repair, transcription, RNA processing, signal transduction, intracellular signalling and mRNA translation). Others, such as the alpha globin genes (HBA1 and HBA2), PDIP and BAIAP3, are specialized tissue-restricted genes. Some of the genes have been previously implicated in the pathophysiology of important human genetic diseases (e.g. asthma, cataracts and the ATR-16 syndrome). Others are known disease genes for alpha thalassaemia, adult polycystic kidney disease and tuberous sclerosis. There is also linkage evidence for bipolar affective disorder, epilepsy and autism in this region. Sixty-three chromosomal deletions reported here and elsewhere allow us to interpret the results of removing progressively larger numbers of genes from this well defined human telomeric region.

Relationship between novel isoforms, functionally important domains, and subcellular distribution of CD164/endolyn.

Functional analyses have indicated that the human CD164 sialomucin may play a key role in hematopoiesis by facilitating the adhesion of human CD34(+) cells to the stroma and by negatively regulating CD34(+)CD38(lo/-) cell proliferation. We have identified three novel human CD164 variants derived by alternative splicing of bona fide exons from a single genomic transcription unit. The predominant CD164(E1-6) isoform, encoded by six exons, is a type I transmembrane protein containing two extracellular mucin domains (I and II) interrupted by a cysteine-rich non-mucin domain. The 103B2/9E10 and 105A5 epitopes, which specify ligand binding characteristics, are located on the exon 1-encoded mucin domain I. Three human CD164(E1-6) mRNA species, exhibiting differential polyadenylation site usage, are differentially expressed in hematopoietic and non-hematopoietic tissues. This study provides additional evidence that human CD164(E1-6) represents the ortholog of murine MGC-24v and rat endolyn. Comparative analysis of murine MGC-24v/CD164(E1-6) with human CD164(E1-6) revealed two potential splice variants and a similar genomic structure. Whereas the human CD164 gene is located on chromosome 6q21, the mouse gene occurs in a syntenic region on chromosome 10B1-B2. By confocal microscopy, human CD164 in CD34(+)CD38(+) hematopoietic progenitor (KG1B) and epithelial cell lines appears to be localized primarily in endosomes and lysosomes, with low concentrations at the cell surface. However, in a minority of KG1B cells, CD164 is more prominently expressed at the plasma membrane and in the recycling endosomes, suggesting that its distribution is regulated in cells of hematopoietic origin.

Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes.

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

Chromosomal stabilisation by a subtelomeric rearrangement involving two closely related Alu elements.

We have characterised a subtelomeric rearrangement involving the short arm of chromosome 16 that gives rise to alpha-thalassaemia by deleting the major, remote regulatory element controlling alpha-globin expression. The chromosomal breakpoint lies in an Alu family repeat located only approximately 105 kb from the 16p subtelomeric region. The broken chromosome has been stabilised with a newly positioned telomere acquired by recombination between this 16p Alu element and a closely related subtelomeric Alu element of the Sx subfamily. It seems most likely that this abnormal chromosome has been rescued by the mechanism of telomere capture which may reflect a more general process by which subtelomeric sequences are normally dispersed between chromosomal ends.

Radiation-induced genomic instability: delayed cytogenetic aberrations and apoptosis in primary human bone marrow cells.

Transmissible chromosomal instability, characterized by non-clonal cytogenetic aberrations with a high frequency of chromatid-type aberrations together with a lower frequency of chromosome-type aberrations, has been demonstrated in the clonal descendants of human haemopoietic stem cells after alpha- but not X-irradiation. Comparable cytogenetic abnormalities have also been demonstrated in non-clonal cultures of alpha-irradiated primary human bone marrow, but a different pattern of delayed aberrations, mainly of chromosome-type, was found after X-irradiation in non-clonal cultures. In clonal analyses, delayed apoptotic cell death was evident after both X- and alpha-irradiation. It is suggested that the type of radiation exposure, the type of cell and its genetically determined susceptibility are factors that may influence the expression of delayed effects of radiation.

The detection of subtelomeric chromosomal rearrangements in idiopathic mental retardation.

A major challenge for human genetics is to identify new causes of mental retardation, which, although present in about 3% of individuals, is unexplained in more than half of all cases. We have developed a strategy to screen for the abnormal inheritance of subtelomeric DNA polymorphisms in individuals with mental retardation and have detected three abnormalities in 99 patients with normal routine karyotypes. Pulsed-field gel electrophoresis and reverse chromosome painting showed that one case arose from an interstitial or terminal deletion and two from the de novo inheritance of derivative translocation chromosomes. At least 6% of unexplained mental retardation is accounted for by these relatively small chromosomal abnormalities, which will be an important resource in the characterization of the genetic basis of neurodevelopment.

Clinical and hematologic aspects of the X-linked alpha-thalassemia/mental retardation syndrome (ATR-X).

The hallmarks of the X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome are severe psychomotor retardation, minor facial anomalies, genital abnormalities, and an unusual form of alpha-thalassemia. The demonstration of HbH inclusions in red blood cells after incubation with brilliant cresyl blue confirms the diagnosis. We describe 15 previously unreported cases and analyse the phenotypic and hematologic findings in these subjects and compare them with previously published cases. This study demonstrates the consistency of the main characteristics of this syndrome and extends the phenotype. Developmental changes in phenotype, in particular the coarsening of the facial appearance, are illustrated. The hematologic findings are shown to vary widely; in some cases the manifestation of alpha-thalassemia may be subtle and missed without repeated examination.

Alpha-particle-induced chromosomal instability in human bone marrow cells.

alpha-particles, which are ionising radiation of high linear-energy-transfer emitted, for example, from radon or plutonium, pass through tissue as highly structured tracks. Single target cells in the path of the tracks might be damaged by even low-dose alpha-irradiation. We found non-clonal cytogenetic aberrations, characterised by a high frequency of chromatid aberrations with chromosome aberrations, in clonal descendants of haemopoietic stem cells after exposure to alpha-particles of bone marrow cells from two of four haematologically normal individuals (up to 25% abnormal metaphases). The data are consistent with a transmissible genetic instability induced in a stem cell resulting in a diversity of aberrations in its clonal progeny many cell divisions later.

A large duplicated area in the polycystic kidney disease 1 (PKD1) region of chromosome 16 is prone to rearrangement.

An area of 500 kb at the proximal end of the polycystic kidney disease 1 (PKD1) region has been mapped in detail, with 260 kb cloned in cosmids. The area cloned from normal individuals contains two homologous but divergent regions each of 75 kb, including the previously described marker 26-6. Pulsed-field gel electrophoresis identified a duplication of 75 kb of this region, referred to as the OX duplication (OXdup), in three patients with PKD1. The OXdup probably arose by an unequal exchange promoted by misalignment of partially homologous areas. Study of the OXdup in a large PKD1 family showed that it segregated with PKD1 in just one-half of the family, indicating that a recent crossover had occurred between the OXdup and PKD1 and showing that it was not a PKD1 mutation. Further analysis identified an OXdup breakpoint fragment: the OXdup was subsequently identified in 2 normal individuals of 110 assayed. The finding of the OXdup and in other individuals an 11-kb deletion (OXdel) at a similar point within this duplicated area indicates that this is an unusually unstable genomic region.

Dissecting the centromere of the human Y chromosome with cloned telomeric DNA.

We have used telomeric DNA to break the human Y chromosome within the centromeric array of alphoid satellite DNA and have created two derivative chromosomes; one consists of the short arm and 140 kb of alphoid DNA, the other consists of the long arm and 480 kb of alphoid DNA. Both segregate accurately at mitosis. It is known that there is no large scale sequence duplication around the alphoid DNA and so the simplest interpretation of our results is that the sequence responsible for accurate segregation is the alphoid DNA itself. Although the long arm acrocentric derivative segregates accurately it lags with respect to the other chromosomes in about 10% of anaphase cells and thus additional sequences may be required for orderly segregation. The short arm acrocentric chromosome is probably no larger than 12 Mb in size and thus our results also demonstrate that chromosomes of this size are capable of accurate segregation.

New methods in cytogenetics.

Developments in the technique of fluorescence in situ hybridization (FISH) now permit hybridization of sequences ranging from 1 kb to whole genomes. The technique can be used in applications from coarse mapping of whole chromosomes to high-resolution analysis of extended strands of DNA. The complexity, and hence the coverage, of 'paints' prepared by amplification is being improved to the extent that such methods are used in cloning strategies for the generation of region-specific probes. Interphase analysis and comparative genomic hybridization are becoming important tools in cancer cytogenetics, and the potential for routine analysis of fetal cells obtained from maternal blood may provide a fresh approach to prenatal cytogenetic screening. Functional studies of gene activity and nuclear organization are now also possible.

DNA rearrangements proximal to the EVI1 locus associated with the 3q21q26 syndrome.

Specific rearrangements involving 3q21 and 3q26 are well documented in acute myeloid leukemia (AML). Aberrant expression of the Ecotropic virus integration-1 (EVI1) gene, located at 3q26, has been reported in individuals with AML and translocations or inversions of chromosome 3 long arm. We have studied six individuals with AML and inv(3)(q21q26) for disruptions to the EVI1 locus by in situ hybridization and long-range mapping. EVI1 transcripts have been detected in the blast cells of the two individuals available for expression studies. We derived a YAC containing the EVI1 gene and showed that it crossed the 3q26 inversion breakpoints in three of four cases examined. Pulsed field analysis detected aberrant fragments 3' of the EVI1 gene in all six patients. The orientation of the gene was established and the locations of the breakpoints were refined by in situ hybridization using phage clones from this region.

Molecular mapping of uncharacteristically small 5q deletions in two patients with the 5q- syndrome: delineation of the critical region on 5q and identification of a 5q- breakpoint.

Molecular mapping techniques have defined the region of gene loss in two patients with the 5q- syndrome and uncharacteristically small 5q deletions (5q31-q33). The allelic loss of 10 genes localized to 5q23-qter (centromere-CSF2-EGR1-FGFA-GRL-ADRB2-CS F1R-SPARC-GLUH1-NKSF1-FLT4-telomere) was investigated in peripheral blood cell fractions. Gene dosage experiments demonstrated that CSF2, EGR1, NKSF1, and FLT4 were retained on the 5q- chromosome in both patients and that FGFA was retained in one patient, thus placing these genes outside the critical region. GRL, ADRB2, CSF1R, SPARC, and GLUH1 were shown to be deleted in both patients. The proximal breakpoint is localized between EGR1 and FGFA in one patient and between FGFA and ADRB2 in the other, and the distal breakpoint is localized between GLUH1 and NKSF1 in both patients. Pulsed-field gel electrophoresis was used to map the 5q deletion breakpoints, and breakpoint-specific fragments were detected with FGFA in the granulocyte but not the lymphocyte fraction of one patient. This study has established the critical region of gene loss of the 5q- chromosome in the 5q- syndrome, giving the location for a putative tumor-suppressor gene in the 5.6-Mb region between FGFA and NKSF1.

Allelic loss of IRF1 in myelodysplasia and acute myeloid leukemia: retention of IRF1 on the 5q- chromosome in some patients with the 5q- syndrome.

Acquired interstitial deletions of the long arm of chromosome 5 occur frequently in the myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Recently IRF1, a putative tumor suppressor gene localized to the long arm of chromosome 5, has been shown to be deleted from the 5q- chromosome in a group of patients with MDS and AML. It has been suggested that the loss of IRF1 may be critical to the development of the 5q- syndrome. We have investigated the allelic loss of IRF1 in a group of 12 patients with MDS and a 5q deletion and 2 patients with AML and a 5q deletion. Gene dosage experiments demonstrated that 12 of 14 patients had loss of one allele of the IRF1 gene but no evidence of homozygous loss and that 2 patients with 5q- syndrome retained both copies of the gene. The retention of IRF1 on the 5q- chromosome in these two cases has been confirmed by fluorescent in situ hybridization localization using an IRF1 cosmid. Pulsed field gel electrophoresis was used to determine whether there was any evidence for structural rearrangement in the region encompassing the IRF1 gene in these two patients. No aberrant bands were detected with a range of rare cutter enzyme digests. We conclude that IRF1 maps outside the commonly deleted segment of the 5q- chromosome and that loss of IRF1 is not solely responsible for the development of the 5q- syndrome.

Characterisation of molecular DNA rearrangements within the Xq12-q13.1 region, in three patients with X-linked hypohidrotic ectodermal dysplasia (EDA).

A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal dysplasia (EDA) patient-derived cell lines, containing different rearranged X chromosomes, have been used to refine the physical map of the Xq12-q13.1 region. The patient-derived material included genomic DNA from an EDA male (EDA family 1015) with an interstitial deletion, and a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, derived from this cell line. This map subdivides the region into at least 6 mapping-intervals. DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to identify homologous Yeast Artificial Chromosome (YAC) clones. Two of the DXS732-specific YACs were shown by fluorescent in situ hybridisation (FISH) analysis to bridge the (X;9) translocation breakpoint. These two YACs were also screened against the ICRF human X chromosome cosmid library and identified 36 cosmid clones. Direct cosmid-cosmid hybridisation analysis placed subsets of these clones within four different cosmid contigs. Mapping of anchor clones from each contig, against the mapping panel, localised all these contigs within the Xq12-q13.1 region. One cosmid, ICRFc104C03.184, identified potential junctional-fragments in several restriction digests of AnLy hybrid DNA. This was confirmed by FISH analysis of the GM0705A cell line with total cosmid ICRFc104C03.184, in which both chromosomal elements of the (X;9) translocation were identified. A single-copy probe pC03.184E2, derived from this cosmid, also identified the der(9)-derived junctional fragment when hybridised against AnLy DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the FRAXE fragile site in two families ascertained for X linked mental retardation.

Chromosome fragility in two families not exhibiting amplification of the CGG trinucleotide associated with the fragile X site has been examined. Fluorescence in situ hybridisation with cosmid DNA from loci immediately flanking FRAXA and other distal loci have confirmed that cytogenetic fragility in these subjects is the result of expression of a new folate sensitive fragile X site, FRAXE.