An Langeveld

Heterochromatin Effects on the Frequency and Duration of LCR-Mediated Gene Transcription

Locus control regions (LCRs) are responsible for initiating and maintaining a stable tissue-specific open chromatin structure of a locus. In transgenic mice, LCRs confer high level expression on linked genes independent of position in the mouse genome. Here we show that an incomplete LCR loses this property when integrated into heterochromatic regions. Two disruption mechanisms were observed. One is classical position-effect variegation, resulting in continuous transcription in a clonal subpopulation of cells. The other is a novel mechanism resulting in intermittent gene transcription in all cells. We conclude that only a complete LCR fully overcomes heterochromatin silencing and that it controls the level of transcription by ensuring activity in all cells at all times rather than directly controlling the rate of transcription.

An intrinsic but cell-nonautonomous defect in GATA-1-overexpressing mouse erythroid cells

GATA-1 is a tissue-specific transcription factor that is essential for the production of red blood cells. Here we show that overexpression of GATA-1 in erythroid cells inhibits their differentiation, leading to a lethal anaemia. Using chromosome-X-inactivation of a GATA-1 transgene and chimaeric animals, we show that this defect is intrinsic to erythroid cells, but nevertheless cell nonautonomous. Usually, cell nonautonomy is thought to reflect aberrant gene function in cells other than those that exhibit the phenotype. On the basis of our data, we propose an alternative mechanism in which a signal originating from wild-type erythroid cells restores normal differentiation to cells overexpressing GATA-1 in vivo. The existence of such a signalling mechanism indicates that previous interpretations of cell-nonautonomous defects may be erroneous in some cases and may in fact assign gene function to incorrect cell types.

In vivo transposition of Minos, a Drosophila mobile element, in mammalian tissues

Transposable elements have been used widely in the past 20 years for gene transfer and insertional mutagenesis in Drosophila. Transposon-based technology for gene manipulation and genomic analysis currently is being adopted for vertebrates. We tested the ability of Minos, a DNA transposon from Drosophila hydei, to transpose in mouse tissues. Two transgenic mouse lines were crossed, one expressing Minos transposase in lymphocytes under the control of the CD2 promoter/locus control region and another carrying a nonautonomous Minos transposon. Only mice containing both transgenes show excision of the transposon and transposition into new chromosomal sites in thymus and spleen cells. In addition, expression of Minos transposase in embryonic fibroblast cell lines derived from a transposon-carrying transgenic mouse resulted in excision of the transposon. These results are a first step toward a reversible insertional mutagenesis system in the mouse, opening the way to develop powerful technologies for functional genomic analysis in mammals.

Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.

The analysis of a number of cases of β‐globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the β‐globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal γ‐ to adult β‐globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and δβ–thalassemia, located 5′ of the δ‐globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human β‐globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the β‐globin gene is decreased, but the developmental silencing of the γ‐globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the β‐globin gene perhaps through the loss of a structural function required for gene activation by long‐range interactions.

Chromosome 12q heterozygosity is retained in i(12p)-positive testicular germ cell tumor cells

Restriction fragment length polymorphism analysis is used to demonstrate that formation of the i(12p) chromosome, characteristic of testicular germ cell tumors, does not lead to loss of heterozygosity of various loci on the q arm of chromosome 12. This result suggests that during the etiology of these tumors, aneuploidization precedes the formation of the i(12p) marker chromosome.

Transposition of the Drosophila hydei Minos transposon in the mouse germ line

We tested the suitability of the fly transposon Minos, a member of the Tc1/mariner superfamily, for insertional mutagenesis in the mouse germ line. We generated a transgenic mouse line expressing Minos transposase in growing oocytes and another carrying a tandem array of nonautonomous transposons. The frequency of transposition in the progeny derived from oocytes carrying both transgenes is 8.2%. Analysis of the new integration sites shows a high frequency of transpositions to a different chromosome. Thus Minos transposition could be an effective system for insertional mutagenesis and functional genomic analysis in the mouse.

Positional mapping of loci in the DiGeorge critical region at chromosome 22q11 using a new marker (D22S183)

The majority of patients with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) and a minority of patients with non-syndromic conotruncal heart defects are hemizygous for a region of chromosome 22q11. The chromosomal region that is commonly deleted is larger than 2 Mb. It has not been possible to narrow the smallest region of overlap (SRO) of the deletions to less than ca 500 kb, which suggests that DGS/VCFS might be a contiguous gene syndrome. The saturation cloning of the SRO is being carried out, and one gene (TUPLE1) has been identified. By using a cosmid probe (M51) and fluorescence in situ hybridization, we show here that the anonymous DNA marker locus D22S183 is within the SRO, between TUPLE1 and D22S75 (probe N25). A second locus with weak homology to D22S183, recognized by cosmid M56, lies immediately outside the common SRO of the DGS and VCFS deletions, but inside the SRO of the DGS deletions. D22S183 sequences are strongly conserved in primates and weaker hybridizing signals are found in DNA of other mammalian species; no transcripts are however detected in polyA+ RNA from various adult human organs. Probe M51 allows fast reliable screening for 22q11 deletions using fluorescence in situ hybridization. A deletion was found in 11 out of 12 DGS patients and in 3 out of 7 VCFS patients. Two patients inherited the deletion from a parent with mild (atypical) symptoms.

Homotypic signalling regulates Gata1 activity in the erythroblastic island

Gata1 is a transcription factor essential for erythropoiesis. Erythroid cells lacking Gata1 undergo apoptosis, while overexpression of Gata1 results in a block in erythroid differentiation. However, erythroid cells overexpressing Gata1 differentiate normally in vivo when in the presence of wild-type cells. We have proposed a model, whereby a signal generated by wild-type cells (red cell differentiation signal; REDS) overcomes the intrinsic defect in Gata1-overexpressing erythroid cells. The simplest interpretation of this model is that wild-type erythroid cells generate REDS. To substantiate this notion, we have exploited a tissue specific Cre/loxP system and the process of X-inactivation to generate mice that overexpress Gata1 in half the erythroid cells and are Gata1 null in the other half. The results show that the cells supplying REDS are erythroid cells. This study demonstrates the importance of intercellular signalling in regulating Gata1 activity and that this homotypic signalling between erythroid cells is crucial to normal differentiation.

Tagged Mutagenesis by Efficient Minos-Based Germ Line Transposition

ABSTRACT Germ line gene transposition technology has been used to generate “libraries” of flies and worms carrying genomewide mutations. Phenotypic screening and DNA sequencing of such libraries provide functional information resulting from insertional events in target genes. There is also a great need to have a fast and efficient way to generate mouse mutants in vivo to model developmental defects and human diseases. Here we describe an optimized mammalian germ line transposition system active during early mouse spermatogenesis using the Minos transposon. Transposon-positive progeny carry on average more than 2 new transpositions, and 45 to 100% of the progeny carry an insertion in a gene. The optimized Minos-based system was tested in a small rapid dominant functional screen to identify mutated genes likely to cause measurable cardiovascular “disease” phenotypes in progeny/embryos. Importantly this system allows rapid screening for modifier genes.