Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice.

The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. We have carried out gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, we assessed the effect of compound Lmo1/Lmo3 null mutations. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. Our results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways.

The crystal structure of the PX domain from p40(phox) bound to phosphatidylinositol 3-phosphate.

More than 50 human proteins with a wide range of functions have a 120 residue phosphoinositide binding module known as the PX domain. The 1.7 A X-ray crystal structure of the PX domain from the p40(phox) subunit of NADPH oxidase bound to PtdIns(3)P shows that the PX domain embraces the 3-phosphate on one side of a water-filled, positively charged pocket and reveals how 3-phosphoinositide specificity is achieved. A chronic granulomatous disease (CGD)-associated mutation in the p47(phox) PX domain that abrogates PtdIns(3)P binding maps to a conserved Arg that does not directly interact with the phosphoinositide but instead appears to stabilize a critical lipid binding loop. The SH3 domain present in the full-length protein does not affect soluble PtdIns(3)P binding to the p40(phox) PX domain.

The mll-AF9 gene fusion in mice controls myeloproliferation and specifies acute myeloid leukaemogenesis.

The MLL gene from human chromosome 11q23 is involved in >30 different chromosomal translocations resulting in a plethora of different MLL fusion proteins. Each of these tends to associate with a specific leukaemia type, for example, MLL-AF9 is found mainly in acute myeloid leukaemia. We have studied the role of the Mll-AF9 gene fusion made in mouse embryonic stem cells by an homologous recombination knock-in. Acute leukaemias developed in heterozygous mice carrying this fusion as well as in chimeric mice. As with human chromosomal translocation t(9;11), the majority of cases were acute myeloid leukaemias (AMLs) involving immature myeloblasts, but a minority were acute lymphoblastic leukaemia. The AMLs were preceded by effects on haematopoietic differentiation involving a myeloproliferation resulting in accumulation of Mac-1/Gr-1 double-positive mature myeloid cells in bone marrow as early as 6 days after birth. Therefore, non-malignant expansion of myeloid precursors is the first stage of Mll-AF9-mediated leukaemia followed by accumulation of malignant cells in bone marrow and other tissues. Thus, the late onset of overt tumours suggests that secondary tumorigenic mutations are necessary for malignancy associated with MLL-AF9 gene fusion and that myeloproliferation provides the pool of cells in which such events can occur.

The oncogenic T cell LIM-protein Lmo2 forms part of a DNA-binding complex specifically in immature T cells.

The LIM-only protein LMO2 is expressed aberrantly in acute T-cell leukaemias as a result of the chromosomal translocations t(11;14) (p13;q11) or t(7;11) (q35;p13). In a transgenic model of tumorigenesis by Lmo2, T-cell acute leukaemias arise after an asymptomatic phase in which an accumulation of immature CD4(-) CD8(-) double negative thymocytes occurs. Possible molecular mechanisms underlying these effects have been investigated in T cells from Lmo2 transgenic mice. Isolation of DNA-binding sites by CASTing and band shift assays demonstrates the presence of an oligomeric complex involving Lmo2 which can bind to a bipartite DNA motif comprising two E-box sequences approximately 10 bp apart, which is distinct from that found in erythroid cells. This complex occurs in T-cell tumours and it is restricted to the immature CD4(- )CD8(-) thymocyte subset in asymptomatic transgenic mice. Thus, ectopic expression of Lmo2 by transgenesis, or by chromosomal translocations in humans, may result in the aberrant protein interactions causing abnormal regulation of gene expression, resulting in a blockage of T-cell differentiation and providing precursor cells for overt tumour formation.

Chromosomal translocations and leukaemia: a role for LMO2 in T cell acute leukaemia, in transcription and in erythropoiesis.

The LMO2 gene associated with T cell acute leukaemia has been used as an example of a gene activated by association with the T cell receptor genes after chromosomal translocations. The gene is shown to encode a LIM protein which is involved in protein interactions and during normal haematopoiesis is necessary for erythroid development. LMO2 has been shown to cause tumours when aberrantly expressed and to be able to heterodimerise with TAL1 to facilitate tumour development.

An Mll-AF9 fusion gene made by homologous recombination causes acute leukemia in chimeric mice: a method to create fusion oncogenes.

Homologous recombination in embryonal stem cells has been used to produce a fusion oncogene, thereby mimicking chromosomal translocations that frequently result in formation of tumor-specific fusion oncogenes in human malignancies. AF9 sequences were fused into the mouse Mll gene so that expression of the Mll-AF9 fusion gene occurred from endogenous Mll transcription control elements, as in t(9;11) found in human leukemias. Chimeric mice carrying the fusion gene developed tumors, which were restricted to acute myeloid leukemias despite the widespread activity of the Mll promoter. Onset of perceptible disease was preceded by expansion of ES cell derivatives in peripheral blood. This novel use of homologous recombination formally proves that chromosomal translocations contribute to malignancy and provides a general strategy to create fusion oncogenes for studying their role in tumorigenesis.

Protein dimerization between Lmo2 (Rbtn2) and Tal1 alters thymocyte development and potentiates T cell tumorigenesis in transgenic mice.

The LMO2 and TAL1 genes were first identified via chromosomal translocations and later found to encode proteins that interact during normal erythroid development. Some T cell leukaemia patients have chromosomal abnormalities involving both genes, implying that LMO2 and TAL1 act synergistically to promote tumorigenesis after their inappropriate co-expression. To test this hypothesis, transgenic mice were made which co-express Lmo2 and Tal1 genes in T cells. Dimers of Lmo2 and Tal1 proteins were formed in thymocytes of double but not single transgenic mice. Furthermore, thymuses of double transgenic mice were almost completely populated by immature T cells from birth, and these mice develop T cell tumours approximately 3 months earlier than those with only the Lmo2 transgene. Thus interaction between these two proteins can alter T cell development and potentiate tumorigenesis. The data also provide formal proof that TAL1 is an oncogene, apparently acting as a tumour promoter in this system.

The oncogenic LIM protein Rbtn2 causes thymic developmental aberrations that precede malignancy in transgenic mice.

RBTN2 is activated by the chromosomal translocation t(11;14) (P13;p11) in some T cell leukaemias. Histologically similar T cell tumours develop with long latency in transgenic mice when either CD2 or thy1.1 promoters control rbtn2 expression. During the asymptomatic period, perturbation of T cell differentiation occurs in the thymus. The major anomalies present during this phase are an increase in the percentage of large thymocytes lacking CD4 and CD8 markers and also of small thymocytes express both the T cell marker CD3 and the B cell-specific form of CD45. These abnormal T cell populations can be clonal and thus a primary result of aberrant expression of the LIM-protein Rbtn2 is alteration of T cell differentiation preceding overt malignancy. These data provide a biological explanation for the role of Rbtn2 in tumorigenesis and presumably RBTN2 expression in T cells after the translocation t(11;14) in children has the same effect.

The Hox11 gene is essential for cell survival during spleen development.

The HOX11 homeobox gene was identified via the translocation t(10;14) in T cell leukaemia. To determine the function of this gene in mice, null mutations were made using homologous recombination in ES cells to incorporate lacZ into the hox11 transcription unit. Production of beta-galactosidase from the recombinant hox11 allele in +/- mutants allowed identification of sites of hox11 expression which included the developing spleen. Newborn hox11 -/- mice exhibit asplenia. Spleen formation commences normally at E11.5 in hox11 -/- mutant embryos but the spleen anlage undergoes rapid and complete resorption between E12.5 and E13.5. Dying spleen cells exhibit molecular features of apoptosis, suggesting that programmed cell death is initiated at this stage of organ development in the absence of hox11 protein. Thus hox11 is not required to initiate spleen development but is essential for the survival of splenic precursors during organogenesis. This function for hox11 suggests that enhanced cell survival may result from the t(10;14) which activates HOX11 in T cell leukaemias, further strengthening the association between oncogene-induced cell survival and tumorigenesis.

T cell tumours of disparate phenotype in mice transgenic for Rbtn-2.

RBTN2 is a LIM domain protein which can be activated by the translocation t(11;14)(p13;q11) in childhood T cell acute leukaemia. Transgenic mice were examined in which rbtn2 protein is expressed in the T cell lineage. An average of 72% of these mice developed T cell tumours before 18 months of age, compared with 9% in transgenic mice expressing the related gene Rbtn-1. Rbtn2-induced tumours first appeared at 5 months of age and were clonal. They displayed a range of phenotypes, the most notable being CD3/CD45R double-positive cells. Tumours expressing either T cell receptor alpha/beta or gamma/delta heterodimers were found. Thus rbtn2 can promote tumours within a range of T cell types and maturities. The latency period before tumour development indicates that secondary events must occur before the onset of overt malignancy.

T-cell acute lymphoblastic lymphoma induced in transgenic mice by the RBTN1 and RBTN2 LIM-domain genes.

Two members of the RBTN gene family, RBTN1/Ttg-1 and RBTN2/Ttg-2, were found by their association with T-cell tumour-specific chromosomal translocations and are thought to be involved in the aetiology of such T-cell tumours. Here a transgenic mouse model is described in which T-cell tumours are induced by the presence of RBTN1 and RBTN2 transgenes that direct expression in thymus-derived cells. The latency period for lymphoid tumour appearance is variable, and tumours occur in a small proportion of transgenic animals that develop T-cell acute lymphoblastic malignancies. No significant increase in the rate of tumour development was observed in RBTN1 transgenic mice infected with Moloney murine leukaemia virus, nor did tumours arise in mice bearing a construct in which RBTN1 was expressed from the insulin transcriptional promoter. These data, which provide formal proof of the oncogenic activity of these genes, suggest that aberrant expression of transcription factor genes, such as RBTN1 and RBTN2, functions in tumour aetiology by disturbing some aspect of T-cell differentiation.

Immunoglobulin VH-T cell receptor C alpha fusion mRNA resulting from chromosome inversion include the T cell-associated 5' exon ET.

A human T cell lymphoma has been described in which an inversion of chromosome 14 results in fusion of an immunoglobulin heavy chain VH with a T cell receptor J alpha segment, potentially resulting in a chimeric protein with immunoglobulin VH region recognition plus T cell receptor effector functions. Examination of the mRNA species expressed from the IgT gene in this lymphoma shows a variety of forms but all IgT mRNA include the T cell-specific exon, ET, previously located in the distal part of the VH locus. In such mRNA species, the normal leader exon of the Ig VH segment, which encodes most of the hydrophobic signal peptide, is replaced by the short ET exon encoding mainly non-hydrophobic residues. Two forms of this mRNA exist which lack the Ig VH leader sequence and thus potentially yield non-membrane proteins in the T cell lymphoma.

An unusual structure of a putative T cell oncogene which allows production of similar proteins from distinct mRNAs.

We previously identified a putative T cell oncogene on chromosome 11 near a translocation t(11;14)(p15;q11) in a human T cell tumour. The gene is transcribed from distinct promoters which have unrelated sequences, which occur within close but distinct methylation-free islands and which allow cell specific production of mRNA. The alternative first exons each contain a protein initiation codon from which two species of protein can be made, differing by only a single amino acid. The protein sequence is highly conserved between man and mouse (98%) and the same single codon difference between alternative first exons is also conserved. This is, therefore, a new form of eukaryotic gene organization from which similar proteins can be made from distinct mRNA species.

Alternating purine-pyrimidine tracts may promote chromosomal translocations seen in a variety of human lymphoid tumours.

Chromosomal abnormalities which are prevalent in human lymphoid tumours are believed to be involved in tumour pathogenesis and their formation may be the result of erroneous activity by the V-D-J recombinase. Frequently, recombinase accessibility is provided by prior transcription of the chromosomal regions involved. However, this may not always be so and in those cases DNA structural features must be involved. Here we examine the breakpoints of three different tumour-specific translocations in the proximity of which we can detect no transcription; two of the translocations involve regions of chromosome 11, (t[11;14] [p13;q11] and t[11;14] [q13;q32]), and the third is a newly described translocation, t[7;10] [q35;q24], involving the T cell receptor beta-gene on chromosome 7. In each case, a purine--pyrimidine tract (potential Z-DNA) occurs near the translocation breakpoints. Four independent tumours with translocation t[11;14] [p13;q11] reveal a 2 kb breakpoint cluster region at 11p13 with an adjacent potential Z-DNA region of 62 bp in length; the analogous purine--pyrimidine tract at 10q24 is 32 bp long. The purine--pyrimidine tract at the 11q13 chromosome breakpoint, however, is very large as it covers approximately 800 bp. The position, surrounding sequence and potential Z-DNA tract of the human 11p13 TALLber is conserved in rodents. These results suggest that the purine--pyrimidine tracts, presumably in the Z-DNA form, can influence chromatin structure giving access for recombinase-mediated translocations. Such putative alterations of chromatin organization are supported by the observation of DNase I hypersensitive sites near to translocation breakpoints on 10q24 and 11p13.

The T-ALL specific t(11;14)(p13;q11) translocation breakpoint cluster region is located near to the Wilms' tumour predisposition locus.

A breakpoint cluster region (T-ALLbcr) has been previously described on 11p13 for T-ALL carrying t(11;14)(p13;q11). One further T-ALL breakpoint is described bringing to 5 out of 6 such translocations which are found to break within a maximum of 6.7 kb on chromosome 11p13. Studies of somatic cell hybrids derived from t(11;14)(p13;q11) T-ALL placed the T-ALLbcr between the genes for catalase (CAT) and the beta-subunit of follicle stimulating hormone (FSHB). This suggested a link between the T-ALLbcr and the Wilms' tumour predisposition locus (WT) since constitutional 11p13 deletions predispose to Wilms' tumour. Utilising somatic cell hybrids from patients with Wilms' tumours and aniridia, we show that while the T-ALLbcr maps distal to the catalase gene at 11p13, it maps outside the shortest region of overlap of a series of 11p13 deletions associated with Wilms'-Aniridia. The data suggest the order of genes at 11p13 to be: centromere-CAT-T-ALLbcr-WT-aniridia-FSHB-telomere. Therefore, the T-ALLbcr must lie very close to but may be distinct from the Wilms' predisposition locus at 11p13.

Immunoglobulin VH genes are transcribed by T cells in association with a new 5' exon.

We previously detected mRNAs in a number of human T cell lines with a probe from within the Ig VH gene locus. We now show these mRNAs consist of Ig VH genes expressed in T cells. In one human T cell line, two RNA species have been studied and found to come from transcripts of unrearranged VH segments in which the leader exon, normally associated with VH transcripts in B cells, is replaced by a novel 5' exon (ET) not encoding a hydrophobic leader peptide. In genomic DNA, this new ET exon is adjacent to a pseudo-VH gene that has not been observed in mature mRNA. This implies that RNA splicing controls association of the new exon with the expressed VH segments. Hence, VH transcription does indeed occur in T cells, but is qualitatively different from that in B cells.

The mechanism of chromosomal translocation t(11;14) involving the T-cell receptor C delta locus on human chromosome 14q11 and a transcribed region of chromosome 11p15.

A chromosomal translocation t(11;14) (p15;q11) is described in a human acute T-cell leukaemia of immature phenotype (CD3-, CD4-, CD8-). The translocation occurs at a T-cell receptor joining J delta segment, 12 kb upstream of the constant C delta gene and 98 kb upstream of the C alpha gene at chromosome band 14q11. Nucleotide sequencing shows that both J delta and C delta are very conserved between mouse and man. The region of chromosome 11 involved in the translocation is transcriptionally active and produces a 4-kb mRNA. The DNA sequence at the chromosome 11 junction shows a perfect match to a recombinase signal sequence implying that this translocation occurred by recombinase error. The occurrence of the translocation breakpoint at the C delta locus, normally rearranged in immature T cells, and the structure of the translocation junctions suggests that the translocation occurred during an attempt at normal rearrangement of the J delta segment in an early thymocyte.

Unusual forms of T cell gamma mRNA in a human T cell leukemia cell line: implications for gamma gene expression.

The expression and rearrangement of T cell rearranging (TRG) gamma genes in human leukemic cell lines has been examined. The cell line MOLT-17 produces abundant gamma mRNA which is translated into a protein found on the cell surface which is associated with the CD3 molecule. The analysis of the gamma mRNA sequences in MOLT-17, by cDNA cloning, shows transcripts of aberrantly rearranged genes as well as the productively rearranged allele. The productive allele consists of a rearranged V gamma 8 gene joined to J gamma 2. Two forms of aberrant transcript originate from the other rearranged gamma allele. One of these initiates just upstream of the unrearranged J gamma 2 segment, and the other initiates from a V gamma 8 gene segment joined to another J gamma segment, upstream of J gamma 2. An unusual feature of the latter transcript is that polyadenlyation has occurred at the end of the first exon of C gamma 2, where two conserved poly(A) addition signals occur. The MOLT-4 cell line, on the other hand, has productively and nonproductively rearranged gamma alleles, from which relatively little transcription occurs. These results define new J gamma segments in the human TRG gamma locus and suggest that positive activation of the gamma locus is necessary for high level transcription after rearrangement.