Isabelle Lavenir

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.

The Crystal Structure of the Px Domain from P40Phox 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.

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 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 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.

Stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy

The accumulation of insoluble proteins is a pathological hallmark of several neurodegenerative disorders. Tauopathies are caused by the dysfunction and aggregation of tau protein and an impairment of cellular protein degradation pathways may contribute to their pathogenesis. Thus, a deficiency in autophagy can cause neurodegeneration, while activation of autophagy is protective against some proteinopathies. Little is known about the role of autophagy in animal models of human tauopathy. In the present report, we assessed the effects of autophagy stimulation by trehalose in a transgenic mouse model of tauopathy, the human mutant P301S tau mouse, using biochemical and immunohistochemical analyses. Neuronal survival was evaluated by stereology. Autophagy was activated in the brain, where the number of neurons containing tau inclusions was significantly reduced, as was the amount of insoluble tau protein. This reduction in tau aggregates was associated with improved neuronal survival in the cerebral cortex and the brainstem. We also observed a decrease of p62 protein, suggesting that it may contribute to the removal of tau inclusions. Trehalose failed to activate autophagy in the spinal cord, where it had no impact on the level of sarkosyl-insoluble tau. Accordingly, trehalose had no effect on the motor impairment of human mutant P301S tau transgenic mice. Our findings provide direct evidence in favour of the degradation of tau aggregates by autophagy. Activation of autophagy may be worth investigating in the context of therapies for human tauopathies.

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 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 ∼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.

“Prion‐Like” Templated Misfolding in Tauopathies

The soluble microtubule‐associated protein tau forms hyperphosphorylated, insoluble and filamentous inclusions in a number of neurodegenerative diseases referred to as “tauopathies.” In Alzheimers disease, tau pathology develops in a stereotypical manner, with the first lesions appearing in the locus coeruleus and entorhinal cortex, from where they appear to spread to the hippocampus and neocortex. Propagation of tau pathology is also a characteristic of argyrophilic grain disease, where the tau lesions spread throughout the limbic system. Significantly, isoform composition and morphology of tau filaments can differ between tauopathies, suggesting the existence of distinct tau strains. Extensive experimental findings indicate that prion‐like mechanisms underly the pathogenesis of tauopathies.

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

ABSTRACT 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.