Terence H. Rabbitts

The LIM‐only protein Lmo2 is a bridging molecule assembling an erythroid, DNA‐binding complex which includes the TAL1, E47, GATA‐1 and Ldb1/NLI proteins

The LIM‐only protein Lmo2, activated by chromosomal translocations in T‐cell leukaemias, is normally expressed in haematopoiesis. It interacts with TAL1 and GATA‐1 proteins, but the function of the interaction is unexplained. We now show that in erythroid cells Lmo2 forms a novel DNA‐binding complex, with GATA‐1, TAL1 and E2A, and the recently identified LIM‐binding protein Ldb1/NLI. This oligomeric complex binds to a unique, bipartite DNA motif comprising an E‐box, CAGGTG, followed ∼9 bp downstream by a GATA site. In vivo assembly of the DNA‐binding complex requires interaction of all five proteins and establishes a transcriptional transactivating complex. These data demonstrate one function for the LIM‐binding protein Ldb1 and establish a function for the LIM‐only protein Lmo2 as an obligatory component of an oligomeric, DNA‐binding complex which may play a role in haematopoiesis.

The Oncogenic Cysteine-rich LIM domain protein Rbtn2 is essential for erythroid development

The LIM domain protein rbtn2 is associated with T cell acute leukemias. We demonstrate that rbtn2 is a nuclear protein expressed in the erythroid lineage in vivo, and using homologous recombination, we show that it is essential for erythroid development in mice. The homozygous rbtn2 null mutation leads to failure of yolk sac erythropoiesis and embryonic lethality around E10.5. Moreover, in vitro differentiation of yolk sac tissue from homozygous mutant mice and sequentially targeted double-mutant ES cells demonstrates a block to erythroid development. This shows a pivotal role for a LIM domain protein in lineage specification during mammalian development and suggests that RBTN2 and GATA-1 are critical at similar stages of erythroid differentiation.

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.

Diversity and rearrangement of the human T cell rearranging γ genes: Nine germ-line variable genes belonging to two subgroups

We describe nine T cell gamma variable (V) gene segments isolated from human DNA. These genes, which fall into two subgroups, are mapped in two DNA regions covering 54 kb and probably represent the majority of human V gamma genes. One subgroup (V gamma I) contains eight genes, consisting of four active genes and four pseudogenes. The single V gamma II gene is potentially active. Sequence analysis of the V gamma I genes shows variation clustered in hypervariable regions, but somatic variability is restricted to N-region diversity. Studies on rearrangement in T cell lines and in thymic DNA show that major rearrangements can be observed that are attributable to the five active V gamma genes. In addition, human cells with the phenotype of helper T cells can undergo productive V gamma-J gamma joining.

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.

Lmo2 and Scl/Tal1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata1.

The LIM domain protein Lmo2 and the basic helix-loop-helix transcription factor Scl/Tal1 are expressed in early haematopoietic and endothelial progenitors and interact with each other in haematopoietic cells. While loss-of-function studies have shown that Lmo2 and Scl/Tal1 are essential for haematopoiesis and angiogenic remodelling of the vasculature, gain-of-function studies have suggested an earlier role for Scl/Tal1 in the specification of haemangioblasts, putative bipotential precursors of blood and endothelium. In zebrafish embryos, Scl/Tal1 can induce these progenitors from early mesoderm mainly at the expense of the somitic paraxial mesoderm. We show that this restriction to the somitic paraxial mesoderm correlates well with the ability of Scl/Tal1 to induce ectopic expression of its interaction partner Lmo2. Co-injection of lmo2 mRNA with scl/tal1 dramatically extends its effect to head, heart, pronephros and pronephric duct mesoderm inducing early blood and endothelial genes all along the anteroposterior axis. Erythroid development, however, is expanded only into pronephric mesoderm, remaining excluded from head, heart and somitic paraxial mesoderm territories. This restriction correlates well with activation of gata1 transcription and co-injection of gata1 mRNA along with scl/tal1 and lmo2 induces erythropoiesis more broadly without ventralising or posteriorising the embryo. While no ectopic myeloid development from the Scl/Tal1-Lmo2-induced haemangioblasts was observed, a dramatic increase in the number of endothelial cells was found. These results suggest that, in the absence of inducers of erythroid or myeloid haematopoiesis, Scl/Tal1-Lmo2-induced haemangioblasts differentiate into endothelial cells.

Mechanisms of divergence and convergence of the human immunoglobulin α1 and α2 constant region gene sequences

Abstract Nucleotide sequences of the human α1 and two allelic α2 immunoglobulin heavy chain constant region genes are presented. The genes contain three exons, each encoding a single constant region protein domain. The protein hinge region is encoded at the 5′ end of the second exon, and the rapid evolutionary changes in length of the hinge correspond to duplications or deletions within the hinge-coding region, probably facilitated by repeats in the DNA sequence. Alignment of the α1 and α2 gene sequences reveals an unusual coupled deletion-duplication in the 5′-flanking region, which can be explained in terms of a slipped-strand mispairing model. Comparison of nucleotide sequences of the α1 gene and two alleles of the α2 gene indicates a localized transfer of genetic information from the 3′ end of the α1 gene to one of the α2 alleles, probably by a gene conversion. At one end of the region within which conversion apparently occurred, there is a 40 bp sequence of the type that can form Z-DNA.

The Lmo2 Oncogene Initiates Leukemia in Mice by Inducing Thymocyte Self-Renewal

Its All About Self-Renewal The Lmo2 oncogene was identified as a contributing factor in human T cell acute lymphoblastic leukemia (T-ALL) nearly two decades ago, but the gene rose to prominence in 2003 when its inadvertent activation by a retroviral vector was shown to cause leukemia in two patients in a gene therapy trial. The cellular mechanism by which the gene product of Lmo2, a transcriptional regulator, induces T-ALL is poorly understood. Studying transgenic mice, McCormack et al. (p. 879, published online 21 January) now show that Lmo2 confers self-renewal activity to committed T cells in the thymus without affecting their capacity for T cell differentiation. These self-renewing cells, which were detectable 8 months prior to the onset of overt leukemia in the mice, expressed genes in common with hematopoietic stem cells (HSCs), suggesting that Lmo2 might reactivate an HSC-specific transcriptional program. Expression of an oncogene confers self-renewal activity to committed T cells in the thymus long before disease onset. The LMO2 oncogene causes a subset of human T cell acute lymphoblastic leukemias (T-ALL), including four cases that arose as adverse events in gene therapy trials. To investigate the cellular origin of LMO2-induced leukemia, we used cell fate mapping to study mice in which the Lmo2 gene was constitutively expressed in the thymus. Lmo2 induced self-renewal of committed T cells in the mice more than 8 months before the development of overt T-ALL. These self-renewing cells retained the capacity for T cell differentiation but expressed several genes typical of hematopoietic stem cells (HSCs), suggesting that Lmo2 might reactivate an HSC-specific transcriptional program. Forced expression of one such gene, Hhex, was sufficient to initiate self-renewal of thymocytes in vivo. Thus, Lmo2 promotes the self-renewal of preleukemic thymocytes, providing a mechanism by which committed T cells can then accumulate additional genetic mutations required for leukemic transformation.