Oncogene database

4005

LMO2

LIM domain only 2 (rhombotin-like 1);LMO2;LIM domain only 2 (rhombotin-like 1)

LIM domain only protein 2|LMO-2|T-cell translocation gene 2|T-cell translocation protein 2|cysteine-rich protein TTG-2|rhombotin 2|rhombotin-2|rhombotin-like 1

11p13

protein-coding

The LMO2 gene is activated by chromosomal translocations in human T cell acute leukemias, but in mouse embryogenesis, Lmo2 is essential for initiation of yolk sac and definitive hematopoiesis. The LMO2 protein comprises two LIM-zinc-finger-like protein interaction modules and functions by interaction with specific partners in DNA-binding transcription complexes. We have now investigated the role of Lmo2-associated transcription complexes in the formation of the vascular system by following the fate of Lmo2-null embryonic stem (ES) cells in mouse chimeras. Lmo2 is expressed in vascular endothelium, and Lmo2-null ES cells contributed to the capillary network normally until around embryonic day 9. However, after this time, marked disorganization of the vascular system was observed in those chimeric mice that have a high contribution of Lmo2-null ES cells. Moreover, Lmo2-null ES cells do not contribute to endothelial cells of large vessel walls of surviving chimeric mice after embryonic day 10. These results show that Lmo2 is not needed for de novo capillary formation from mesoderm but is necessary for angiogenic remodeling of the existing capillary network into mature vasculature. Thus, Lmo2-mediated transcription complexes not only regulate distinct phases of hematopoiesis but also angiogenesis, presumably by Lmo2 interacting with distinct partners in the different settings.

The LIM only protein Lmo2 plays an important role in hematopoiesis and leukemogenesis. Lmo2 acts as a bridging molecule between components of hematopoietic gene regulatory protein complexes. We used the yeast two-hybrid system to identify novel Lmo2 interacting proteins and found that the AF6 protein binds to Lmo2. AF6 is a recurrent fusion partner of MLL, the human homolog of Drosophila trithorax chromatin remodeling protein that is involved in childhood leukemia and mixed lineage leukemia. Our data support the notion that recurrent fusion partners of chimeric MLL proteins recruit hematopoietic gene regulatory complexes.

The LMO2 gene encodes a LIM-only protein and is a target of chromosomal translocations in human T-cell leukemia. Recently, two X-SCID patients treated by gene therapy to rescue T-cell lymphopoiesis developed T-cell leukemias with retroviral insertion into the LMO2 gene causing clonal T-cell proliferation. In view of the specificity of LMO2 in T-cell tumorigenesis, we investigated a possible role for Lmo2 in T-lymphopoiesis, using conditional knockout of mouse Lmo2 with loxP-flanked Lmo2 and Cre recombinase alleles driven by the promoters of the lymphoid-specific genes Rag1, CD19, and Lck. While efficient deletion of Lmo2 was observed, even in the earliest detectable lymphoid cell progenitors of the bone marrow, there was no disturbance of lymphopoiesis in either T- or B-cell lineages, and in contrast to Lmo2 transgenic mice, there were normal distributions of CD4- CD- thymocytes. We conclude that there is no mandatory role for LMO2 in lymphoid development, implying that its specific role in T-cell tumorigenesis results from a reprogramming of gene expression after enforced expression in T-cell precursors.

The activation of oncogenic transcription factors defines distinct molecular subsets of T-cell acute lymphoblastic leukaemia and has prognostic relevance in children. We investigated the prognostic effect of the expression levels of eight oncogenic transcription factors--TLX1 (HOX11), TLX3 (HOX11L2), TAL1, TAL2, LYL1, OLIG2 (BHLHB1), LMO1, and LMO2--in 52 adults with T-cell acute lymphoblastic leukaemia. The leukaemia-specific survival rate for the 16 TLX1-positive patients was 88% (90% CI 73-100%), compared with 56% (42-70%) for ALL other cases (p=0.019). Only the TLX1 oncogene expression subgroup showed difference in leukaemia-specific survival. Our results suggest that overexpression of TLX1 confers a good outlook for adults with T-cell acute lymphoblastic leukaemia. Furthermore, our findings lead to questions about whether stem-cell transplantation in first remission is necessary for effective treatment of patients in the low-risk subgroup of patients with TLX1 oncogene expression.

Ectopic expression of LMO2 occurs in approximately 45% of T-lineage acute lymphoblastic leukemias (T-ALL), sometimes in association with chromosomal translocations. Recently, a lymphoproliferative disorder developed in two participants in a gene therapy trial due to LMO2 activation via integration of the retroviral vector. To investigate these regulatory disruptions, we analyzed the promoter region and identified a tissue-specific repressor. The fragment containing this element could also produce tissue-specific suppression of transcription from the SV40 promoter. This suppression involves histone acetylation which can be relieved with Trichostatin A (TSA). The negative element is in a region consistently removed from LMO2 in the known chromosomal translocations.

To identify new cytogenetic abnormalities associated with leukemogenesis or disease outcome, T-cell acute lymphoblastic leukemia (T-ALL) patient samples were analyzed by means of the array-comparative genome hybridization technique (array-CGH). Here, we report the identification of a new recurrent and cryptic deletion on chromosome 11 (del(11)(p12p13)) in about 4% (6/138) of pediatric T-ALL patients. Detailed molecular-cytogenetic analysis revealed that this deletion activates the LMO2 oncogene in 4 of 6 del(11)(p12p13)-positive T-ALL patients, in the same manner as in patients with an LMO2 translocation (9/138). The LMO2 activation mechanism of this deletion is loss of a negative regulatory region upstream of LMO2, causing activation of the proximal LMO2 promoter. LMO2 rearrangements, including this del(11)(p12p13) and t(11;14) (p13;q11) or t(7;11)(q35;p13), were found in the absence of other recurrent cytogenetic abnormalities involving HOX11L2, HOX11, CALM-AF10, TAL1, MLL, or MYC. LMO2 abnormalities represent about 9% (13/138) of pediatric T-ALL cases and are more frequent in pediatric T-ALL than appreciated until now.

We previously developed a multivariate model based on the RNA expression of 6 genes (LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2) that predicts survival in diffuse large B-cell lymphoma (DLBCL) patients. Since LMO2 emerged as the strongest predictor of superior outcome, we generated a monoclonal anti-LMO2 antibody in order to study its tissue expression pattern. Immunohistologic analysis of over 1200 normal and neoplastic tissue and cell lines showed that LMO2 protein is expressed as a nuclear marker in normal germinal-center (GC) B cells and GC-derived B-cell lines and in a subset of GC-derived B-cell lymphomas. LMO2 was also expressed in erythroid and myeloid precursors and in megakaryocytes and also in lymphoblastic and acute myeloid leukemias. It was rarely expressed in mature T, natural killer (NK), and plasma cell neoplasms and was absent from nonhematolymphoid tissues except for endothelial cells. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of the LMO2 protein was similar to that of other GC-associated proteins (HGAL, BCL6, and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2). Our results warrant inclusion of LMO2 in multivariate analyses to construct a clinically applicable immunohistologic algorithm for predicting survival in patients with DLBCL.

The nuclear proteins TAL1 (T-cell acute leukaemia protein 1) and LMO2 (LIM-only protein 2) have critical roles in haematopoietic development, but are also often aberrantly activated in T-cell acute lymphoblastic leukaemia. TAL1 and LMO2 operate within multifactorial protein-DNA complexes that regulate gene expression in the developing blood cell. TAL1 is a tissue-specific basic helix-loop-helix (bHLH) protein that binds bHLH domains of ubiquitous E-proteins, (E12 and E47), to bind E-box (CANNTG) DNA motifs. TAL1(bHLH) also interacts specifically with the LIM domains of LMO2, which in turn bind Ldb1 (LIM-domain binding protein 1). Here we used biophysical methods to characterize the assembly of a five-component complex containing TAL1, LMO2, Ldb1, E12, and DNA. The bHLH domains of TAL1 and E12 alone primarily formed helical homodimers, but together preferentially formed heterodimers, to which LMO2 bound with high affinity (K(A) approximately 10(8) M(-1)). The resulting TAL1/E12/LMO2 complex formed in the presence or absence of DNA, but the different complexes preferentially bound different Ebox-sequences. Our data provide biophysical evidence for a mechanism, by which LMO2 and TAL1 both regulate transcription in normal blood cell development, and synergistically disrupt E2A function in T-cells to promote the onset of leukaemia.

Lmo2 is a T cell leukemia-related proto-oncogene, which belongs to the LIM protein family. Previous work has established its key role in yolk sac erythropoiesis and adult hematopoiesis, and it is also necessary for regulating angiogenesis. It has been demonstrated that this gene encodes a protein of 158 amino acids, consisting of two tandem cysteine-rich LIM domains, but the detailed mechanism of its transcriptional regulation remains to be elucidated. To further investigate the mechanism of transcriptional regulation of Lmo2, we combined SMART PCR technology with 5 RACE and found a novel post-transcriptional splicing form of Lmo2 in adult human kidney. This alternative transcript contains only two exons, encoding a smaller protein of 151 amino acids. Interestingly, it shares the same reading frame as the original Lmo2, but differs in 7 amino acids at the N-terminus. A genomic DNA fragment (from -294 nts to +180 nts) containing the putative promoter region has been inserted into the luciferase reporter gene vector pGL3-basic and showed stable promoter activity when transfected into COS7. RT-PCR analysis revealed that this variant transcript was expressed widely in human tissues and cell lines, suggesting its potential basic functional importance.

The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. ALL candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.

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.

The T-cell oncogene rhombotin was first identified as a gene near a chromosomal translocation breakpoint in a human T-cell tumour and represents the first example of an oncogene carrying the duplicated cysteine-rich regions (CRR or LIM domains). Transgenic expression of a reporter gene under the control of one of the rhombotin gene promoters subsequently showed high levels of expression in the developing brain. These disparate sites of transcriptional activity suggested that the gene may have been activated de novo specifically in the T cell tumour via the translocation. Here, we assess this possibility by analysing rhombotin gene expression in mouse development by in situ hybridization of whole embryos, Northern filter hybridization, and a sensitive semiquantitative PCR method. The results show that the central nervous system is the major site of rhombotin mRNA production. Low level expression does, however, occur in other tissues including thymus. Furthermore, both promoters are active and differentially regulated during mouse embryogenesis in both brain and thymus. In subregions of the adult brain, different levels of rhombotin activity can be observed, with evidence for regional variation in promoter usage. A detailed analysis of mouse and human T-cell differentiation suggests that fluctuating promoter activities are related to a general T-cell differentiation process rather than to the differentiation of functionally distinct subsets of T-cells. These data suggest that the transforming activity of rhombotin in the T-cell with the chromosomal translocation was not due to de novo transcriptional activation, but rather to a quantitative or qualitative change in expression levels of this CRR-containing oncogene after chromosomal translocation.

The oncogenic fusion protein E2A-HLF is a chimeric transcription factor that arises from the t(17;19) translocation in childhood B-cell acute lymphoblastic leukemias (B-precursor ALL) and is associated with very poor outcome. We show that retroviral-mediated expression of E2A-HLF alone is sufficient to immortalize primary lymphoid progenitors. We identify Lmo2 and Bcl-2 as direct target genes downstream of E2A-HLF. We use real-time PCR analysis to show that LMO2 and BCL-2 expression is preferentially upregulated both in biopsy material from t(17;19) B-precursor ALL patients and lymphoid cell lines derived from t(17;19) leukemias. Co-expression of Lmo2 and Bcl-2 was sufficient to immortalize lymphoid progenitor cells resulting in a similar phenotype to that induced by E2A-HLF alone. Both shRNA-mediated knockdown of Lmo2 expression and pharmacological inhibition of BCL-2 function in E2A-HLF immortalized cells severely compromised their viability. These data suggest that both Lmo2 and Bcl-2 are required for the action of E2A-HLF in leukemogenesis.

The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 A resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.

LMO2 was discovered via chromosomal translocations in T-cell leukaemia and shown normally to be essential for haematopoiesis. LMO2 is made up of two LIM only domains (thus it is a LIM-only protein) and forms a bridge in a multi-protein complex. We have studied the mechanism of formation of this complex using a single domain antibody fragment that inhibits LMO2 by sequestering it in a non-functional form. The crystal structure of LMO2 with this antibody fragment has been solved revealing a conformational difference in the positioning and angle between the two LIM domains compared with its normal binding. This contortion occurs by bending at a central helical region of LMO2. This is a unique mechanism for inhibiting an intracellular protein function and the structural contusion implies a model in which newly synthesized, intrinsically disordered LMO2 binds to a partner protein nucleating further interactions and suggests approaches for therapeutic targeting of LMO2.

Rhombotin-2 (RBTN-2) is a LIM domain protein that, with the exception of thymocytes, is widely expressed during fetal development. Although RBTN-2 is crucial for normal erythropoiesis, the ectopic expression of RBTN-2 in T lymphocytes results in T-cell proliferation and leukemogenesis. Thus, while a proliferative function for RBTN-2 has been established in T-cells, neither its role in erythropoiesis nor its function(s) in other tissues are known. We have examined the expression and location of RBTN-2 in normal and malignant cells. Similar to fetal development, RBTN-2 RNA was detected in ALL normal adult tissues tested with the exception of colon and thymocytes. RBTN-2 RNA was not detected in ALL primary tumors and tumor cell lines, indicating RBTN-2 expression is not ubiquitous in proliferating cells. Using polyclonal antisera, RBTN-2 was detected predominantly in the nucleus of human hematopoietic cells. Significantly, human leukemic T cells with disruption of the RBTN-2 locus and thymocytes from transgenic mice with enforced expression of RBTN-2 showed similar nuclear location of RBTN-2 protein, consistent with the notion that RBTN-2 acts as a transcriptional regulator in T-cell proliferation. Surprisingly, in normal tissues, RBTN-2 showed a strikingly similar distribution to that of metallothionein-1, having both nuclear and cytoplasmic localization that suggested that RBTN-2 may be involved in the acute phase response. Indeed, similar to metallothionein-1, RBTN-2 mRNA was induced in thymocytes of mice exposed to zinc and in human thymocytes treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Since the LIM domain permits binding of multiple protein partners, the specific function of RBTN-2 may depend upon subcellular sequestration through interaction with different cofactors. Thus, in addition to its roles in erythropoiesis and T-cell leukemia, RBTN-2 may also be involved in the acute phase response.

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 TTG-2 gene has been identified at the site of chromosomal translocations in acute T-cell leukemia s (T-ALL). These breakpoints map to a region between 2 and 30 kb upstream of TTG-2 in chromosome 11p13. To establish the role of these translocation breakpoints in the deregulation of TTG-2 in T-ALL we have determined the complete structure of this gene. Isolation of new TTG-2 cDNA clones from fetal liver identified an alternative transcript (TTG-2a) containing two new noncoding 5 exons. Analysis of exon/intron boundaries, identified 6 exons spread over 35 kb in 11p13. The gene encodes two alternative transcripts initiating from two promoters. TTG-2a, from promoter 1 (P1) and TTG-2b, from promoter 2 (P2) differ in the length of the 5 untranslated region, but encode the same protein. A high level of TTG-2a was present in fetal liver and spleen, whereas in adult kidney a low level of TTG-2a and a high level of TTG-2b was found. The transcription start site for TTG-2a was identified by RNase protection experiments and it displayed sequence homology to an initiator element (inr). P1 lacks a TATA box, but binding sites for SP1 and GATA-1 are present. This new genomic organisation revealed that ALL known chromosomal translocations map upstream of P2, removing P1 and putative upstream regulatory sequences leaving P2 intact. These results show that chromosomal translocations disrupt the TTG-2 gene itself, further confirming its role in the development of T-ALL.

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.

The LIM domain protein rhombotin-2 (RBTN-2/TTG-2/LMO2) is involved in many processes, including leukemogenesis and erythropoiesis. It is thought that the principle role of RBTN-2 in these processes is to regulate transcription. To examine the potential for RBTN-2 to modulate transcription, we constructed RBTN-2/GAL4 DNA-binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct. From these studies we identified a transcription activation domain within the NH2 terminus of RBTN-2. This activation domain was further localized within a proline-rich 19-amino acid region. A second activation domain of 11 amino acids was also identified. This domain was located within the COOH terminus of RBTN-2, and functioned in mammalian cells but not in yeast. Furthermore, the two LIM domains of RBTN-2 were shown to function as transcription repression domains. Each individual LIM domain acted as an independent transcription repression domain on a heterologous activation domain. However, in context of full-length RBTN-2, the LIM domains selectively repressed the NH2-terminal activation domain, but had no effect on the COOH-terminal domain. Overall, these results demonstrate that the T-cell oncogene RBTN-2 is a complex transcription factor possessing multiple transcription regulatory modules, including two activation domains and two repression domains.

The LIM domain protein rhombotin-2 (RBTN-2/TTG-2/Lmo2) has distinct functions in erythropoiesis and in T-cell leukemogenesis. Additional functions for RBTN2 are indicated by its expression in non-hematopoietic tissues. These diverse functions of RBTN2 are presumed to be accomplished through physical interaction with different protein partners that bind the LIM domains of RBTN2. To identify these proteins which may modulate the activity of RBTN2, a human cDNA library was screened using the yeast two-hybrid assay. Using the RBTN2 LIM domain region as bait , the retinoblastoma-binding protein 2 (RBP2) was identified as a partner for RBTN2. The interaction between RBTN2 and RBP2 was confirmed using in vitro binding assays, and by co-immunoprecipitation of the two proteins. Deletion analysis showed the second LIM domain of RBTN2 was necessary and sufficient for binding to the last 69 amino acids of RBP2. The interaction between RBTN2 and RBP2 had a functional consequence: the combination of RBP2 and RBTN2 gave higher transcription in vitro, than RBTN2 alone. The interaction with RBP2 suggests two additional functions for RBTN2: (i) RBTN2 may directly affect the activity of RBP2, and/or (ii) RBTN2 may indirectly modulate the functions of the retinoblastoma protein by binding to RBP2.

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.