IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem-epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem-epithelial-B-cell phenotype that underlies high-risk B-ALL.

We review here the development of the hematopoietic system and its relationship to the endothelium, with a special focus on the characterisation of the hemangioblast, the putative ancestor for endothelial cells and hematopoietic cells. Using the avian model, we have traced in vivo the progeny of embryonic endothelial cells and shown that aortic-born hematopoietic cells (known to generate the definitive hematopoietic lineage) derive from endothelial cells in the floor of the aorta. During this process, endothelial cells undergo a switch from endothelial cells to hematopoietic cells characterised by a downgrading of endothelial cell-specific genes and the parallel upgrading of hematopoietic cell-specific genes. Using a similar approach, we have shown that generation of hematopoietic cells from endothelial cells also takes place during mouse embryonic development. We have thoroughly characterised the dynamics of key molecules (several of which we have cloned) specifically expressed by the yolk sac or aortic hemangioblast. The yolk sac hemangioblast is characterized by the specific expression of SCL/Tal-1 and Lmo2, whereas the aortic hemangioblast expresses Runx-1 (a runt domain transcription factor). Finally, we have demonstrated the existence of a new site for hematopoiesis, namely the allantois. Using quail/chick grafts, we show that this embryonic appendage autonomously produces endothelial cells and hematopoietic cells, these latter being endowed with the attributes of the definitive hematopoietic lineage.

The transcription factor LMO2 is involved in vascular and hematopoietic development and hematolymphoid neoplasia. We have demonstrated that LMO2 is expressed nearly ubiquitously in native and neoplastic vasculature, including lymphatics. LMO2 reactivity is otherwise virtually absent in nonhematolymphoid tissues except in breast myoepithelium, prostatic basal cells, and secretory phase endometrial glands. Vasculature is LMO2- in adult and fetal heart, brain of older adults, hepatic sinusoids, and hepatocellular carcinoma. LMO2 is uniformly expressed in benign vascular and lymphatic neoplasms and in most malignant vascular neoplasms with the exception of epithelioid vascular neoplasms of pleura and bone. Among nonvascular neoplasms, LMO2 reactivity is present in giant cell tumor of tendon sheath, juvenile xanthogranuloma, a subset of gastrointestinal stromal tumors, small round blue cell tumors, and myoepithelial-derived neoplasms. The restricted expression pattern, nuclear localization, and crisp staining of LMO2 in paraffin blocks make it an attractive candidate for the diagnostic immunohistochemistry laboratory.

Mutations in the DYNAMIN2 (DNM2) gene are frequently detected in human acute T-cell lymphoblastic leukemia (T-ALL), although the mechanisms linking these mutations to disease pathogenesis remain unknown. Using an ENU-based forward genetic screen for mice with erythroid phenotypes, we identified a heterozygous mouse line carrying a mutation in the GTPase domain of Dnm2 (Dnm2V265G) that induced a microcytic anemia. In vitro assays using the V265G mutant demonstrated loss of GTPase activity and impaired endocytosis that was comparable to other DNM2 mutants identified in human T-ALL. To determine the effects of DNM2 mutations in T-ALL, we bred the Dnm2V265G mice with the Lmo2 transgenic mouse model of T-ALL. Heterozygous Dnm2 mutants lacking the Lmo2 transgene displayed normal T-cell development, and did not develop T-ALL. In contrast, compound heterozygotes displayed an accelerated onset of T-ALL compared with mice carrying the Lmo2 oncogene alone. The leukemias from these mice exhibited a more immature immunophenotype and an expansion in leukemic stem cell numbers. Mechanistically, the Dnm2 mutation impaired clathrin-mediated endocytosis of the interleukin (IL)-7 receptor resulting in increased receptor density on the surface of leukemic stem cells. These findings suggest that DNM2 mutations cooperate with T-cell oncogenes by enhancing IL-7 signalling.

The most frequent oncogenic activation events characterized in childhood T acute lymphoblastic leukemia (T-ALL) result in the transcriptional activation of genes coding for transcription factors. The main genes are TAL1/SCL, a member of the basic region helix-loop-helix gene family, and HOX11L2, a member of the homeobox-containing protein family. To gain insight into the pathogenesis of this type of hematologic malignancy, we analyzed 28 T-ALL samples. SIL-TAL1/SCL fusion was detected in 6 patients; expression of HOX11L2 was observed in 6 patients and of HOX11 in 3 patients. With one exception, these activations did not occur simultaneously in the same patients, and they allowed the subclassification of 50% of the patients. SIL-TAL1 fusion was detected in association with HOX11 expression in one patient and with a t(8;14) (q24;q11) in another. High expression of LYL1, LMO2, or TAL1 was observed mainly in samples negative for HOX11L2 expression. HOX11L1 and HOX11 expression were observed in one instance each, in the absence of detectable chromosomal abnormality of their respective loci, on chromosomes 2 and 10, respectively. HOX11L2 expression was associated with a chromosome 5q abnormality, the location of the HOX11L2 locus in each case tested. Finally, our data show that HOX11L2 expression was a suitable marker for minimal residual disease follow-up and was significantly associated with relapse (P =.02).

Prognostically relevant risk factors in patients with diffuse large B-cell lymphoma (DLBCL) have predominantly been evaluated in elderly populations. We tested whether previously described risk factors are also valid in younger, poor-prognosis DLBCL patients. Paraffin-embedded samples from 112 patients with de novo DLBCL, enrolled in the R-MegaCHOEP trial of the German High Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) were investigated using immunohistochemistry (MYC, FOXP1, LMO2, GCET1, CD5, CD10, BCL2, BCL6, IRF4/MUM1) and fluorescence in situ hybridization (MYC, BCL2, BCL6). MYC, BCL2 and BCL6 breaks occurred in 14, 21 and 31%, respectively. In the majority of cases, MYC was simultaneously rearranged with BCL2 and/or BCL6. The adverse impact of MYC rearrangements was confirmed, but the sole presence of BCL2 breaks emerged as a novel prognostic marker associated with inferior overall survival (OS) (P=0.002). Combined overexpression of MYC and BCL2 showed only limited association with inferior OS. All immunohistochemical cell of origin classifiers applied failed to predict survival time. DLBCL tumors with significant proportion of immunoblastic and/or immunoblastic-plasmacytoid cells had inferior OS, independently from from BCL2 break. Younger, poor-prognosis DLBCL patients, therefore, display different biological risk factors compared with an elderly population, with BCL2 translocations emerging as a powerful negative prognostic marker.

The Notch receptor pathway provides a paradigm for juxtacrine signaling pathways and controls stem cell function, developmental cell fate decisions, and cellular differentiation. The Notch pathway is constitutively activated in human cancers by chromosomal rearrangements, activating point mutations, or altered expression patterns. Therefore, the Notch pathway is the subject of chemotherapeutic intervention in a variety of human cancers. Notch receptor activation results in the gamma-secretase dependent proteolytic cleavage of the receptor to liberate the Notch intracellular domain that acts to mediate co-activator recruitment to the DNA binding transcription factor, CSL (CBF-1/RBP-Jκ, Su(H), Lag-1). Therapeutic targeting of the Notch pathway by gamma-secretase inhibitors prevents NICD production and regulates CSL-dependent transcriptional activity. To interrogate the loss of CSL activity in breast and prostate cancer cells, we used lentiviral-based shRNA knockdown of CSL. Knockdown of CSL expression was assessed by decreased DNA binding activity and resulted in decreased cell proliferation. In contrast, gamma-secretase inhibitor (GSI) treatment of these prostate and breast cancer cell lines resulted in minimal growth effects. PCR profiling of Notch pathway genes identified expression changes in few genes (Delta-like-1, Deltex-1, LMO2, and SH2D1A) after CSL knockdown. Consistent with differential effects of GSI on cell survival, GSI treatment failed to recapitulate the gene expression changes observed after CSL knockdown. Thus, CSL inhibition may provide a more effective mechanism to inhibit Notch-pathway dependent cancer cell proliferation as compared to GSI treatment.

The generation of platelets from megakaryocytes in the steady state is regulated by a variety of cytokines and transcription factors, including thrombopoietin (TPO), GATA-1, and NF-E2. Less is known about platelet production in the setting of stress thrombopoiesis, a pivotal event in the context of cytotoxic chemotherapy. Here we show in mice that the transcription factor Scl is critical for platelet production after chemotherapy and in thrombopoiesis induced by administration of TPO. Megakaryocytes from these mice showed appropriate increases in number and ploidy but failed to shed platelets. Ultrastructural examination of Scl-null megakaryocytes revealed a disorganized demarcation membrane and reduction in platelet granules. Quantitative real-time polymerase chain reaction showed that Scl-null platelets lacked NF-E2, and chromatin immunoprecipitation analysis demonstrated Scl binding to the NF-E2 promoter in the human megakaryoblastic-cell line Meg-01, along with its binding partners E47, Lmo2, and the cofactors Ldb1 and GATA-2. These findings suggest that Scl acts up-stream of NF-E2 expression to control megakaryocyte development and platelet release in settings of thrombopoietic stress.

Janus kinase 2 (Jak2) transduces signals from hematopoietic cytokines, and a gain-of-function mutation (Jak2(617V>F)) is associated with myeloproliferative diseases, particularly polycythemia vera. In this study, we examined the role of jak2a in zebrafish embryos in knock-down and overexpression studies using morpholinos (MOs) targeting the 5' untranslated region (UTR) (jak2a(UTR)-MO) and splice-site junction (jak2a(SS)-MO) of jak2a, a Jak inhibitor AG490 and a constitutive-active form of jak2a (jak2a(ca)). At 18 and 24 hours after fertilization (hpf), jak2a is expressed predominantly in the intermediate cell mass (ICM; site of primitive hematopoiesis) of wild-type and chordin morphant embryos (characterized by expansion of ICM). Both jak2a MOs and AG490 reduced gata1(+) (erythroid) cells in Tg(gata1:GFP) embryos, signal transducer and activation of transcription 5 (stat5) phosphorylation, and gene expression associated with early progenitors (scl and lmo2) and erythroid (gata1, alphahe1 and betahe1) and myeloid (spi1 [early] and mpo [late]) lineages. The chordin morphant is associated with increased stat5 phosphorylation, and both jak2a MOs and treatment with AG490 significantly ameliorated ICM expansion and hematopoietic gene up-regulation in these embryos. Injection of plasmid encoding jak2a(ca) significantly increased erythropoiesis and expression of gata1, alphahe1 and betahe1, spi1, mpo, and l-plastin. In conclusion, zebrafish jak2a is involved in primitive hematopoiesis under normal and deregulated conditions.

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.

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.

LMO2 regulates gene expression by facilitating the formation of multipartite DNA-binding complexes. In B cells, LMO2 is specifically up-regulated in the germinal center (GC) and is expressed in GC-derived non-Hodgkin lymphomas. LMO2 is one of the most powerful prognostic indicators in diffuse large B-cell (DLBCL) patients. However, its function in GC B cells and DLBCL is currently unknown. In this study, we characterized the LMO2 transcriptome and transcriptional complex in DLBCL cells. LMO2 regulates genes implicated in kinetochore function, chromosome assembly, and mitosis. Overexpression of LMO2 in DLBCL cell lines results in centrosome amplification. In DLBCL, the LMO2 complex contains some of the traditional partners, such as LDB1, E2A, HEB, Lyl1, ETO2, and SP1, but not TAL1 or GATA proteins. Furthermore, we identified novel LMO2 interacting partners: ELK1, nuclear factor of activated T-cells (NFATc1), and lymphoid enhancer-binding factor1 (LEF1) proteins. Reporter assays revealed that LMO2 increases transcriptional activity of NFATc1 and decreases transcriptional activity of LEF1 proteins. Overall, our studies identified a novel LMO2 transcriptome and interactome in DLBCL and provides a platform for future elucidation of LMO2 function in GC B cells and DLBCL pathogenesis.

The non-coding regions of tumour cell genomes harbour a considerable fraction of total DNA sequence variation, but the functional contribution of these variants to tumorigenesis is ill-defined. Among these non-coding variants, somatic insertions are among the least well characterized due to challenges with interpreting short-read DNA sequences. Here, using a combination of Chip-seq to enrich enhancer DNA and a computational approach with multiple DNA alignment procedures, we identify enhancer-associated small insertion variants. Among the 102 tumour cell genomes we analyse, small insertions are frequently observed in enhancer DNA sequences near known oncogenes. Further study of one insertion, somatically acquired in primary leukaemia tumour genomes, reveals that it nucleates formation of an active enhancer that drives expression of the LMO2 oncogene. The approach described here to identify enhancer-associated small insertion variants provides a foundation for further study of these abnormalities across human cancers.

Two serious adverse events involving activation of the LMO2 oncogene through retrovirus vector insertion in the otherwise extremely successful first gene therapy trial for X-linked severe combined immunodeficieny type 1 (SCID-X1) had initially caused widespread concern in the patient and research communities. Careful consideration 1 year after diagnosis of the second case still finds 12 of the treated patients clearly benefiting from gene therapy (freedom from treatment failure, 80%; survival 100%), a situation that should not portend the end of gene therapy for this disease, and is, in fact encouraging. While current approaches are justified to treat patients with otherwise life-threatening disorders, a broad consensus has developed that systematic basic research is required to further understand the pathophysiology of these serious adverse events and to provide new insights, enabling safer and more effective gene therapy strategies. With the continued success of SCID-X1 gene therapy in the majority of patients treated, it is of even greater importance to understand exactly which vector element or combination of elements predispose to toxicity. An in-depth study of the mechanisms behind the activation of the LMO2 and gammac genes will be highly instructive for the development of safer procedures and vectors. We summarize the central observations, ongoing experimental approaches, new concepts, and developments relevant to understanding, interpreting, and eventually overcoming the real and perceived obstacles posed by insertional mutagenesis due to gene transfer vectors.

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.

The t(11;14)(p13;q11) is presumed to arise from an erroneous T-cell receptor delta TCRD V(D)J recombination and to result in LMO2 activation. However, the mechanisms underlying this translocation and the resulting LMO2 activation are poorly defined. We performed combined in vivo, ex vivo, and in silico analyses on 9 new t(11;14)(p13;q11)-positive T-cell acute lymphoblastic leukemia (T-ALL) as well as normal thymocytes. Our data support the involvement of 2 distinct t(11;14)(p13;q11) V(D)J-related translocation mechanisms. We provide compelling evidence that removal of a negative regulatory element from the LMO2 locus, rather than juxtaposition to the TCRD enhancer, is the main determinant for LMO2 activation in the majority of t(11;14)(p13;q11) translocations. Furthermore, the position of the LMO2 breakpoints in T-ALL in the light of the occurrence of TCRD-LMO2 translocations in normal thymocytes points to a critical role for the exact breakpoint location in determining LMO2 activation levels and the consequent pressure for T-ALL development.

LMO2 is a transcription regulator involved in human T-cell leukemia, including some occurring in X-SCID gene therapy trials, and in B-cell lymphomas and prostate cancer. LMO2 functions in transcription complexes via protein-protein interactions involving two LIM domains and causes a preleukemic T-cell development blockade followed by clonal tumors. Therefore, LMO2 is necessary but not sufficient for overt neoplasias, which must undergo additional mutations before frank malignancy. An open question is the importance of LMO2 in tumor development as opposed to sustaining cancer. We have addressed this using a peptide aptamer that binds to the second LIM domain of the LMO2 protein and disrupts its function. This specificity is mediated by a conserved Cys-Cys motif, which is similar to the zinc-binding LIM domains. The peptide inhibits Lmo2 function in a mouse T-cell tumor transplantation assay by preventing Lmo2-dependent T-cell neoplasia. Lmo2 is, therefore, required for sustained T-cell tumor growth, in addition to its preleukemic effect. Interference with LMO2 complexes is a strategy for controlling LMO2-mediated cancers, and the finger structure of LMO2 is an explicit focus for drug development.

Interfering intracellular antibodies are valuable for biological studies as drug surrogates and as potential macromolecular drugs per se. Their application is still limited because of the difficulty of acquisition of functional intracellular antibodies. We describe the use of the new intracellular antibody capture procedure (IAC(3)) to facilitate direct isolation of functional single domain antibody fragments using four independent target molecules (LMO2, TP53, CRAF1, and Hoxa9) from a set of diverse libraries. Initially, these have variability in only one of the three antigen-binding CDR regions of VH or VL and first round single domains are affinity matured by iterative randomization of the two other CDRs and reselection. We highlight the approach using a single domain binding to LMO2 protein. Our results show that interfering with LMO2 protein function demonstrates a role specifically in erythroid differentiation, confirm a necessary and sufficient function for LMO2 as a cancer therapy target in T-cell neoplasia and allowed for the first time production of soluble recombinant LMO2 protein by co-expression with intracellular domain antibodies. Co-crystallization of LMO2 and the anti-LMO2 VH protein was successful. These results demonstrate that this third generation IAC(3) offers a robust toolbox for various biomedical applications and consolidates functional features of the LMO2 protein complex, which includes the importance of Lmo2-Ldb1 protein interaction.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the hematopoietic transcription factors LYL1 and LMO2. In contrast, ERG, FLI1, TAL1, and RUNX1 bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. We find that expression of wild-type transcription factors RUNX1 and ERG in AML is required to prevent this oncogene overexpression. Together our results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells.

The c-Myb transcription factor is an important regulator of hematopoietic cell development. c-Myb is expressed in immature hematopoietic cells and plays a direct role in lineage fate selection, cell cycle progression, and differentiation of myeloid as well as B- and T-lymphoid progenitor cells. As a DNA-binding transcription factor, c-Myb regulates specific gene programs through activation of target genes. Still, our understanding of these programs is incomplete. Here, we report a set of novel c-Myb target genes, identified using a combined approach: specific c-Myb knockdown by 2 different siRNAs and subsequent global expression profiling, combined with the confirmation of direct binding of c-Myb to the target promoters by ChIP assays. The combination of these 2 approaches, as well as additional validation such as cloning and testing the promoters in reporter assays, confirmed that MYADM, LMO2, GATA2, STAT5A, and IKZF1 are target genes of c-Myb. Additional studies, using chromosome conformation capture, demonstrated that c-Myb target genes may directly interact with each other, indicating that these genes may be coordinately regulated. Of the 5 novel target genes identified, 3 are transcription factors, and one is a transcriptional co-regulator, supporting a role of c-Myb as a master regulator controlling the expression of other transcriptional regulators in the hematopoietic system.

The most common translocation in childhood T-cell acute lymphoblastic leukemia (T-ALL) involves the LMO2 locus, resulting in ectopic expression of the LMO2 gene in human thymocytes. The LMO2 gene was also activated in patients with X-linked Severe Combined Immune Deficiency treated with gene therapy because of retroviral insertion in the LMO2 locus. The LMO2 insertions predisposed these children to T-ALL, yet how LMO2 contributes to T cell transformation remains unclear. The LIM (Lin 11, Isl-1, Mec-3) domain containing LMO2 protein regulates erythropoiesis as part of a large transcriptional complex consisting of LMO2, TAL1, E47, GATA1 and LDB1 that recognizes bipartite E-box-GATA1 sites on target genes. Similarly, a TAL1/E47/LMO2/LDB1 complex is observed in human T-ALL and Tal1 and Lmo2 expression in mice results in disease acceleration. To address the mechanism(s) of Tal1/Lmo2 synergy in leukemia, we generated Lmo2 transgenic mice and mated them with mice that express wild-type Tal1 or a DNA-binding mutant of TAL1. Tal1/Lmo2 and MutTAL1/Lmo2 bitransgenic mice exhibit perturbations in thymocyte development due to reduced E47/HEB transcriptional activity and develop leukemia with identical kinetics. These data demonstrate that the DNA-binding activity of Tal1 is not required to cooperate with Lmo2 to cause leukemia in mice and suggest that Lmo2 may cooperate with Tal1 to interfere with E47/HEB function(s).

Ocular coloboma is a potentially blinding congenital eye malformation caused by failure of optic fissure closure during early embryogenesis. The optic fissure is a ventral groove that forms during optic cup morphogenesis, and through which hyaloid artery and vein enter and leave the developing eye, respectively. After hyaloid artery and vein formation, the optic fissure closes around them. The mechanisms underlying optic fissure closure are poorly understood, and whether and how this process is influenced by hyaloid vessel development is unknown. Here we show that a loss-of-function mutation in lmo2, a gene specifically required for hematopoiesis and vascular development, results in failure of optic fissure closure in zebrafish. Analysis of ocular blood vessels in lmo2 mutants reveals that some vessels are severely dilated, including the hyaloid vein. Remarkably, reducing vessel size leads to rescue of optic fissure phenotype. Our results reveal a new mechanism leading to coloboma, whereby malformed blood vessels interfere with eye morphogenesis.