Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

Abstract

Genetic studies have demonstrated an essential role for nuclear LIM domain-containing proteins in embryonic development. Disruption of the rhombotin 2 (Rbtn2) gene in mice resulted in embryonic lethality at day 10.5 due to a lack of erythropoeisis (7). Misexpression of Rbtn2 and the related Rbtnl in T cells due to T-cell translocation events leads to T-cell acute lymphoblastic leukemia in children (8-10) or to a leukemia with similar properties in transgenic mice (11-13). The leukemias apparently result from progression of lymphocytes through an inappropriate differentiation pathway, eventually leading to the malignant phenotype (14).The disruption of several LIM homeodomain genes has demonstrated their importance predominately in the development of neuronal lineages. Mutation of the Caenorhabditis elegans Mec3 gene prevented the generation of touch receptor neurons (15), while deletion of Lhxi (Liml) in mice precluded head structure formation (16). These mouse embryos died around E10 and lacked forebrain, midbrain, and some hind- While the biological importance of the LIM domaincontaining transcription factors is clear, comparatively little is known about their gene targets and the molecular mechanisms through which they function. Interaction between Rbtn2 and the erythroid specific basic helix-loop-helix factor Tall and the zinc-finger protein GATAl has been observed (3,21,22). Further evidence for the assembly of LIM domain proteins into nuclear complexes that regulate gene expression is the association of the LIM homeodomain protein Lhx3 with the POU-domain protein Pit-1 (23). In addition to interacting, these proteins synergize in transcriptional activation of the Pit-i, prolactin, and thyroid stimulating hormone 13 subunit promoters. To date, no common binding partners or shared transcriptional mechanisms for the nuclear LIM proteins have been identified.In search of new binding partners for Rbtn2, we identified a nuclear protein that associates with the LIM domains of every rhombotin and LIM homeodomain protein analyzed. Nuclear LIM interactor (NLI) is found in the nuclei of developing embryonic neuronal cells and is coexpressed with Isll early in motor neuron differentiation. The synchronous expression of both proteins in such cells during the initial stages of differentiation suggests a role for NLI in the Islldependent development of motor neurons. MATERIALS AND METHODSIsolation of Rbtn2 cDNA. The 474-bp human Rbtn2 coding sequence was amplified from a Agtl 1 SK-N-MC cell cDNA library provided by E. Turner (University of California at San Diego). Each primer (100 pmol) was incubated with 5 ,ul library for 5 min at 94°C, and 35 cycles of synthesis were carried out in the presence of 2 ,utM tetraethylmethylammonium chloride, 200 ,uM of each dNTP, and 2.5 units Taq DNA polymerase (Promega). The PCR product was initially cloned into pEG202 (24), sequenced in its entirety, and then subcloned into pGEX-2TK (Pharmacia) for expression library screening.Protein Purification. For library screening, glutathione S...

The LIM-only protein, LMO4, and the LIM domain-binding protein, LDB1, expression in squamous cell carcinomas of the oral cavity

“…The LIM domain of LDBs contributes to the binding of transcription factors, including LIM-homeodomain, zinc-finger and basic helixloop -helix (bHLH) proteins (Agulnick et al, 1996;Jurata et al, 1996;Bach et al, 1997;Morcillo et al, 1997). Formation of protein complexes synergistically activates the expression of target genes Milán et al, 1998).…”

“…The LMO4 gene is widely distributed in embryonic tissues (Kenny et al, 1998;Sugihara et al, 1998), and involved in negative regulation of breast carcinoma cell differentiation (Visvader et al, 2001). LMO4 binds with a high affinity to the LIM domain-binding proteins, LDB1 (CLIM2, NLI1) and LDB2 (CLIM1) (Agulnick et al, 1996;Jurata et al, 1996;Sugihara et al, 1998). LDB proteins bind to transcription factors directly or indirectly mediated through LMO proteins, and then bridge a unique bipartite DNA sequence separated by about one helix turn from each other (Agulnick et al, 1996;Jurata et al, 1996;Wadman et al, 1997;Rabbitts, 1998).…”

“…LMO4 binds with a high affinity to the LIM domain-binding proteins, LDB1 (CLIM2, NLI1) and LDB2 (CLIM1) (Agulnick et al, 1996;Jurata et al, 1996;Sugihara et al, 1998). LDB proteins bind to transcription factors directly or indirectly mediated through LMO proteins, and then bridge a unique bipartite DNA sequence separated by about one helix turn from each other (Agulnick et al, 1996;Jurata et al, 1996;Wadman et al, 1997;Rabbitts, 1998). Both LMOs and LDBs are widely expressed during development (Kenny et al, 1998;Millan et al, 1998;Toyama et al, 1998;Hermanson et al, 1999) and appear to have essential functions in cell proliferation and lineage determination, and oncogenesis Milán et al, 1998;Rabbitts, 1998;Thaler et al, 2002).…”

The LMO1 and LDB1 proteins interact in human T cell acute leukaemia with the chromosomal translocation t(11;14)(p15;q11)

“…Thus far, the identity of the 46 Kd protein has not been determined because it is obscured by the similarity in its molecular weight of TAL1, which also binds LMO1 (Valge-Archer et al, 1994). A candidate for the 46 Kd protein is the recently identi®ed LIM-binding protein LDB1 or NLI (Agulnick et al, 1996;Jurata et al, 1996) (herein called LDB1). This protein has been shown to interact in vitro with LMO1 and LMO2 (Agulnick et al, 1996;Jurata et al, 1996) and in vivo LMO2 has been found in complexes with LDB1 in erythroid cells (Wadman et al, 1997) and in T cells from Lmo2-transgenic mice (Grutz et al, 1998).…”

“…A candidate for the 46 Kd protein is the recently identi®ed LIM-binding protein LDB1 or NLI (Agulnick et al, 1996;Jurata et al, 1996) (herein called LDB1). This protein has been shown to interact in vitro with LMO1 and LMO2 (Agulnick et al, 1996;Jurata et al, 1996) and in vivo LMO2 has been found in complexes with LDB1 in erythroid cells (Wadman et al, 1997) and in T cells from Lmo2-transgenic mice (Grutz et al, 1998). We have assessed the interaction of LMO1 and LDB1 in the human T cell acute leukaemia cell line RPMI8402 (which has been t(11;14)(p15;q11)) using a two-step immunoprecipitation protocol described previously (Valge-Archer et al, 1994).…”