LIM homeodomain codes regulate the development of many cell types, though it is poorly understood how these factors control gene expression in a cell-specific manner. Lhx3 is involved in the generation of two adjacent, but distinct, cell types for locomotion, motor neurons and V2 interneurons. Using in vivo function and protein interaction assays, we found that Lhx3 binds directly to the LIM cofactor NLI to trigger V2 interneuron differentiation. In motor neurons, however, Isl1 is available to compete for binding to NLI, displacing Lhx3 to a high-affinity binding site on the C-terminal region of Isl1 and thereby transforming Lhx3 from an interneuron-promoting factor to a motor neuron-promoting factor. This switching mechanism enables specific LIM complexes to form in each cell type and ensures that neuronal fates are tightly segregated.

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Electroconvulsive therapy (ECT) remains the treatment of choice for drug-resistant patients with depressive disorders, yet the mechanism for its efficacy remains unknown. Gene transcription changes were measured in the frontal cortex and hippocampus of rats subjected to sham seizures or to 1 or 10 electroconvulsive seizures (ECS), a model of ECT. Among the 3500 -4400 RNA sequences detected in each sample, ECS increased by 1.5-to 11-fold or decreased by at least 34% the expression of 120 unique genes. The hippocampus produced more than three times the number of gene changes seen in the cortex, and many hippocampal gene changes persisted with chronic ECS, unlike in the cortex. Among the 120 genes, 77 have not been reported in previous studies of ECS or seizure responses, and 39 were confirmed among 59 studied by quantitative real time PCR. Another 19 genes, 10 previously unreported, changed by Ͻ1.5-fold but with very high significance. Multiple genes were identified within distinct pathways, including the BDNF-MAP kinase-cAMP-cAMP response element-binding protein pathway (15 genes), the arachidonic acid pathway (5 genes), and more than 10 genes in each of the immediate-early gene, neurogenesis, and exercise response gene groups. Neurogenesis, neurite outgrowth, and neuronal plasticity associated with BDNF, glutamate, and cAMP-protein kinase A signaling pathways may mediate the antidepressant effects of ECT in humans. These genes, and others that increase only with chronic ECS such as neuropeptide Y and thyrotropin-releasing hormone, may provide novel ways to select drugs for the treatment of depression and mimic the rapid effectiveness of ECT.

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...

LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn 2؉binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified, widely expressed protein, NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. In this study, a 38-amino-acid fragment of NLI was found to be sufficient for the association of NLI with nuclear LIM domains. In addition, NLI was shown to form high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI was not required for association with the LIM homeodomain protein Lmx1. Chemical crosslinking analysis revealed higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. Additionally, NLI formed complexes with Lmx1 on the rat insulin I promoter and inhibited the LIM domain-dependent synergistic transcriptional activation by Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I minienhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses which require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions.Cell fate decisions made throughout development result in diverse, differentiated cell types capable of unique functions. One class of transcription factors known to influence the developmental programs of undifferentiated precursor cells consists of the LIM domain-containing transcription factors (for reviews, see references 13 and 20). Some nuclear LIM domain proteins contain a DNA binding homeodomain; others, known as LIM-only proteins (LMO), lack an obvious DNA binding element but associate with DNA binding factors (37, 51, 52). LIM transcription factors are required for head formation (44), neurogenesis (39, 55), pituitary development (46), erythropoiesis (54), pancreas development (2), and the determination of other cell types (9,12,14,15). However, few gene targets and transcriptional mechanisms have been identified which account for the phenotypes resulting from targeted deletion of these genes.The basic helix-loop-helix (bHLH) proteins constitute a second class of transcription factors involved in cellular differentiation (33). bHLH proteins are involved in muscle formation (36), B-cell development (6, 59), neurogenesis (7, 10, 22, 31), hematopoiesis (40, 42, 47), and myeloid differentiation (27), and they appear to function biologically by directing precursor cells to exit the cell cycle (38) and to activate cell-type-specific genes. In general, cell-type-restricted bHLH factors (B class) require heterodimerization with t...

The underlying transcriptional mechanisms that establish the proper spatial and temporal pattern of gene expression required for specifying neuronal fate are poorly defined. We have characterized how the Hb9 gene is expressed in developing motoneurons in order to understand how transcription is directed to specific cells within the developing CNS. We found that non-specific general-activator proteins such as E2F and Sp1 are capable of driving widespread low level transcription of Hb9 in many cell types throughout the neural tube; however, their activity is modulated by specific repressor and activator complexes. The general-activators of Hb9 are suppressed from triggering inappropriate transcription by repressor proteins Irx3 and Nkx2.2. High level motoneuron expression is achieved by assembling an enhancesome on a compact evolutionarily-conserved segment of Hb9located from –7096 to –6896. The ensemble of LIM-HD and bHLH proteins that interact with this enhancer change as motoneuron development progresses, facilitating both the activation and maintenance of Hb9expression in developing and mature motoneurons. These findings provide direct support for the derepression model of gene regulation and cell fate specification in the neural tube, as well as establishing a role for enhancers in targeting gene expression to a single neuronal subtype in the spinal cord.

LIM homeodomain (LIM-HD) proteins play key roles in a variety of developmental processes throughout the animal kingdom. Here we show that the LIM-binding protein Chip acts as a cofactor for the Drosophila LIM-HD family member Apterous (Ap) in wing development. We define the domains of Chip required for LIM-HD binding and for homodimerization and show that mutant proteins deleted for these domains act in a dominant-negative fashion to disrupt Ap function. Our results support a model for multimeric complexes containing Chip and Ap in transcriptional regulation. This model is confirmed by the activity of a chimeric fusion between Chip and Ap that reconstitutes the complex and rescues the ap mutant phenotype.

LMO4 is a novel member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors. LMO1, LMO2, and LMO4 have distinct expression patterns in adult tissue, and we demonstrate that nuclear retention of LMO proteins is enhanced by the nuclear LIM interactor (NLI). In situ hybridization to early mouse embryos of 8-14.5 days revealed a complex pattern of LMO4 expression spatially overlapping with NLI and LHX genes. LMO4 expression in somite is repressed in mice mutant for the segment polarity gene Mesp2 and expanded in Splotch mutants. During jaw and limb outgrowth, LMO4 and LMO2 expression define mesenchyme that is uncommitted to regional fates. Although both LMO2 and LMO4 are activated in thymic blast cells, only LMO4 is expressed in mature T cells. Mesenchymal and thymic blast cell expression patterns of LMO4 and LMO2 are consistent with the suggestion that LMO genes inhibit differentiation.The LIM domain, an approximately 55-residue, cysteine-rich zinc-binding motif, is present in a variety of proteins including LIM homeobox (LHX) proteins that contain two LIM domains and one homeodomain. LHX genes are expressed in many types of neurons and other cell types, and deletion of LHX genes results in the loss of cell fate (1). Mice mutant for LHX1 have diminished organizer activity that results in lack of head structures anterior to rhombomere 3 (2). In the central nervous system, development of forebrain and pituitary derivatives are defective in mice mutant for LHX2, LHX3, or LHX4 (1), while activation of the LHX gene Isl1 is essential for the survival of motor neurons and neighboring interneurons (3).LMO2 represents a family of nuclear LIM-only (LMO) proteins that lack a DNA-binding homeodomain (4, 5). Unregulated LMO2 expression induces T cell tumors (6), while deletion blocks hematopoietic development (7,8). The mechanism of LMO2 activity is thought to be the LIM domaindependent assembly of transcription complexes and transcription regulation (9).LIM domains of nuclear proteins bind with high affinity to the widely expressed nuclear LIM interactor (NLI) and with lesser affinity to other transcription factors (10-12). Dimeric NLI supports assembly of heteromeric complexes of LIM proteins (13), and CHIP, the Drosophila ortholog of NLI, mediates enhancer-promoter interactions of the cut and ultrabithorax genes, presumably by complex formation with transcription factors (14).To identify novel LIM domain transcription factors, we screened two mouse embryonic expression libraries by using the LIM interaction domain (LID) of NLI. We report the isolation and characterization of LMO4, a novel LIM-only gene, which is highly expressed in the T lymphocyte lineage, cranial neural crest cells, somite, dorsal limb bud mesenchyme, motor neurons, and Schwann cell progenitors. Somitic expression of LMO4 is repressed in mice mutant for the segment polarity gene Mesp2. LMO4 and LMO2 expression in the jaw, limb, and thymus defines cells that are uncommitted to cell fates. Interaction with NLI mediates the n...