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...

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

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...

PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein ␣-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal ␤-TM). The interaction between Enigma and skeletal ␤-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal ␤-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal ␤-TM in transfected cells. The association of Enigma with skeletal ␤-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells. INTRODUCTIONConserved protein interaction domains found in many proteins with diverse functions provide molecular recognition essential for assembling multiprotein complexes (Pawson and Scott, 1997). LIM and PDZ domains are two protein interaction motifs that are widely distributed in cells of both plants and animals (Gill, 1995;Fanning and Anderson, 1996). LIM domains, named for the three homeodomain proteins in which they were first recognized (lin-11, isl-1, and mec-3) (Jurata and Gill, 1998) are cysteine-rich modules that contain two coordinated Zn 2ϩ atoms (PerezAlverado et al., 1994). Nuclear LIM domains interact with nuclear LIM interactor (Agulnick et al., 1996, Jurata et al., 1996 and transcription factors (Wadman et al., 1994), whereas cytoplasmic LIM domains bind to protein kinases (Wu and Gill, 1994;Kuroda et al., 1996;Wu et al., 1996), other LIM domains (Schmeichel and Beckerle, 1994), and cytoskeletal targets (Crawford and Bekerle, 1992;Arber and Caroni, 1996;Pomies et al., 1997). PDZ domains, named for the three proteins in which they were first recognized (postsynaptic density-95, discs large, and zo1 tight junction protein) (Fanning and Anderson, 1996), are ϳ85 amino acid ␤-barrel structures (Doyle et al., 1996) that bind to the consensus sequence (Ser/Thr)-X-(Val/Leu/Ile) contained in targets, most commonly at the carboxyl terminus (Kim et al., 1995;Kornau et al., 1995;Songyang et al., 1997). PDZ domains are also reported to recognize internal consensus sites (Shieh and Zhu, 1996), other PDZ domains , spectrinlike repeats (Xia et al., 1997), LIM domains (Cuppen et al., 1998), and unspecified sites (Tsunoda et al., 1997). Several PDZ ...

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