Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart.

N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.

ZBP-89, Sp1, and nuclear factor-kappa B regulate epithelial neutrophil-activating peptide-78 gene expression in Caco-2 human colonic epithelial cells.

We reported previously that human colonic epithelial cells produce the C-X-C chemokine epithelial neutrophil-activating peptide-78 (ENA-78) and that its expression is up-regulated in ulcerative colitis. The aim of this study was to investigate the transcriptional regulation of ENA-78 gene expression in Caco-2 intestinal epithelial cells. Reporter gene transfection and electrophoretic mobility shift assay studies demonstrated that cooperation between two regions of the ENA-78 promoter were required for maximal gene expression in interleukin-1beta-stimulated Caco-2 cells. Binding of activated p50/p65 nuclear factor-kappaB to nucleotides -82 to -91 was essential for interleukin-1beta-dependent gene transcription, whereas binding of constitutively expressed zinc-requiring nuclear factors to nucleotides -125 to -134 (site A) was required for basal gene expression. Scanning mutagenesis of site A demonstrated overlapping binding elements at this locus. One site (CTCCCCC) bound Sp1 and Sp3, and overexpression of Sp1 (but not Sp3) up-regulated basal ENA-78 transcription. Another site (CCCCTCCCCC) was found to bind the zinc finger nuclear factor ZBP-89, and overexpression of this protein significantly repressed ENA-78 reporter gene activity. This study demonstrates that ENA-78 gene expression in Caco-2 intestinal epithelial cells is subject to complex regulation involving the coordinate binding of ZBP-89, Sp1, and nuclear factor-kappaB to the ENA-78 promoter.

The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1.

Vimentin is a component of the eukaryotic cytoskeleton belonging to the family of intermediate filament proteins. It exhibits a complex pattern of tissue- and development-specific expression. It is also a marker of the metastatic potential of many tumor cells. Previously, the human vimentin promoter was shown to contain several regions for the binding of positive and negative acting regulatory factors. Until now, the silencer element, which shuts down vimentin synthesis in selected tissues during development, was not precisely localized; nor was its binding protein known. In vivo DMS footprinting by ligation-mediated PCR delineated the position of guanine residues important to vimentin expression. Transient transfection assays in HeLa cells of various vimentin 5'-end promoter sequences and mutants thereof precisely defined two regulatory elements, a negative element and an adjoining positive acting element. Band shift assays, UV cross-linking, and Southwestern blot analysis confirm that the silencer element specifically binds a protein. Several lines of evidence show that ZBP-89, a zinc finger, Kruppel-like repressor protein is vimentin's silencer element binding factor. Co-immunoprecipitation and DNA affinity chromatography prove that Sp1 heterodimerizes with ZBP-89 when bound to the silencer element to yield a DNA-protein complex whose mobility is indistinguishable from that displayed by HeLa nuclear extract in band shift assays.

Terminal regions of mouse nebulin: sequence analysis and complementary localization with N-RAP.

The regions of mouse nebulin extending from the ends of the super repeats to the C-terminus and N-terminus were cloned and sequenced. Comparison of the mouse sequence with the previously published human sequence shows that the terminal regions of nebulin are highly conserved. The four phosphorylation motifs and SH3 domain found at the C-terminus of mouse nebulin are identical to those found in human nebulin, with the exception of four conservative substitutions. The modules linking this C-terminal region to the super repeats have deletions relative to both fetal and adult human nebulins that correspond to integral numbers of modules, making the mouse C-terminal simple repeat region among the shortest observed to date. The N-terminal region and the C-terminal modules were expressed in Escherichia coli and used for antibody production. Immunofluorescent labeling of these regions of nebulin in isolated myofibrils demonstrates that they are located near the center of the sarcomere and near the Z-line, respectively. Immunogold labeling with antibodies raised against the N-terminal nebulin sequence localizes this region in the A-band near the tips of the thin filaments. Nebulin localization is complementary to that of N-RAP, another muscle-specific protein containing nebulin-like super repeats; nebulin is exclusively found in the sarcomeres, while N-RAP is confined to the terminal bundles of actin filaments at the myotendinous junction. Cell Motil. Cytoskeleton 3:211-222, 2000 Published 2000 Wiley-Liss, Inc.

ZBP-99 defines a conserved family of transcription factors and regulates ornithine decarboxylase gene expression.

Among transcription factors that regulate ornithine decarboxylase (ODC) gene expression are those that interact with GC-rich promoters, including Sp1 and ZBP-89. Sp1 functions as a transactivator and ZBP-89 as a transrepressor of both the ODC and gastrin promoters. This study reports the cloning and characterization of a second member of the ZBP family that also binds GC boxes. ZBP-99 contains four Krüppel-type zinc fingers that collectively share 91% amino acid sequence similarity and 79% sequence identity with those found in ZBP-89. In addition, there are highly conserved amino acid sequences in the carboxy-terminal segments of the two genes. In spite of their structural similarities, the two proteins are encoded at distinct loci, ZBP-89 on chromosome 3q21 and ZBP-99 on 1q32.1. The predicted open reading frame of ZBP-99 cDNA encodes a 99-kDa protein. Electrophoretic mobility shift assays showed that ZBP-99 protein specifically binds to the GC-rich promoter elements of gastrin and ODC genes. Northern blot analysis showed that a major ZBP-99 transcript of 5.6 kb is expressed ubiquitously at low levels, with elevated expression levels in placenta and in adult kidney, liver, and lymphocytes. Cotransfection of AGS gastric adenocarcinoma and HT-29 colon adenocarcinoma cells with a ZBP-99 expression construct and with an ODC reporter construct show that ZBP-99 repressed basal expression in the two cell lines by 80 and 60%, respectively. Collectively, the data suggest that ZBP-99 binds GC-rich promoters and may complement the activities mediated by ZBP-89.

An integrated physical and gene map of human distal chromosome 17q24-proximal 17q25 encompassing multiple disease loci.

Genetic mapping studies suggest that a small interval on human chromosome distal 17q24-proximal 17q25 harbors genes involved in sporadic breast and ovarian tumorigenesis and in the autosomal dominant disorders hereditary neuralgic amyotrophy and tylosis with esophageal cancer. Prior to this study, isolated genomic clones and markers were assigned to this interval but integrated physical maps were not available. We improved resolution by isolating 52 additional clones and developing 24 additional markers. Genomic clones spanning distal 17q24-proximal 17q25 were organized into a contig with two gaps that encompassed 14 existing genetic markers, 8 known genes (GALR2, AANAT, ENVL, SFRS2, SEC14L, DNAH17, API4, and TK1), and 11 previously identified expressed sequence tags. This integrated map provides a foundation for identifying additional candidate genes for the disorders mapped to this interval.

Basic fibroblast growth factor does not prevent heparan sulphate proteoglycan catabolism in intact cells, but it alters the distribution of the glycosaminoglycan degradation products.

Heparan sulphate proteoglycans on cell surfaces have been shown to mediate the degradation or recycling of several ligands. Since the interaction with ligand may affect proteoglycan catabolism once the complex is internalized, this could alter the cellular pool of heparan sulphate chains, with possible consequences for heparan sulphate-mediated cellular processes. We have recently demonstrated that the specific binding of basic fibroblast growth factor (bFGF) to heparan sulphate chains prevents the glycosaminoglycan from being degraded by partially purified heparanases from Chinese hamster ovary (CHO) cells [Tumova and Bame (1997) J. Biol. Chem. 272, 9078-9085]. The present study examines the effect of bFGF on heparan sulphate catabolism in intact cells. The distribution and size of the heparan sulphate degradation products in CHO cells was analysed in the presence and absence of bFGF using pulse-chase protocols. Although heparan sulphate molecules and bFGF are internalized through the same pathway, even relatively high concentrations of the growth factor do not have any inhibitory effects on glycosaminoglycan degradation. However, the interaction with the growth factor alters the distribution of heparan sulphate-degradation products, presumably by preventing secretion of the short heparanase-derived species. Our findings show that most of the free and bFGF-bound heparan sulphate chains are destined for lysosomes, which would be consistent with a recent hypothesis that the primary role of proteoglycan-mediated internalization of the growth factor is to remove bFGF from its site of action at the cell surface. However, in the presence of bFGF, a fraction of intracellular, heparanase-degraded heparan sulphate chains is delivered to the nucleus, suggesting that the glycosaminoglycan accompanies the growth factor to the organelle. It may be important for bFGF activity that the growth factor is protected from proteolytic degradation by its interaction with heparan sulphate. This work demonstrates that the internalization of a ligand along with the proteoglycan can affect the sorting of heparan sulphate-degradation products in endosomes, and the ultimate destination of the short glycosaminoglycan. It also provides evidence that formation of heparan sulphate-ligand complexes may regulate the recycling and degradation of both ligands and heparan sulphate chains and, consequently, affect their biological activities.

Transcription factor ZBP-89 regulates the activity of the ornithine decarboxylase promoter.

Appropriate cellular levels of polyamines are required for cell growth and differentiation. Ornithine decarboxylase is a key regulatory enzyme in the biosynthesis of polyamines, and precise regulation of the expression of this enzyme is required, according to cellular growth state. A variety of mitogens increase the level of ornithine decarboxylase activity, and, in most cases, this elevation is due to increased levels of mRNA. A GC box in the proximal promoter of the ornithine decarboxylase gene is required for basal and induced transcriptional activity, and two proteins, Sp1 and NF-ODC1, bind to this region in a mutually exclusive manner. Using a yeast one-hybrid screening method, ZBP-89, a DNA-binding protein, was identified as a candidate for the protein responsible for NF-ODC1 binding activity. Three lines of evidence verified this identification; ZBP-89 copurified with NF-ODC1 binding activity, ZBP-89 antibodies specifically abolished NF-ODC1 binding to the GC box, and binding affinities of 12 different double-stranded oligonucleotides were indistinguishable between NF-ODC1, in nuclear extract, and in vitro translated ZBP-89. ZBP-89 inhibited the activation of the ornithine decarboxylase promoter by Sp1 in Schneider's Drosophila line 2, consistent with properties previously attributed to NF-ODC1.

Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome.

Ulnar-mammary syndrome is a rare pleiotropic disorder affecting limb, apocrine gland, tooth and genital development. We demonstrate that mutations in human TBX3, a member of the T-box gene family, cause ulnar-mammary syndrome in two families. Each mutation (a single nucleotide deletion and a splice-site mutation) is predicted to cause haploinsufficiency of TBX3, implying that critical levels of this transcription factor are required for morphogenesis of several organs. Limb abnormalities of ulnar-mammary syndrome involve posterior elements. Mutations in TBX5, a related and linked gene, cause anterior limb abnormalities in Holt-Oram syndrome. We suggest that during the evolution of TBX3 and TBX5 from a common ancestral gene, each has acquired specific yet complementary roles in patterning the mammalian upper limb.

Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family.

Holt-Oram syndrome is a developmental disorder affecting the heart and upper limb, the gene for which was mapped to chromosome 12 two years ago. We have now identified a gene for this disorder (HOS1). The gene (TBX5) is a member of the Brachyury (T) family corresponding to the mouse Tbx5 gene. We have identified six mutations, three in HOS families and three in sporadic HOS cases. Each of the mutations introduces a premature stop codon in the TBX5 gene product. Tissue in situ hybridization studies on human embryos from days 26 to 52 of gestation reveal expression of TBX5 in heart and limb, consistent with a role in human embryonic development.

Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation.

Epigenetic alterations in the genome of tumor cells have attracted considerable attention since the discovery of widespread alterations in DNA methylation of colorectal cancers over 10 years ago. However, the mechanism of these changes has remained obscure. el-Deiry and coworkers [el-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R. C., Falco, J. P., Hamilton, S. R. & Baylin, S. B. (1991) Proc. Natl. Acad. Sci. USA 88, 3470-3474], using a quantitative reverse transcription-PCR assay, reported 15-fold increased expression of DNA methyltransferase (MTase) in colon cancer, compared with matched normal colon mucosa, and a 200-fold increase in MTase mRNA levels compared with mucosa of unaffected patients. These authors suggested that increases in MTase mRNA levels play a direct pathogenetic role in colon carcinogenesis. To test this hypothesis, we developed a sensitive quantitative RNase protection assay of MTase, linear over three orders of magnitude. Using this assay on 12 colorectal carcinomas and matched normal mucosal specimens, we observed a 1.8- to 2.5-fold increase in MTase mRNA levels in colon carcinoma compared with levels in normal mucosa from the same patients. There was no significant difference between the normal mucosa of affected and unaffected patients. Furthermore, when the assay was normalized to histone H4 expression, a measure of S-phase-specific expression, the moderate increase in tumor MTase mRNA levels was no longer observed. These data are in contrast to the previously reported results, and they indicate that changes in MTase mRNA levels in colon cancer are nonspecific and compatible with other markers of cell proliferation.

Detailed deletion analysis of sporadic breast tumors defines an interstitial region of allelic loss on 17q25.

Whole genome analyses of breast tumors with polymorphic markers have detected nonrandom loss of heterozygosity on multiple chromosomes, providing clues to the locations of suspected tumor suppressor genes. Tumors are thought to initiate, progress, and metastasize as mutations accumulate in multiple growth-regulatory genes; thus, identification and characterization of these genes are critical to understanding and controlling breast tumorigenesis. To map more precisely a novel breast tumor suppressor gene that has been localized previously to distal 17q, we constructed a detailed deletion map of 17q25 by analyzing eight microsatellite markers on 39 sporadic primary breast tumors. The smallest overlapping region of interstitial loss was narrowed to approximately 3 cM and included D17S937/AFM107ye3, which showed the highest percentage of allelic loss (41%). These results provide a framework from which a genomic contig will be constructed and candidate transcripts will be analyzed.

FISH localization of the soluble thymidine kinase gene (TK1) to human 17q25, a region of chromosomal loss in sporadic breast tumors.

Soluble thymidine kinase (TK1) is an important 17q marker in somatic cell genetics. Its activity is increased in many malignancies, including breast cancer. Through somatic cell hybrid and fluorescence in situ hybridization studies, we mapped TK1 to 17q25.2-->25.3, in region demonstrating loss of heterozygosity in primary breast tumors. It lies near D17S836 and is proximal to the avian erythroblastic leukemia viral oncogene homolog 2-like gene (ERBA2L).

Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments.

Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on 11p15.

Identification, characterization, and localization to chromosome 17q21-22 of the human TBX2 homolog, member of a conserved developmental gene family.

The T-box motif is present in a family of genes whose structural features and expression patterns support their involvement in developmental gene regulation. Previously, sequence comparisons among the T-box domains of ten vertebrate and invertebrate T-box (Tbx) genes established a phylogenetic tree with three major branches. The Tbx2-related branch includes mouse Mm-Tbx2 and Mm-Tbx3, Drosophila optomotor-blind (Dm-Omb), and Caenorhabditis elegans Ce-Tbx2 and Ce-Tbx2 and Ce-Tbx7 genes. From the localization of Mm-Tbx2 to Chromosome (Chr) 11, we focused our search for the human homolog, Hs-TBX2, within a region of synteny conservation on Chr 17q. We used Dm-Omb polymerase chain reaction (PCR) primers to amplify a 137-basepair(bp)_ product from human genomic, Chr 17 monochromosome hybrid, and fetal kidney cDNA templates. The human PCR product showed 89% DNA sequence identity and 100% peptide sequence identity to the corresponding T-box segment of Mm-Tbx2. The putative Hs-TBX2 locus was isolated within a YAC contig that included three anonymous markers, D17S792, and D17S948, located at Chr 17q21-22. Hybridization-and PCR-based screening of a 15-week fetal kidney cDNA library yielded several TBX2 clones. Sequence analysis of clone lambda omicron TBX2-1 confirmed homology to Mm-TBx2-90% DNA sequence identity over 283 nt, and 96% peptide sequence identity over 94 amino acids. Similar analysis of Hs-TBX2 cosmid 154F11 confirmed the cDNA coding sequence and also identified a 1.7-kb intron located at the same relative position as in Mm-Tbx2. Phylogenetic analyses of the T-box domain sequences found in several vertebrate and invertebrate species further suggested that the putative human TBX2 and mouse Tbx2 are true homologs. Northern blot analysis identified two major TBX2 expression fetal kidney and lung; and in adult kidney, lung, ovary, prostate, spleen, and testis. Reduced expression levels were seen in heart, white blood cells, small intestine, and thymus. These results suggest that Hs-TBX2 could play important roles in both developmental and postnatal gene regulation.

Site and mechanics of failure in normal and dystrophin-deficient skeletal muscle.

Partial or complete tearing of skeletal muscle occurs both in acute muscle injury and in some pathological muscle conditions. Despite the impact of such tearing on normal muscle function, few studies have examined the site or mechanism of muscle injury at cellular or subcellular levels of organization. The present study determines the ultrastructural location and mechanical conditions of tensile failure in normal mouse extensor digitorum longus muscles. All of these muscles failed near a myotendinous junction (MTJ), but within the muscle fibers, in a transverse plane coincident with the edge of an A-band. The breaking stress averaged 5.71 x 10(5) N/m2 for muscles stimulated tetanically during lengthening, and 5.01 x 10(5) N/m2 for unstimulated muscles. Breaking strain averaged 135%, and showed no dependence on the state of activation of the muscle. The energy absorbed by each muscle averaged 362.5 mJ/g in unstimulated muscle, and 613.2 mJ/g in the stimulated samples. In addition to the failure properties of normal muscle, the effect of dystrophin deficiency on the site and conditions of failure was determined using muscle from mdx mice, which lack dystrophin. The absence of dystrophin had no detectable effect on the stress, strain, or energy absorbed, regardless of the state of muscle activation. Unstimulated mdx muscle failed in the plane of an A-band, but tetanic stimulation produced failure in the reticular lamina of the tendon, just external to the MTJ, in 75% of the fibers in the mdx muscles. Although dystrophin's cytoplasmic location makes it unlikely that this unique mode of failure is due directly to the absence of the protein, failure in the reticular lamina may result from a difference in load distribution that accompanies the response of mdx muscle to dystrophin deficiency.

Modifications in myotendinous junction surface morphology in dystrophin-deficient mouse muscle.

Single muscle fibers from mdx mouse muscle, which is deficient in dystrophin, and control mouse muscle, containing dystrophin, were compared by scanning electron microscopy. In particular, comparisons were made of the surface morphology at myotendinous junctions and costameres, sites at the muscle cell surface that are enriched in dystrophin and where force is transmitted across the cell membrane. Muscle fibers from 4- and 6-week-old controls display nearly uniform surface morphology characterized by numerous digit-like processes at the myotendinous junction and nonjunctional surface membrane possessing distinct grooves at sites corresponding to underlying costameres. Mdx fibers at this stage showed blunted myotendinous junctions with few digit-like processes, infrequent indistinct costameric markings, and holes in the cell membrane. Cells from peak regenerating mdx muscle (6 weeks) showed surface morphology similar to 4-week mdx fibers, although the proportion of fibers displaying extensive structural defects was reduced at 6 weeks. Completely regenerated mdx fibers (23 weeks) were indistinguishable from fibers of 6-week-old mdx mice. In control mice, only approximately 6% of the fibers examined from 4- or 6-week-old mice showed any of the structural defects characteristic of the majority of mdx fibers. However, fibers from 23-week-old control mice displayed an increased frequency of cells with poorly defined junctional processes and surface striations. These findings indicate that the fibers displaying extensive disruption of surface features, which are most commonly observed in 4-week mdx mice at peak necrosis, are necrotic fibers. Specific defects, such as the reduction in myotendinous junction folding, loss of costameres, and increased occurrence of membrane holes, are observed in the majority of mdx fibers at all ages. Thus, these defects are more directly attributable to dystrophin's absence because their frequency of occurrence is independent of the stage of necrosis and regeneration.