Petra Kioschis

Dickkopf genes are co-ordinately expressed in mesodermal lineages.

Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.

Regulation of DMBT1 via NOD2 and TLR4 in Intestinal Epithelial Cells Modulates Bacterial Recognition and Invasion

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-α, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-κB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn’s disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-κB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn’s disease.

Comparative sequence analysis of the MECP2-locus in human and mouse reveals new transcribed regions

Abstract. Comparative sequence analysis facilitates the identification of evolutionarily conserved regions, that is, gene-regulatory elements, which can not be detected by analyzing one species only. Sequencing of a 152-kb region on human Chromosome (Chr) Xq28 and of the synthenic 123 kb on mouse Chr XC identified the MECP2/Mecp2 locus, which is flanked by the gene coding for Interleukin-1 receptor associated kinase (IRAK/Il1rak) and the red opsin gene (RCP/Rsvp). By comparative sequence analysis, we identified a previously unknown, non-coding 5′ exon embedded in a CpG island associated with MECP2/Mecp2. Thus, the MECP2/Mecp2 gene is comprised of four exons instead of three. Furthermore, sequence comparison 3′ to the previously reported polyadenylation signal revealed a highly conserved region of 8.5 kb terminating in an alternative polyadenylation signal. Northern blot analysis verified the existence of two main transcripts of 1.9 kb and ∼10 kb, respectively. Both transcripts exhibit tissue-specific expression patterns and have almost identical short half-lifes. The ∼10-kb transcript corresponds to a giant 3′ UTR contained in the fourth exon of MECP2. The long 3′ UTR and the newly identified first intron of MECP2/Mecp2 are highly conserved in human and mouse. Furthermore, the human MECP2 locus is heterogeneous with respect to its DNA composition. We postulate that it represents a boundary between two H3 isochores that has not been observed previously.

The Human Glycine Receptor Subunit α3 GLRA3 GENE STRUCTURE, CHROMOSOMAL LOCALIZATION, AND FUNCTIONAL CHARACTERIZATION OF ALTERNATIVE TRANSCRIPTS

The neuronal glycine receptor is a ligand-gated chloride channel composed of ligand binding α and structural β polypeptides. Homology screening of a human fetal brain cDNA library resulted in the identification of two alternative splice variants of the glycine receptor α3 subunit. The amino acid sequence predicted for the α3L variant was largely identical to the corresponding rat subunit. In contrast, the novel splice variant α3K lacked the coding sequence for 15 amino acids located within the cytoplasmic loop connecting transmembrane spanning region 3 (TM3) and TM4. Using P1 artificial chromosome (PAC) clones, the structure of theGLRA3 gene was elucidated and its locus assigned to human chromosomal bands 4q33-q34 by fluorescence in situhybridization. Two transcripts of 2.4 and 9 kilobases, corresponding to α3L and α3K, respectively, were identified and found to be widely distributed throughout the human central nervous system. Structural analysis of the GLRA3 gene revealed that the α3K transcript resulted from a complex splice event where excision of the novel exon 8A comprising the alternative sequence of 45 base pairs coincides with the persistence of a large intronic sequence in the 3′-untranslated region. Functional expression in HEK 293 cells of α3L and α3K subunits resulted in the formation of glycine-gated chloride channels that differed significantly in desensitization behavior, thus defining the cytoplasmic loop as an important determinant of channel inactivation kinetics.

The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability

Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3 – q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.

Identification of Tctex2β, a Novel Dynein Light Chain Family Member That Interacts with Different Transforming Growth Factor-β Receptors

Endoglin is a membrane-inserted protein that is preferentially synthesized in angiogenic vascular endothelial and smooth muscle cells. Endoglin associates with members of the transforming growth factor-β (TGF-β) receptor family and has been identified as the gene involved in hereditary hemorrhagic telangiectasia. Although endoglin is known to affect cell responses to TGF-β, its mode of action is largely unknown. We performed yeast two-hybrid screening of a human placental cDNA library and isolated a new endoglin-binding partner, a novel 221-amino acid member of the Tctex1/2 family of cytoplasmic dynein light chains named Tctex2β, as the founder of a new Tctex1/2 subfamily. The interaction was localized exclusively to the cytoplasmic domain of endoglin. Reverse transcription-PCR showed expression of Tctex2β in a wide range of tissues, including vascular endothelial and smooth muscle cells, placenta, and testis, as well as in several tumor cell lines. High expression levels were found in human umbilical vein endothelial cells and the large cell lung cancer cell line. Forced expression of Tctex2β had a profound inhibitory effect on TGF-β signaling. Additional Tctex2β-interacting receptors were identified to be the TGF-β type II receptor and most likely beta-glycan, but not ALK5, ALK1, or the bone morphogenetic protein type II receptor. Upon fluorescence tagging, co-localization of Tctex2β and endoglin, as well as Tctex2β, endoglin, and the TGF-β type II receptor, was observed by different microscopy techniques. Our findings link endoglin for the first time to microtubule-based minus end-directed transport machinery, suggesting that some endoglin functions might be regulated and directed by its interaction with the cytoplasmic dynein light chain Tctex2β.

DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands.

Deleted in malignant brain tumors 1 (DMBT1) is a secreted glycoprotein displaying a broad bacterial‐binding spectrum. Recent functional and genetic studies linked DMBT1 to the suppression of LPS‐induced TLR4‐mediated NF‐κB activation and to the pathogenesis of Crohns disease. Here, we aimed at unraveling the molecular basis of its function in mucosal protection and of its broad pathogen‐binding specificity. We report that DMBT1 directly interacts with dextran sulfate sodium (DSS) and carrageenan, a structurally similar sulfated polysaccharide, which is used as a texturizer and thickener in human dietary products. However, binding of DMBT1 does not reduce the cytotoxic effects of these agents to intestinal epithelial cells in vitro. DSS and carrageenan compete for DMBT1‐mediated bacterial aggregation via interaction with its bacterial‐recognition motif. Competition and ELISA studies identify poly‐sulfated and poly‐phosphorylated structures as ligands for this recognition motif, such as heparansulfate, LPS, and lipoteichoic acid. Dose–response studies in Dmbt1−/− and Dmbt1+/+ mice utilizing the DSS‐induced colitis model demonstrate a differential response only to low but not to high DSS doses. We propose that DMBT1 functions as pattern‐recognition molecule for poly‐sulfated and poly‐phosphorylated ligands providing a molecular basis for its broad bacterial‐binding specificity and its inhibitory effects on LPS‐induced TLR4‐mediated NF‐κB activation.

X-linked myotubular myopathy: refinement of the gene to a 280-kb region with new and highly informative microsatellite markers

We have recently refined the localization of the myotubular myopathy (MTM1) gene to a 430-kb region between DXS304 and DXS1345 in proximal Xq28. We report two new polymorphic microsatellite markers, DXS8377 and DXS7423, that were physically mapped within the critical interval. A recombination event in a family segregating for MTM1 placed the disease gene telomeric to the trinucleotide polymorphism DXS8377. Together with the recent mapping of two microdeletions associated with MTM1, the recombination refines the critical region to 280 kb. A second recombination event was observed distal to the tetranucleotide repeat DXS7423. This recombination has occurred in the offspring of a female with a more than 67% probability of being a carrier and very likely restricts the MTM1 gene to a 130-kb region. This physical refinement is significant for positional cloning of the disease gene. The highly polymorphic markers and the precise localization of the MTM1 gene will facilitate genetic diagnosis of the disorder.

Assignment of CASP8 to human chromosome band 2q33-->q34 and Casp8 to the murine syntenic region on chromosome 1B-proximal C by in situ hybridization.

Supported by the Resource Center/Primary Database of the German Human Genome Initiative (BMB+F). We thank Dr. Hans Lehrach for collaboration and the Reference Library, ICRF as source of the genomic clones, Ute Satorius for fruitful discussions, Katrin Krieger and Christiane Rutenberg for excellent technical support, and Marcus Peter and Ruthild Weber for critically reading the manuscript. F.C.K. and P.K. contributed equally to this work.

Identification of tissue-specific expressed sequences in human band Xq28 with complex pig cDNA probes

As a part of the functional analysis of the region from the position of the fragile X mutation to the telomere of the long arm of the human X Chromosome (Chr), we have developed a number of different approaches to identify genes located in this area. We describe here a procedure allowing the rapid identification of expressed sequences based on the hybridization of radioactively labeled complex cDNA probes derived from different pig and human tissues to cosmid clones gridded onto nylon filters and to restriction fragments of these clones. This technique has allowed the identification of a number of differentially expressed sequences in cosmid clones covering most of the Xq27.3 to Xqter region. Using these sequences as hybridization probes, cDNA clones for new genes expressed in a tissue-specific manner were isolated. Applied to genomic regions defined by overlapping cosmid clones, this method will serve as a major component in our strategy to establish integrated physical and transcription maps.