Expression profiling of Wnt family of genes in normal and inflammatory bowel disease primary human intestinal myofibroblasts and normal human colonic crypt epithelial cells.

BACKGROUND: Wnt signaling regulates intestinal epithelial stem cell function. Wnt ligands bind Frizzled (Fz) receptors and low-density lipoprotein-receptor-related protein (LRP) 5 and 6. Secreted Frizzled-related protein (SFRP) and Dickkopf families inhibit Wnt signaling. Our aim was to study expression of Wnt family of genes in isolated intestinal myofibroblasts and crypt epithelial cells. METHODS: Myofibroblasts were isolated from normal colonic and small intestinal mucosal samples and those affected by ulcerative colitis (UC) and Crohn's disease. Expression of the Wnt family of genes was studied by polymerase chain reaction (PCR) array. Epithelial proliferation was studied using IEC-6 cells. RESULTS: Most of the myofibroblast isolates expressed Wnt2, Wnt5A, Wnt5B, Fzd1, Fzd2, Fzd4, Fzd6, Fzd7, Fzd8, LRP6, Dickkopf1, and SFRP1. Compared to myofibroblasts isolated from normal colonic mucosal samples, real-time reverse transcription-PCR studies (using additional isolates) showed significantly reduced expression of SFRP1 in UC myofibroblasts (3.34-fold reduction, P < 0.01). Recombinant SFRP1 inhibited proliferation of IEC-6 epithelial cells. In colonic crypt epithelial cells, expression of Wnt ligands and their inhibitors was generally either absent or very weak. By contrast, all the crypt epithelial preparations expressed Fzd1, Fzd5, Fzd7, Fzd8, and LRP6. CONCLUSIONS: Human intestinal myofibroblasts expressed a number of Wnt ligands, their receptors, and inhibitors. In contrast, colonic crypt epithelial cells predominantly expressed Wnt receptors. Compared to myofibroblasts isolated from normal colonic mucosa, those affected by UC showed significantly reduced expression of SFRP1. Since reduced SFRP1 expression has been associated with malignancy, low myofibroblast expression of this Wnt inhibitor may be implicated in increased risk of cancer in UC.

Cre-mediated transgene activation in the developing and adult mouse brain.

The neuron-specific rat enolase (NSE) promoter was employed to establish transgenic mice expressing Cre recombinase in the central nervous system. Founders were crossed with dormant lacZ indicator mice and specificity as well as efficiency of Cre-mediated transgene activation was determined by PCR and/or X-gal staining. Whereas most transgenic lines exhibited Cre activity in early development resulting in widespread Cre activity, one line (NSE-Cre26) expressed high levels of Cre in the developing and adult brain. With the exception of kidney, which showed occasionally low level of Cre activity, Cre recombination in double transgenics was restricted to the nervous system. Whole-mount X-gal staining of 9.5 dpc embryos indicated Cre-mediated lacZ expression in forebrain, hindbrain, and along the midbrain flexure. A similar expression pattern was observed during later stages of embryogenesis (11.5-13.5 dpc). In adult mice, Cre recombinase was expressed in cerebral cortex and cerebellum and high levels of Cre-mediated lacZ expression were observed in hippocampus, cortex, and septum. The NSE-Cre26 transgenic mouse line thus provides a useful tool to specifically overexpress and/or inactivate genes in the developing and adult brain.

Def-2, -3, -6 and -8, novel mouse genes differentially expressed in the haemopoietic system.

To identify developmentally regulated genes during myeloid differentiation, a self-inactivating retroviral gene-trap vector carrying a beta-galactosidase-neomycin (SA/lacZ/neo) fusion gene was constructed and used to infect myeloid progenitor cells (FDCP-Mix A4). G418-resistant and beta-galactosidase positive cell lines (gene-trap integration [GTI] clones) were established and induced to differentiate in vitro into either macrophages or granulocytes. Expression of the trapped loci was monitored at a single-cell level by analysing the mature cell types for beta-galactosidase activity. All 37 GTI clones tested showed down-regulation either during granulocyte or both granulocytic and macrophage differentiation. The endogenous coding regions fused to the SA/lacZ/neo reporter gene were isolated from eight clones. Molecular analysis revealed that half of them represented novel mouse genes (def-2, -3, -6 and -8) which we confirmed to be differentially expressed in primary haemopoietic tissues. Database searches revealed no significant similarities for def-2 (associated with haemopoietic progenitors) and def-8 (expressed most strongly in peripheral leucocytes). Def-6, which is down-regulated upon the differentiation into myeloid as well as erythroid lineages, was found to be closely related but not identical with the recently described B-cell-specific switch recombinase SWAP-70. Def-3, which is down-regulated upon differentiation into granulocytes but expressed in progenitor cells and macrophages, defines a novel family of RNA binding proteins.

DEF-3(g16/NY-LU-12), an RNA binding protein from the 3p21.3 homozygous deletion region in SCLC.

DEF-3(g16/NY-LU-12) encodes a novel RNA binding protein isolated by positional cloning from an SCLC homozygous deletion region in 3p21.3 and, in parallel, as a differentially expressed gene during myelopoiesis from FDCPmix-A4 cells. DEF-3(g16/NY-LU-12) is ubiquitously expressed during mouse embryogenesis and in adult organs while human hematopoietic tissues showed differential expression. The mouse and human proteins are highly conserved containing two RNA recognition motifs (RRMs) and other domains associated with RNA binding and protein-protein interactions. A database search identified related proteins in human, rat, C. elegans and S. pombe including the 3p21.3 co-deleted gene, LUCA15. Recombinant proteins containing the RRMs of DEF-3(g16/NY-LU-12) and LUCA15 specifically bound poly(G) RNA homopolymers in vitro. These RRMs also show similarity to those of the Hu protein family. Since anti-Hu RRM domain antibodies are associated with an anti-tumor effect and paraneoplastic encephalomyelitis, we tested sera from Hu syndrome patients with the RRMs of DEF-3(g16/NY-LU-12) and LUCA15. These were non-reactive. Thus, DEF-3(g16/NY-LU-12) and LUCA15 represent members of a novel family of RNA binding proteins with similar expression patterns and in vitro RNA binding characteristics. They are co-deleted in some lung cancers and immunologically distinct from the Hu proteins.

Structure, chromosomal localisation and expression of the murine dominant negative helix-loop-helix Id4 gene.

Id proteins antagonise the functional properties of DNA-binding, basic helix-loop-helix transcription factors. Id proteins inhibited cell differentiation in various model systems, both in vitro and in vivo. They are transcriptionally and post-transcriptionally regulated during cell cycle progression and promote cell proliferation. In order to establish the molecular and functional properties of Id4, we analysed structure, chromosomal localisation and expression of the murine Id4 gene. Sequence analysis indicated that the Id4 gene consists of three exons. Multiple transcription start sites map about 300 bp upstream of the ATG translational start codon within a 30-bp region of the Id4 promoter, which lacks a classic TATA box. Expression of the Id4 gene results in four major transcripts, most likely generated by differential use of polyadenylation sites. Abundance of the four transcripts varies across tissues, suggesting tissue-specific regulation of polyadenylation and/or post-transcriptional regulation of Id4 expression. However, the Id4 gene seems to be expressed as a single protein. Id4 expression is switched on during embryogenesis between day 7.5 and 9.5 of gestation and is most abundant in adult brain, kidney and testis. Id4 maps to chromosome 13 of the mouse.

Stage- and subcellular-specific expression of Id proteins in male germ and Sertoli cells implicates distinctive regulatory roles for Id proteins during meiosis, spermatogenesis, and Sertoli cell function.

Immunohistological detection of each of the four Id proteins (Id1-Id4) in sections of mouse testis revealed a unique temporal and spatial expression pattern for each Id protein during spermatogenesis. Furthermore, each Id protein displayed a distinctive, dynamic pattern of subcellular distribution. Id1 was uniquely expressed in MI/MII spermatocytes undergoing meiotic division. Id4 protein was detectable in the cytoplasm of type A1 spermatogonia, as well as in late pachytene and in diplotene spermatocytes. Id2 protein, which was most abundant in Sertoli cell nuclei, was also detectable in pachytene and diplotene spermatocytes, but as with Id4, it was absent from MI/MII cells. In postmeiotic spermatids, Id1, Id2, and Id4 proteins were expressed in a stage- and subcellular-specific manner. Expression of Id3 was restricted to Sertoli cell cytoplasm. In malignant seminoma cells, all four Id proteins were abundantly expressed with accompanying changes in their subcellular distribution. The observed expression of Id proteins in postproliferative Sertoli cells and spermatids and during specific stages of meiosis implies novel functional roles for this class of transcriptional regulator during spermatogenesis.

Id helix-loop-helix proteins in cell growth and differentiation.

Id helix-loop-helix proteins function at a general level as positive regulators of cell growth and as negative regulators of cell differentiation. They act as dominant-negative antagonists of other helix-loop-helix transcription factors, which drive cell lineage commitment and differentiation in diverse cell types of higher eukaryotes. In addition, the functions of Id proteins are integrated with cell-cycle-regulatory pathways orchestrated by cyclin-dependent kinases and the retinoblastoma protein. Here, some of the recent advances that highlight the importance of Id proteins as regulatory intermediates for coordinating differentiation-linked gene expression with cell-cycle control in response to extracellular signalling are reviewed.

Deletion of the IgH intronic enhancer and associated matrix-attachment regions decreases, but does not abolish, class switching at the mu locus.

The IgH locus intronic enhancer (Emu), located in the intron between the J(H) segments and the Cmu gene, and flanked by two matrix attachment regions (MAR), has been shown to be a major regulator of IgH gene transcription and VDJ recombination. To define the potential role of Emu plus MAR in class switch recombination (CSR), we generated IgG-expressing hybridomas from B cells heterozygous for mutations that delete all of these elements or replace them with a neo(r) gene and analyzed the switch status of the mutated IgH loci. Emu/MAR-deleted IgH loci displayed a highly significant, although not complete, decrease in CSR when compared to unmutated loci in normal hybridomas. Surprisingly, mutant loci with a pgk promoter-driven neo(r) gene replacing the Emu/MAR showed relatively normal switch frequency. These findings indicate that the Emu/MAR region plays a significant, but not necessary role in facilitating class switching at the mu locus. Potential mechanisms for these findings are discussed.

Cloning and targeted deletion of the mouse fetuin gene.

We proposed that the alpha2-Heremans Schmid glycoprotein/fetuin family of serum proteins inhibits unwanted mineralization. To test this hypothesis in animals, we cloned the mouse fetuin gene and generated mice lacking fetuin. The gene consists of seven exons and six introns. The cystatin-like domains D1 and D2 of mouse fetuin are encoded by three exons each, whereas a single terminal exon encodes the carboxyl-terminal domain D3. The promoter structure is well conserved between rat and mouse fetuin genes within the regions shown to bind transcription factors in the rat system. Expression studies demonstrated that mice homozygous for the gene deletion lacked fetuin protein and that mice heterozygous for the null mutation produced roughly half the amount of fetuin protein produced by wild-type mice. Fetuin-deficient mice were fertile and showed no gross anatomical abnormalities. However, the serum inhibition of apatite formation was compromised in these mice as well as in heterozygotes. In addition, some homozygous fetuin-deficient female ex-breeders developed ectopic microcalcifications in soft tissues. These results corroborate a role for fetuin in serum calcium homeostasis. The fact that generalized ectopic calcification did not occur in fetuin-deficient mice proves that additional inhibitors of phase separation exist in serum.

Inducible site-directed recombination in mouse embryonic stem cells.

The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly, the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins, tamoxifen can efficiently induce Cre-mediated recombination, thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen, recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein, the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals.

Mutually exclusive expression of two dominant-negative helix-loop-helix (dnHLH) genes, Id4 and Id3, in the developing brain of the mouse suggests distinct regulatory roles of these dnHLH proteins during cellular proliferation and differentiation of the nervous system.

The dominant-negative helix-loop-helix (dnHLH) proteins Id1 and Id2 have been implicated in the regulation of cell proliferation and differentiation in myogenesis, neurogenesis, and/or hematopoiesis. To further investigate the functional role of dnHLH proteins, we have performed in situ hybridization analysis on serial sections of mouse embryos from days 9.5 to 17.5 postcoitus to establish the spatial and temporal expression patterns of Id3 (HLH462) and Id4, a recently isolated fourth member of the mammalian dnHLH gene family. Id3 transcripts are present throughout embryogenesis and are found in neural cells as well as in cartilage primordia and in epithelial cells lining a variety of organs. The spatial expression pattern of Id3 overlaps considerably with the previously determined pattern of Id1. Id4 expression, which is up-regulated during embryogenesis, is restricted to specific cells of the central and peripheral nervous system. Within the detection limits of in situ hybridization, Id4 and Id3 expression is mutually exclusive in neural precursor cells of the developing brain, suggesting distinct regulatory functions for these dnHLH proteins during neurogenesis.

A homologue of the human MSS1 gene, a positive modulator of HIV-1 gene expression, is massively expressed in Xenopus oocytes.

Here the nucleotide sequence of a Xenopus homologue of the human MSS1 gene, a positive modulator of the HIV-1 Tat mediated transactivation in mammalian cells, is presented. This gene is highly conserved and almost exclusively expressed in Xenopus oocytes. We speculate about a possible role of this gene in the HIV-1 Tat/TAR mediated transactivation in Xenopus oocytes.

The expression pattern of Id4, a novel dominant negative helix-loop-helix protein, is distinct from Id1, Id2 and Id3.

Molecular interaction between transcription factors containing an basic-helix-loop-helix (bHLH) domain is known to regulate differentiation in several cellular systems including myogenesis, neurogenesis and haematopoiesis. DNA-binding activity of the bHLH proteins is mediated via the basic region and is dependent upon formation of homo- and/or heterodimers of these transcription factors. Dominant negative (dn) HLH proteins (Id1, Id2, Id3 and emc) also contain the HLH-dimerization domain but lack the DNA-binding basic region. Formation of heterodimers between dnHLH and bHLH proteins abolishes the DNA-binding activity of the latter. Concordantly, it was shown that the dnHLH protein Id1 inhibits differentiation of muscle and myeloid cells in vitro. Therefore, it was postulated that dnHLH proteins serve as general antagonists of cell differentiation. We have isolated and characterized a novel mouse dnHLH gene, designated Id4. The Id4 protein contains a HLH domain highly conserved among the dnHLH proteins from mouse and drosophila. Outside of the HLH domain, three additional short regions of the dnHLH proteins show some degree of homology. DNA-binding of E47 homo- as well as E47/MyoD heterodimers is inhibited by Id4. Transcription of the Id4 gene results in three RNA molecules of 3.7, 2.0 and 1.7 kb which are presumably a result of differential splicing and/or alternatively used polyadenylation sites within the 3' untranslated region. During embryogenesis, Id4 expression is up-regulated between day 9.5 and 13.5 of gestation. The highest expression in adult tissues was detected in testis, brain and kidney. Comparison of the expression patterns of the four mouse dnHLH genes revealed that Id4 expression differs from the more restricted expression of Id2 as well as from the widespread expression of Id1 and Id3.

V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer.

We have assessed the importance of the immunoglobulin heavy chain (IgH) intron enhancer for recombination of variable gene segments (V, D and J) during B cell development. We generated chimeric mice with embryonic stem cells lacking the intron enhancer from one of their IgH loci. The IgH intron enhancer was substituted by a short oligonucleotide through homologous recombination using the 'Hit and Run' procedure. V(D)J recombination occurred less frequently on mutant alleles, but was not blocked completely. Quantitative polymerase chain reaction analyses demonstrated that 15-30% of the mutated loci in mature B cells were unrearranged, in striking contrast to the wild-type alleles. The remainder of the mutated loci underwent D-J (65-80%) as well as V-DJ rearrangements, although the latter were less frequent (3-6%). These results are in line with previous data which showed that the V(D)J recombination machinery is modulated through cis-regulatory elements within the intron enhancer. However, our data predict the existence of additional cis-regulatory element(s) which, together with the intron enhancer, are required to activate the V(D)J recombination machinery fully. Such cis-regulatory element(s) might function as an enhancer of recombination or as a locus control region regulating the accessibility of the IgH locus.

High frequency expression of integrated proviruses derived from enhancer trap retroviruses.

Since retroviruses integrate preferentially into transcriptionally active loci, the provirus may come under the control of regulatory elements of the gene into which it integrated and thus become a functional tag for that gene. In order to determine the frequency of retroviral integration near active endogenous enhancer elements, a retroviral enhancer trap vector was constructed. Lacking the long terminal repeat enhancer, expression of the neomycin resistance (neo) gene, used as a reporter, is dependent upon endogenous enhancer elements able to activate the long terminal repeat promoter. Infection of murine fibroblast cells indicated that a high proportion of the proviral copies expressed the neo gene. Infection of hematopoietic lines confirmed this high frequency of expression of integrated proviruses. Overall, between 43 and 74% of proviruses integrated into several different cell lines expressed the neo gene. These data suggest that retroviral integration is not only dependent upon transcriptional activity of the genomic target sites, but, more specifically, retroviruses preferentially integrate near active enhancer elements which are often associated with developmentally regulated genes.

V gene rearrangement is required to fully activate the hypermutation mechanism in B cells.

Nonproductively rearranged H and L chain loci of B cell hybridoma lines expressing heavily mutated antibodies were cloned and partially sequenced. The results confirm earlier data showing that somatic point mutations are as frequent in nonproductively rearranged loci containing a rearranged V gene as in productively rearranged loci. They establish in addition that in nonproductive H chain loci which bear a DJH rearrangement the frequency of somatic mutations is more than 10 times lower (0.2%) than in VDJH loci expressed by the same cells (2.5%). Thus, the hypermutation mechanism operating in B cell differentiation is targeted at V genes rearranged to the J locus and may require nucleotide sequences associated with both V and J elements in order to be fully activated. An inversion of the JH2 segment was detected in one DJH locus. This inversion appears to be the result of a secondary joining event occurring occasionally in the course of B cell development.

Antibody engineering for the analysis of affinity maturation of an anti-hapten response.

The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1-expressing anti-NP antibodies from the secondary response, on the affinity of these antibodies was examined by site-specific mutagenesis and recombinant antibody construction. A tryptophan----leucine exchange at position 33 in the VH segment of all but one of the high-affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10-fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high-affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one-codon deletion) in VH and the particular D-JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain-bearing anti-NP antibodies which by itself leads to an increase of hapten-binding affinity. Based on the analysis of two secondary response antibodies from which somatic mutations in VH and VL have been eliminated, it is also concluded that the recruitment of B cell clones into the pathway of hypermutation involves a mechanism which is not based upon affinity differences towards the antigen.

Transfected plasmacytoma cells do not transport the membrane form of IgM to the cell surface.

Expression vectors coding for membrane-bound IgM antibodies were introduced into myeloma and B lymphoma cells. Only the lymphoma but not the myeloma cells were able to express the antibodies on the cell surface, although in both cases, complete antibodies were assembled intracellularly. In myeloma cells, the Ig molecules did not reach the Golgi compartment. Thus, the intracellular transport of membrane-bound antibodies is controlled in the B cell lineages in a developmentally ordered fashion.

Modulation of antibody binding affinity by somatic mutation.

The affinity of hapten binding of monoclonal antibodies (MAbs) specific for 4-hydroxy-3-nitrophenylacetyl (NP) has been investigated at the molecular level by both site-specific mutagenesis and recombinant antibody construction, followed by expression in myeloma cells. We have shown that a single point mutation (trp----leu at codon 33) in the variable region of the heavy chain (VH) is sufficient to endow a primary-response, germline-encoded antibody with an affinity for antigen typical of a secondary-response antibody carrying the same mutation. We have also demonstrated that mutations additional to the trp----leu exchange in the heavy chain and further mutations in the light chain are irrelevant to the high-affinity phenotype of secondary-response antibodies. Since some of these are "parallel" mutations common to clonally unrelated antibodies, this suggests that the mutation rate is not constant across the entire immunoglobulin variable region. Although antibodies with a trp----leu exchange at position 33 are positively selected because of improved hapten binding affinity, we have found that, under rare circumstances, other patterns of mutations may be selected through particular D-JH combinations; we have demonstrated one case where this has generated an antibody with very efficient hapten binding ability.