Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity.

In Wnt-stimulated cells, beta-catenin becomes stabilized in the cytoplasm, enters the nucleus and interacts with HMG box transcription factors of the lymphoid-enhancing factor-1 (LEF-1)/T-cell factor (TCF) family, thereby stimulating the transcription of specific target genes. We recently identified Pontin52 as a nuclear protein interacting with beta-catenin and the TATA-box binding protein (TBP), suggesting its involvement in regulating beta-catenin-mediated transactivation. Here, we report the identification of Reptin52 as an interacting partner of Pontin52. Highly homologous to Pontin52, Reptin52 likewise binds beta-catenin and TBP. Using reporter gene assays, we show that the two proteins antagonistically influence the transactivation potential of the beta-catenin-TCF complex. Furthermore, we demonstrate the evolutionary conservation of this mechanism in Drosophila. dpontin and dreptin are essential genes that act antagonistically in the control of Wingless signalling in vivo. These results indicate that the opposite action of Pontin52 and Reptin52 on beta-catenin-mediated transactivation constitutes an additional mechanism for the control of the canonical Wingless/Wnt pathway.

dlarp, a new candidate Hox target in Drosophila whose orthologue in mouse is expressed at sites of epithelium/mesenchymal interactions.

Hox complex genes are key developmental regulators highly conserved throughout evolution. They encode transcription factors that initiate genetic programs of diversified morphogenesis along the anteroposterior embryonic axis. We report the characterization of the novel Drosophila Hox target gene dlarp, isolated from a further screen of a previously described library of genomic DNA fragments associated in vivo with Ultrabithorax proteins. The dlarp spatio-temporal pattern of transcription in wild-type and homeotic mutant embryos is consistent with a positive regulation by Sex combs reduced and Ultrabithorax in the parasegment 2 ectoderm and the abdominal mesoderm, respectively. The teashirt gene product, thought to act in concert with Hox proteins, is also required for the transcriptional control of this target. Search in databases revealed that dlarp has been highly conserved during evolution. The embryonic expression pattern of the mouse orthologue does not support a function downstream of Hox proteins. It is mainly transcribed in neural structures and in developing organs characterized by epithelial-mesenchymal interactions.

nessy, an evolutionary conserved gene controlled by Hox proteins during Drosophila embryogenesis.

From a library of DNA fragments associated with Ultrabithorax protein in vivo, we have isolated nessy, a new Drosophila gene that encodes a putative transmembrane protein conserved in evolution from Caenorhabditis elegans, to human. Zygotic expression occurs transiently in mesectodermal cells at gastrulation, proceeds in mesoderm and endoderm lineages during germ band movements and becomes then restricted to anterior and posterior domains in the visceral mesoderm. The Hox proteins Ultrabithorax, Antennapedia and AbdominalA are likely acting simultaneously to repress nessy in the other parts of the visceral mesoderm.

Drosophila Hox complex downstream targets and the function of homeotic genes.

Hox complex genes are key developmental regulators highly conserved throughout evolution. The encoded proteins share a 60-amino-acid DNA-binding motif, the homeodomain, and function as transcription factors to control axial patterning. An important question concerns the nature and function of genes acting downstream of Hox proteins. This review focuses on Drosophila, as little is known about this question in other organisms. The noticeable progress gained in the field during the past few years has significantly improved our current understanding of how Hox genes control diversified morphogenesis. Here we summarise the strategies deployed to identify Hox target genes and discuss how their function contributes to pattern formation and morphogenesis. The regulation of target genes is also considered with special emphasis on the mechanisms underlying the specificity of action of Hox proteins in the whole animal.

DWnt-4, a novel Drosophila Wnt gene acts downstream of homeotic complex genes in the visceral mesoderm.

Wnt genes encode putative cell signalling proteins which play crucial roles during development. From a library of DNA fragments associated, in vivo, with Ultrabithorax proteins, we isolated a novel Drosophila Wnt gene, DWnt-4. Neither a paralog nor an ortholog of the gene exist in the current repertoire of full-length Wnt sequences. DWnt-4 maps close (30 kb) to wingless, suggesting that the two Wnt genes derive from a duplication that occurred early in evolution, since they are significantly diverged in sequence and structure. Developmental expression of DWnt-4 partially overlaps that of wingless. The gene is transcribed following a segment polarity-like pattern in the posterior-most cells of each parasegment of the ectoderm, and at two locations that correspond to parasegments 4 and 8 of the visceral mesoderm. The control of DWnt-4 expression in the visceral mesoderm involves a network of regulatory molecules that includes Ultrabithorax and other proteins from the homeotic complex (HOM-C), as well as the TGF-beta decapentaplegic gene product.

The modifier of variegation modulo gene acts downstream of dorsoventral and HOM-C genes and is required for morphogenesis in Drosophila.

Growing evidence involves chromatin structural flexibility in gene regulation during development. modulo is a dominant suppressor of position effect variegation, suggesting the participation of its product in the assembly of higher order chromatin structures. Here we report the patterns of modulo expression and regulation during embryogenesis, analyzed in correlation with phenotypical defects resulting from the amorphic mutation of the gene. Zygotic expression of modulo depends on the activity of genes which pattern the embryo along dorsoventral and anteroposterior axes and specify diversified morphogenesis, dorsal and the mesoderm-specific genes twist and snail direct modulo expression in the presumptive mesoderm. The homeotic genes Sex combs reduced and Ultrabithorax positively regulate the gene in the ectoderm of parasegment 2 and abdominal mesoderm, respectively, modulo mutants exhibit aberrant morphogenesis of tissues originating from those embryonic primordia which normally express the gene, but do not show defect in cell fate specification. We propose that down-stream of pattern-forming genes modulo controls, via chromatin structural changes, genes critical for the process of morphogenesis of several tissue types.

Homeotic control in Drosophila; the scabrous gene is an in vivo target of Ultrabithorax proteins.

The regulatory functions of transcription factors encoded by the Ultrabithorax (Ubx) gene initiate genetic programmes essential for segmental identity and morphogenesis in Drosophila. Based on the formation of DNA-protein adducts in intact nuclei and immunoselection procedure, we cloned genomic targets for Ubx proteins. One clone was studied in detail. It encompasses parts of the last intron and exon of the scabrous (sca) gene, which encodes a secreted protein involved in cellular communication during neurogenesis. Five motifs, presenting the ATTA core, which is shared by most homeodomain binding sites, were found in the nucleotide sequence of this clone. We detail here the dynamic pattern of sca transcript accumulation during embryogenesis and show that mutation of Ubx results in the ectopic transcription of sca in the first abdominal segment. We propose that a direct interaction of Ubx with cis-acting elements in sca negatively regulates the gene. Transcript localization in several combinations of deficiencies in the Bithorax complex (BX-C) indicates that sca is downregulated by abdominal A (abdA) and Abdominal B (AbdB), and suggests that it is a common target of the three genes of BX-C.

A tissue-specific DNase I-hypersensitive site in a class II A alpha gene is under trans-regulatory control.

Class II major histocompatibility complex molecules are integral membrane glycoproteins whose distribution is limited to certain tissues. To identify the molecular basis for such specificity, the chromatin configuration of the class II A alpha gene was examined in intact nuclei from various cell types. We show that there are three DNase I-hypersensitive sites in the A alpha gene. One of these sites, located near the promoter region, is specific to cells that normally express class II molecules at some stage of differentiation. Furthermore, this tissue-specific site appears to be under trans-regulatory control.

Endocytosis of MHC molecules by B cell-B lymphoma and B cell-T lymphoma hybrids.

Different cells and different cell surface determinants of the same cells take up liposomes, bound to them via monoclonal antibodies, with variable efficiency. We have previously reported that T and B lymphocytes differ in the extent to which they take up liposomes bound to MHC class I molecules; T cells endocytose class I molecules rapidly, but B cells endocytose class I molecules much less efficiently, although their endocytosis of class II molecules is rapid. An approach toward understanding the molecular basis for this difference was made by evaluating the internalization patterns of somatic cell hybrids of B and T cells. Hybrid cells were constructed between LPS-stimulated purified B cell blasts from C57BL/6 mice (H-2b) and the HAT-sensitive AKR T lymphoma BW 5147 (H-2k). Hybrids between the BALB/c B lymphoma M12.4.1 (H-2d) and B cell LPS blasts from B10.BR (H-2k) mice were also evaluated. In all cases, for hybrid tumor cells, liposomes that were bound to class I molecules encoded by genes from the B cell donor were endocytosed as efficiently as liposomes bound to the class I molecules of the recipient lymphoid cell. T cell tumors efficiently internalized their own class I molecules and those donated by B cells; B cell tumors internalized liposomes that were bound to their own and the donor B cell class I molecules poorly. Thus, our results suggest that the internalization of an MHC molecule is not an intrinsic property of the molecule, but rather of the cell in which it is found.

Immune clearance of liposomes inhibited by an anti-Fc receptor antibody in vivo.

In a study designed to evaluate the potential for in vivo manipulation of the circulation and tissue distribution of injected liposomes, mice were passively injected with antidinitrophenyl (anti-DNP) monoclonal antibodies of the IgG2a or IgG2b subclasses or were immunized with the nitrophenyl hapten bound to a protein carrier. They were then injected i.v. with 125I- and carboxyfluorescein-labeled, DNP-bearing liposomes. Circulation time of the DNP-bearing liposomes was markedly reduced in actively and passively immune mice, with increased deposition of liposomes in the liver. The increased clearance of liposomes could be abrogated by injection of a monoclonal antibody directed against the murine IgG Fc receptor (2.4G2). The results suggest that clearance of ligand-bearing reagent in the face of an immune response may be modified by specific immunologic manipulation in vivo.