A functional genomic approach, based on systematic data gathering, was used to characterize a family of proteins containing a tripartite motif (TRIM). A total of 37 TRIM genes/proteins were studied, 21 of which were novel. The results demonstrate that TRIM proteins share a common function: by means of homo-multimerization they identify speci®c cell compartments.

Proteins of the Myc and Mad family are involved in transcriptional regulation and mediate cell differentiation and proliferation. These molecules share a basic-helix-loop-helix leucine zipper domain (bHLHZip) and bind DNA at the E box (CANNTG) consensus by forming heterodimers with Max. We report the isolation, characterization and mapping of a human gene and its mouse homolog encoding a new member of this family of proteins, named Rox. Through interaction mating and immunoprecipitation techniques, we demonstrate that Rox heterodimerizes with Max and weakly homodimerizes. Interestingly, bandshift assays demonstrate that the Rox-Max heterodimer shows a novel DNA binding specificity, having a higher affinity for the CACGCG site compared with the canonical E box CACGTG site. Transcriptional studies indicate that Rox represses transcription in both human HEK293 cells and yeast. We demonstrate that repression in yeast is through interaction between the N-terminus of the protein and the Sin3 co-repressor, as previously shown for the other Mad family members. ROX is highly expressed in quiescent fibroblasts and expression markedly decreases when cells enter the cell cycle. Moreover, ROX expression appears to be induced in U937 myeloid leukemia cells stimulated to differentiate with 12-O-tetradecanoylphorbol-13-acetate. The identification of a novel Max-interacting protein adds an important piece to the puzzle of Myc/Max/Mad coordinated action and function in normal and pathological situations. Furthermore, mapping of the human gene to chromosome 17p13.3 in a region that frequently undergoes loss of heterozygosity in a number of malignancies, together with the biochemical and expression features, suggest involvement of ROX in human neoplasia.

Opitz syndrome (OS) is a multiple congenital anomaly manifested by abnormal closure of midline structures. The gene responsible for the X-linked form of this disease, MID1, encodes a protein (midin) that contains a RING, two B-boxes, a coiled-coil (the so-called tripartite motif) and an RFP-like domain. The tripartite motif is characteristic of a family of proteins, named the B-box family, involved in cell proliferation and development. Since the subcellular compartmentalization and the ability to form multiprotein structures both appear to be crucial for the function of this family of proteins, we have studied these properties on the wild-type and mutated forms of midin. We found that endogenous midin is associated with microtubules throughout the cell cycle, co-localizing with cytoplasmic fibres in interphase and with the mitotic spindle and midbodies during mitosis and cytokinesis. Immunoprecipitation experiments demonstrated the ability of the tripartite motif to mediate midin homodimerization, consistent with the evidence, obtained by gel filtration analysis, that midin exists in the form of large protein complexes. Functional characterization of altered forms of midin, resulting from mutations found in OS patients, revealed that association with microtubules is compromised, while the ability to homodimerize and form multiprotein complexes is retained. We suggest that midin is involved in the formation of multiprotein structures acting as anchor points to microtubules and that impaired association with these cytoskeletal structures causes OS developmental defects.

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The B-box family is an expanding new family of genes encoding proteins involved in diverse cellular functions such as developmental patterning and oncogenesis. A member of this protein family, MID1, is the gene responsible for the X-linked form of Opitz G/BBB syndrome, a developmental disorder characterized by defects of the midline structures. We now report the identification of MID2, a new transcript closely related to MID1. MID2 maps to Xq22 in human and to the syntenic region on the mouse X chromosome. The two X-linked genes share the same domains, the same exon-intron organization, a high degree of similarity at the protein level and the same subcellular localization, both being confined to the cytoplasm in association to micro-tubular structures. The expression pattern studied by RNA in situ hybridization in mouse revealed that Mid2 is expressed early in development and the highest level of expression is detected in the heart, unlike Mid1 for which no expression was detected in the developing heart. Together, these data suggest that midin and MID2 have a similar biochemical function but a different physiological role during development.

An obligatory crossing-over event between the X and Y chromosomes in mammals occurs at each male meiosis within the 2.6 Mb of DNA defining the pseudoautosomal region (PAR). Genes located within or near the human PAR have homologous copies on the X and Y chromosomes, escape X inactivation and appear to be highly divergent throughout evolution. We have characterized the genomic structure of two genes from a recently identified cluster of sulfatase genes (ARSD and ARSE) located in the Xp22.3 region, and of their homologs on the Y chromosome. Our results indicate that the ARSD and ARSE genes from within this cluster have a conserved genomic organization, shared also by another Xp22.3 gene, STS, but completely different from that of all the other sulfatase genes. Sequence analysis of the Y-linked homologs indicate that they represent truncated pseudogenes. Sequence identity values between the X and Y copies of each gene is on average 91%, significantly higher than the values obtained by comparing different members of the family. FISH mapping experiments performed in several primate species revealed an identical localization of the X-linked copies to that in man, but different localizations of the Y homologs. Together, our data indicate that the cluster of sulfatase genes on human Xp22.3 was created through duplication events which probably occurred in an ancestral PAR, and support the view that the PAR has undergone multiple changes during recent mammalian evolution.

Molecular analysis has provided important insights into the biochemistry and genetics of the sulphatase family of enzymes. Through bioinformatic searches of the EST database, we have identified a novel gene consisting of 11 exons and encoding a 525 aa protein that shares a high degree of sequence similarity with all sulphatases and in particular with arylsulphatases, hence the tentative name Arylsulfatase G (ARSG). The highest homology is shared with Arylsulfatase A, a lysosomal sulphatase which is mutated in metachromatic leukodistrophy, particularly in the aminoterminal region. The 10 amino acids that form the catalytic site are strongly conserved. The murine homologue of Arylsulfatase G gene product shows 87% identity with the human protein. To test the function of this novel gene we transfected the full-length cDNA in Cos7 cells, and detected an Arylsulfatase G precursor protein of 62 kDa. After glycosylation the precursor is maturated in a 70 kDa form, which localises to the endoplasmic reticulum. Northern blot analysis of Arylsulfatase G revealed a ubiquitous expression pattern. We tested the sulphatase activity towards two different artificial substrates 4-methylumbelliferyl (4-MU) sulphate and p-nitrocatechol sulphate, but no arylsulphatase activity was detectable. Further studies are needed to characterise the function of Arylsulfatase G, possibly revealing a novel metabolic pathway.

The TRIM family members are defined by the presence of the tripartite motif (RING, B-box and coiled-coil domains or RBCC). They have been implicated in a variety of processes, such as regulation of development and oncogenesis. We report the expression analysis of a member of this family, TRIM9. Its expression is mainly confined to the central nervous system. The developing neocortex, the dorsal thalamus, the midbrain, the basal area of the hindbrain and spinal cord show high level of expression during embryogenesis. In adult brain, TRIM9 is detected in the Purkinje cells of the cerebellum, in the hippocampus, and in the cortex.