Katrien Staes

δ-Protocadherins: a gene family expressed differentially in the mouse brain

Abstract.Phylogenetic analysis of protocadherin genes identified a new gene subfamily, the δ-protocadherins, containing several conserved motifs in their cytoplasmic domains. This subfamily can be further subdivided into two subgroups, named δ1-protocadherins (comprising protocadherin-1, -7, -9, and -11 or X/Y) and δ2-protocadherins (comprising protocadherin-8, -10, -17, -18, and -19). The members of the δ1-protocadherin subgroup were analyzed in greater detail here. They share a similar gene structure that results in the expression of multiple alternative transcripts. All members of this subgroup have at least one transcript that contains a binding site for protein phosphatase-1α. Like most classic cadherins, each of three δ1-protocadherins analyzed in this study by in situ hybridization showed a unique expression pattern that differed from the patterns of the other δ1-protocadherins. Together, these results suggest that the members of the δ1-protocadherin subgroup exercise tightly regulated functions in the development, regionalization, and functional differentiation of the mouse brain.

A Constitutional Translocation t(1;17)(p36.2;q11.2) in a Neuroblastoma Patient Disrupts the Human NBPF1 and ACCN1 Genes

The human 1p36 region is deleted in many different types of tumors, and so it probably harbors one or more tumor suppressor genes. In a Belgian neuroblastoma patient, a constitutional balanced translocation t(1;17)(p36.2;q11.2) may have led to the development of the tumor by disrupting or activating a gene. Here, we report the cloning of both translocation breakpoints and the identification of a novel gene that is disrupted by this translocation. This gene, named NBPF1 for Neuroblastoma BreakPoint Family member 1, belongs to a recently described gene family encoding highly similar proteins, the functions of which are unknown. The translocation truncates NBPF1 and gives rise to two chimeric transcripts of NBPF1 sequences fused to sequences derived from chromosome 17. On chromosome 17, the translocation disrupts one of the isoforms of ACCN1, a potential glioma tumor suppressor gene. Expression of the NBPF family in neuroblastoma cell lines is highly variable, but it is decreased in cell lines that have a deletion of chromosome 1p. More importantly, expression profiling of the NBPF1 gene showed that its expression is significantly lower in cell lines with heterozygous NBPF1 loss than in cell lines with a normal 1p chromosome. Meta-analysis of the expression of NBPF and ACCN1 in neuroblastoma tumors indicates a role for the NBPF genes and for ACCN1 in tumor aggressiveness. Additionally, DLD1 cells with inducible NBPF1 expression showed a marked decrease of clonal growth in a soft agar assay. The disruption of both NBPF1 and ACCN1 genes in this neuroblastoma patient indicates that these genes might suppress development of neuroblastoma and possibly other tumor types.

Expression of protocadherin‐1 (Pcdh1) during mouse development

Protocadherin‐1 (Pcdh1) is a member of the δ‐protocadherin subgroup of non‐clustered protocadherins. We studied the expression of Pcdh1 from the early embryonic to the adult stage of mouse development by semi‐quantitative RT‐PCR and in situ hybridization. Pcdh1 can be detected as early as embryonic day 9.5. In early embryogenesis, expression is especially prominent in blood vessels. During later development and in the adult mouse, organs derived from the embryonic gut, such as the esophagus, intestines, liver, lung, and submandibular gland, contain epithelia and other types of tissues that are Pcdh1‐positive. Other positive organs include the brain, spinal cord, retina, peripheral ganglia, the inner ear, hair follicles, kidney, vagina, uterus, placenta, testis, prostate, and the seminal gland. The tight spatial and temporal regulation of Pcdh1 expression suggests that this protocadherin plays multiple roles not only during development but also in mature tissues and organs in the mouse. Developmental Dynamics 237:2496–2505, 2008.

Human alpha-catulin, a novel alpha-catenin-like molecule with conserved genomic structure, but deviating alternative splicing.

A new human cDNA was cloned and termed alpha-catulin, based on sequence similarity with both alpha-CATenins and vincULIN. The mRNA is present ubiquitously, although low expression levels are found in neural tissues. The genomic organization of the alpha-catulin gene CTNNAL1 is closely related to that of the alphaE-catenin gene CTNNA1, but not at all to that of the vinculin gene. Alternative splicing of the last exon generates a frameshift, resulting in a truncated protein with a new carboxy-terminus.

The transcriptional repressor Kaiso localizes at the mitotic spindle and is a constituent of the pericentriolar material.

Kaiso is a BTB/POZ zinc finger protein known as a transcriptional repressor. It was originally identified through its in vitro association with the Armadillo protein p120ctn. Subcellular localization of Kaiso in cell lines and in normal and cancerous human tissues revealed that its expression is not restricted to the nucleus. In the present study we monitored Kaisos subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody. We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex. We analyzed the functions of different domains of Kaiso by visualizing the subcellular distribution of GFP-tagged Kaiso fragments throughout the cell cycle. Our results indicate that two domains are responsible for targeting Kaiso to the centrosomes and microtubules. The first domain, designated SA1 for spindle-associated domain 1, is located in the center of the Kaiso protein and localizes at the spindle microtubules and centrosomes; the second domain, SA2, is an evolutionarily conserved domain situated just before the zinc finger domain and might be responsible for localizing Kaiso towards the centrosomal region. Constructs containing both SA domains and Kaisos aminoterminal BTB/POZ domain triggered the formation of abnormal centrosomes. We also observed that overexpression of longer or full-length Kaiso constructs led to mitotic cell arrest and frequent cell death. Knockdown of Kaiso accelerated cell proliferation. Our data reveal a new target for Kaiso at the centrosomes and spindle microtubules during mitosis. They also strongly imply that Kaisos function as a transcriptional regulator might be linked to the control of the cell cycle and to cell proliferation in cancer.

Localization of the 17q breakpoint of a constitutional 1;17 translocation in a patient with neuroblastoma within a 25-kb segment located between the ACCN1 and TLK2 genes and near the distal breakpoints of two microdeletions in neurofibromatosis type 1 patients.

We have constructed a 1.4‐Mb P1 artificial chromosome/bacterial artificial chromosome (PAC/BAC) contig spanning the 17q breakpoint of a constitutional translocation t(1;17)(p36.2;q11.2) in a patient with neuroblastoma. Three 17q breakpoint‐overlapping cosmids were identified and sequenced. No coding sequences were found in the immediate proximity of the 17q breakpoint. The PAC/BAC contig covers the region between the proximally located ACCN1 gene and the distally located TLK2 gene and SCYA chemokine gene cluster. The observation that the 17q breakpoint region could not be detected in any of the screened yeast artificial chromosome libraries and the localization of the 17q breakpoint in the vicinity of the distal breakpoints of two microdeletions in patients with neurofibromatosis type 1 suggest that this chromosomal region is genetically unstable and prone to rearrangements.

Chibby interacts with NBPF1 and clusterin, two candidate tumor suppressors linked to neuroblastoma

The NBPF genes are members of a gene family that underwent a remarkable increase in their copy number during recent primate evolution. The NBPF proteins contain 5 to 40 copies of a domain known as the NBPF repeat or DUF1220. Very little is known about the function of these domains or about the NBPF proteins. We performed a yeast two-hybrid screening with the aminoterminal domain of NBPF11 and found that Chibby, a documented repressor of Wnt signaling, interacts with multiple NBPF proteins. More specifically, a coiled-coil region in the NBPF proteins interacts with the coiled-coil domain in the carboxyterminal region of Chibby. Nonetheless, this interaction did not influence the repressor function of Chibby in a TOPFLASH reporter assay. Using Chibby as bait in a new yeast two-hybrid screening, we identified clusterin as a binding protein. Chibby and clusterin were co-immunoprecipitated with NBPF1, suggesting the formation of a tri-molecular complex. Although we have not pinpointed the role of these mutual interactions, the possible formation of a macromolecular complex of three candidate tumor suppressor proteins, including the enigmatic NBPF1, points at important functional implications.

NBPF1, a tumor suppressor candidate in neuroblastoma, exerts growth inhibitory effects by inducing a G1 cell cycle arrest

BackgroundNBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved.MethodsExpression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21CIP1/WAF1 were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-of-function approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool.ResultsWe show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21CIP1/WAF1 in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects. In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis.ConclusionsWe demonstrated that NBPF1 exerts different tumor suppressive effects, depending on the cell line analyzed, and provide new clues into the molecular mechanism of the enigmatic NBPF proteins.

The human NANOS3 gene contributes to lung tumour invasion by inducing epithelial–mesenchymal transition

We have explored the role of the human NANOS3 gene in lung tumour progression. We show that NANOS3 is over‐expressed by invasive lung cancer cells and is a prognostic marker for non‐small cell lung carcinomas (NSCLCs). NANOS3 gene expression is restricted in testis and brain and is regulated by epigenetic events. It is up‐regulated in cultured cells undergoing epithelial − mesenchymal transition (EMT). NANOS3 over‐expression in human NSCLC cell lines enhances their invasiveness by up‐regulating EMT, whereas its silencing induces mesenchymal − epithelial transition. NANOS3 represses E‐cadherin at the transcriptional level and up‐regulates vimentin post‐transcriptionally. Also, we show that NANOS3 binds mRNAs encoding vimentin and regulates the length of their poly(A) tail. Finally, NANOS3 can also protect vimentin mRNA from microRNA‐mediated repression. We thus demonstrate a role for NANOS3 in the acquisition of invasiveness by human lung tumour cells and propose a new mechanism of post‐transcriptional regulation of EMT. Copyright

GC content of Early Metazoan genes and its impact on gene expression levels in mammalian cell lines

Abstract With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure–function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines.