Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome.

BACKGROUND: Hypotonia-cystinuria syndrome (HCS) and 2p21 deletion syndrome are two recessive contiguous gene deletion syndromes associated with cystinuria type I. The deletions differ in size and the number of genes involved. In HCS patients, only SLC3A1 and PREPL are disrupted. In the 2p21 deletion syndrome, two additional genes (C2orf34 and PPM1B) are lost. OBJECTIVE: Clinical and molecular analysis of two siblings who presented with an atypical HCS phenotype. METHODS: Molecular analysis of the SLC3A1/PREPL locus was performed in the patients using quantitative polymerase chain reaction (PCR) methods. RESULTS: HCS in both siblings was confirmed with the deletion screen of the SLC3A1/PREPL locus. Fine mapping of the breakpoint revealed a deletion of 77.4 kb, including three genes: SLC3A1, PREPL and C2orf34. Features not present in classical HCS were a mild/moderate mental retardation and a respiratory chain complex IV deficiency documented in patient 2. CONCLUSIONS: We report the first patients with a deletion of SLC3A1, PREPL and C2orf34. They present with a phenotype intermediate between HCS and 2p21 deletion syndrome. These patients facilitate the elucidation of the contribution of each gene to the phenotype in the different 2p21 deletion syndromes.

Regulation of HMGIC expression: an architectural transcription factor involved in growth control and development.

The HMGIC gene has been implicated in the control of cell proliferation and development. We show here that HMGIC has multiple mRNA isoforms that arise by transcription initiation from alternative tandem promoters. These transcripts are not only differentially expressed between cell lines, but they can also differ within an individual cell line, in response to particular stimuli. Whereas quiescent 3T3-L1 preadipocytes express low levels of HMGIC mRNA, stimulation by serum results in a dramatic upregulation with the characteristics of a delayed-early response gene. Characterization of involved signal transduction pathways showed that both FGF-1 and PDGF-BB are strong inducers of HMGIC expression mediated via both the PI-3 kinase and MAP kinase pathways. In order to characterize the regulatory elements, sequences upstream of the translation initiation site of HMGIC were assayed for promoter activity. The HMGIC 5' flanking sequences had constitutive promoter activity in all cell lines tested, suggesting that HMGIC is regulated by negative regulatory elements that were not present in the 5'-flanking regions analysed here.

Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome.

Hypotonia-cystinuria syndrome (HCS) and 2p21 deletion syndrome are two recessive contiguous gene deletion syndromes associated with cystinuria type I. In HCS patients, only SLC3A1 and PREPL are disrupted. In the 2p21 deletion syndrome, two additional genes (C2orf34 and PPM1B) are lost. Molecular analysis of the SLC3A1/PREPL locus was performed in the patients using quantitative polymerase chain reaction (PCR) methods. HCS in both siblings was confirmed with the deletion screen of the SLC3A1/PREPL locus. Fine mapping of the breakpoint revealed a deletion of 77.4 kb, including three genes: SLC3A1, PREPL and C2orf34. Features not present in classical HCS were a mild/moderate mental retardation and a respiratory chain complex IV deficiency. We report the first patients with a deletion of SLC3A1, PREPL and C2orf34. They present with a phenotype intermediate between HCS and 2p21 deletion syndrome.

Neuroendocrine-specific expression of the human prohormone convertase 1 gene. Hormonal regulation of transcription through distinct cAMP response elements.

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 1 (PC1) gene and identified and characterized the PC1 promoter region. We found multiple transcription start sites located within a 15-base pair region, 205 base pairs upstream of the translation start codon. The promoter region is not G+C-rich and does not contain a canonical TATA box nor a CAAT box. Transient expression assays with a set of human PC1 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, the proximal promoter region confers both basal and hormone-regulated promoter activity. Site-specific mutagenesis experiments demonstrate that two closely spaced cAMP response elements within the proximal promoter region direct cAMP-mediated hormonal regulation of transcription of the PC1 gene.

The NNP-1 gene (D21S2056E), which encodes a novel nuclear protein, maps in close proximity to the cystatin B gene within the EPM1 and APECED critical region on 21q22.3.

We have cloned and molecularly characterized a novel human cDNA that encodes a protein of about 52 kDa that was shown by immunocytochemistry to have a nuclear localization. Northern blot analysis of a variety of human tissues revealed that the gene for this novel nuclear protein, designated NNP-1 (HGMW-approved symbol D21S2056E), is ubiquitously expressed as a 2.0-kb transcript. The NNP-1 protein displays significant sequence similarity to the C47E12.7 protein of Caenorhabditis elegans (38% identity and 58% similarity) and the YD78 protein of Saccharomyces cerevisiae (36% identity and 54% similarity). The human NNP-1 gene was mapped to a 15-kb DNA interval on chromosome 21q22.3, close to markers D21S1459 and D21S1953, that is 25 kb distal to the gene encoding cystatin B. Finally, an as-yet unknown gene that is highly related to NNP-1 was found proximal to the gene encoding cystatin B, near marker D21S1458, which is approximately 75 kb centromeric to the NNP-1 gene.

Cell type-specific protein-DNA interactions at the cAMP response elements of the prohormone convertase 1 promoter. Evidence for additional transactivators distinct from CREB/ATF family members.

The proximal promoter region of the neuroendocrine-specific human prohormone convertase 1 (PC1) gene contains two distinct cAMP response elements (CRE-1 and CRE-2). Both elements are essential in directing the cAMP-mediated hormonal regulation of PC1 gene transcription. In this study, we have demonstrated that CRE-1 binds several trans-acting factors. In electrophoretic mobility shift assay experiments with nuclear extracts prepared from neuroendocrine AtT-20 and beta-TC3 cells and non-neuroendocrine COS-1 cells, three specific protein-DNA complexes (I-III) were detected. Complexes II and III were shown to contain CREB-1 and ATF-1, respectively. The most slowly migrating complex I was only detected with the neuroendocrine cell lines and appeared to comprise a c-Jun-containing heterodimer. In addition, CRE-2 was shown to bind a protein that was only detected in nuclear extracts derived from the neuroendocrine cell lines. Antibody supershift experiments indicated that both the c-Jun-interacting protein in CRE-1 complex I and the CRE-2-interacting protein are distinct from known members of the basic domain, leucine zipper family of transcription factors. UV cross-linking experiments demonstrated that these potential novel proteins are approximately 100 and 60 kDa in size, respectively. Site-specific mutagenesis experiments demonstrated that the formation of both CRE-1 and CRE-2 complexes is correlated with the transcriptional activity of the proximal PC1 promoter as has been shown in transient transfections with wild-type and mutant promoter constructs. In addition, it was shown that both CREB-1 and ATF-1 transactivate the human PC1 promoter in transient transfection experiments.

Production of recombinant proteins in Chinese hamster ovary cells overexpressing the subtilisin-like proprotein converting enzyme furin.

The proprotein processing enzyme furin is the mammalian prototype of a novel family of subtilisin-like serine endoproteases which possess cleavage specificity for sites involving multiple basic amino acid residues and are involved in the processing of precursor proteins of a variety of regulatory peptides and proteins. One of the limiting steps in the engineering of mammalian cells designed for the overproduction of secreted proteins is the endoproteolytic cleavage of the precursor molecule to its mature biologically active form. The extremely low level of endogenous furin is likely the reason why cells are not able to fully mature overexpressed precursor proteins to their mature form. Here, we report a CHO-derived cell line genetically engineered for the production of high levels of recombinant proteins that need such endoproteolytic maturation. First, the human furin cDNA under the control of the cytomegalovirus early promoter and enhancer was introduced and overexpressed in a DHFR-deficient CHO cell line. A permanent cell line CHO-D3-FUR was established that expressed biologically active furin. Subsequently, to demonstrate the capacity of CHO-D3-FUR cells to produce recombinant proteins in a fully matured form, two derivative cell lines were established that overexpressed the von Willebrand factor (vWF) and transforming growth factor beta 1 (TGF beta 1); CHO-D3-vWF and CHO-D3-TGF beta 1, respectively. Both derivative cell lines were able to produce relatively high levels of recombinant protein in a fully matured and biologically active form. Our results illustrate the potential of the CHO-D3-FUR cell line in the production of recombinant secretory proteins that need endoproteolytic activation at the consensus furin cleavage sequence Arg-X-Lys/Arg-Arg.

Regulation of human prohormone convertase 2 promoter activity by the transcription factor EGR-1.

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 2 (PC2) gene and established characteristics of the PC2 promoter region. The proximal promoter region is very G+C-rich and does not contain a canonical TATA box or a CAAT box. Transient expression assays with a set of human PC2 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, we show that the transcription factor EGR-1 interacts with two distinct elements within the proximal human PC2 promoter region. Transfection experiments also demonstrate that EGR-1 is able to enhance PC2 promoter activity.