Identification of the gene encoding Brain Cell Membrane Protein 1 (BCMP1), a putative four-transmembrane protein distantly related to the Peripheral Myelin Protein 22 / Epithelial Membrane Proteins and the Claudins

BACKGROUND: A partial cDNA clone from dog thyroid presenting a very significant similarity with an uncharacterized mouse EST sequence was isolated fortuitously. We report here the identification of the complete mRNA and of the gene, the product of which was termed "brain cell membrane protein 1" (BCMP1). RESULTS: The 4 kb-long mRNA sequence exhibited an open-reading frame of only 543 b followed by a 3.2 kb-long 3' untranslated region containing several AUUUA instability motifs. Analysis of the encoded protein sequence identified the presence of four putative transmembrane domains. Similarity searches in protein domain databases identified partial sequence conservations with peripheral myelin protein 22 (PMP22)/ epithelial membrane proteins (EMPs) and Claudins, defining the encoded protein as representative of the existence of a novel subclass in this protein family. Northern-blot analysis of the expression of the corresponding mRNA in adult dog tissues revealed the presence of a huge amount of the 4 kb transcript in the brain. An EGFP-BCMP1 fusion protein expressed in transfected COS-7 cells exhibited a membranous localization as expected. The sequences encoding BCMP1 were assigned to chromosome X in dog, man and rat using radiation hybrid panels and were partly localized in the currently available human genome sequence. CONCLUSIONS: We have identified the existence in several mammalian species of a gene encoding a putative four-transmembrane protein, BCMP1, wich defines a novel subclass in this family of proteins. In dog at least, the corresponding mRNA is highly present in brain cells. The chromosomal localization of the gene in man makes of it a likely candidate gene for X-linked mental retardation

Contribution à l'étude du contrôle transcriptionnel dans la thyroïde

Doctorat en sciences médicalesinfo:eu-repo/semantics/nonPublishe

Cloning and characterization of the cDNA encoding Brain Cell Membrane Protein 1 (BCMP1) identifies a novel subclass of four transmembrane proteins

info:eu-repo/semantics/nonPublishe

Delimitation and functional characterization of the bidirectional THOX-DUOXA promoter regions in thyrocytes.

The THOX and DUOXA genes encode components of the oxidative machinery involved in thyroid hormone biosynthesis. Both of these genes are duplicated in mammalian genomes and are positioned in a head-to-head configuration, THOX1 facing DUOXA1 and THOX2 facing DUOXA2, respectively. The intergenic regions in both couples of genes exhibit dissimilar compositions, being highly GC-rich in the case of THOX1-DUOXA1 but not in the other case. In this study we localized precisely the transcription starts of all four genes using the RLM-RACE technique. It revealed that the distance between THOX1 and DUOXA1 transcription units is of about 70bp only, whereas THOX2 and DUOXA2 transcription starts are separated by 170bp. Analysis of these putative promoter regions revealed the presence of several potential binding sites for transcription factor Sp1 within the THOX1-DUOXA1 intergenic space, and of a TATA box and an Inr element in front of DUOXA2 and THOX2 genes, respectively. The putative promoter regions were inserted into a specifically designed vector harbouring two distinct reporter genes facing each other and their activity was investigated in transient transfection experiments in rat thyroid PCCl3 cells. Both regions exhibited bidirectional promoter activity in the assay. Gel shift experiments using extracts obtained from PCCl3 cells demonstrated the existence of at least one functional Sp1 binding site within the THOX1-DUOXA1 promoter. When Sp1 binding was abolished by mutation of the DNA sequence, a clear reduction in promoter activity in both THOX1 and DUOXA1 directions was observed in the functional assay. As these promoter sequences are well conserved in mammalian genomes, it appears very likely that the results we obtained here in the rat may be extended to the other species.Journal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe

The homopurine-homopyrimidine DNA sequence flanking the thyroglobulin gene does not behave as a transcriptional diode in living cells

info:eu-repo/semantics/publishe

Identification of a short basic peptide motif able to drive copy-number dependent nuclear accumulation of a linked protein.

The repetitive [RTRG](6) peptide was fortuitously identified as a potent nuclear localization signal when linked to the green fluorescent reporter protein. Replacing the arginines by lysines, or the threonines by glycines, both resulted in a decreased nuclear targeting ability of the peptide within this context. By contrast, the sequence [RT](12) proved able to drive nuclear accumulation of the linked protein as efficiently as the starting peptide. Remarkably, [RTRG](n) peptides where n=2 to 6 showed a gradual, copy-number dependent, increase in their ability to target the green fluorescent protein to the cell nucleus. As a consequence, the nuclear to cytoplasmic concentration ratio of the linked protein within the cell could be adjusted to different values depending on the number of repeats used in the fusion. Our observation may open the way to the use of [RTRG](n) repeats of given lengths (n=2 to 6) for fixing the nuclear-cytoplasmic partition of shuttling protein domains in the course of their functional study.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

The cloned promoter sequences of thyroid oxidase genes do not respond to TTF-1 or Pax8

info:eu-repo/semantics/nonPublishe

Human Thyroid Oxidases genes promoter activity in thyrocytes does not appear to be functionally dependent on Thyroid Transcription Factor-1 or Pax8.

Thyroid Oxidases (ThOX/DUOX) genes encode proteins that are thought to play a crucial role in the biosynthesis of thyroid hormone by providing the oxidizing agent required to allow the organification of iodine. The expression of these genes is not restricted to the thyroid, but the corresponding mRNAs are found in the thyrocyte more abundantly than in several other cell types. It raises the question whether the same transcription factors, namely Thyroid Transcription Factor-1 (TTF-1) and Pax8, that control the expression of other genes involved in the differentiated thyroid function, also regulate ThOX/DUOX gene transcription in the thyrocyte. We set up a functional co-transfection assay in which fusion proteins composed of the DNA-binding domain of either TTF-1 or Pax8 fused to the repressive domain of the drosophila engrailed protein were used to competitively counteract the activity of endogenous TTF-1 or Pax8 factor in the differentiated thyroid cell line PCCl3. Contrary to the Thyroglobulin or Thyroid Peroxidase promoter, the known regulatory elements of the human ThOX/DUOX genes displayed no reduction in transcriptional activity when either TTF-1 or Pax8 competitor was produced in the cell, indicating that the presently characterized control elements of human ThOX/DUOX genes are not responsive to these thyroid-specific transcription factors.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Delimitation and functional characterization of the bidirectional THOX-DUOXA promoter regions

34th Annual meeting of the European Thyroid Association, Lisbon (Portugal), September 5-9, 2009;info:eu-repo/semantics/publishe

Two binding sites for thyroid transcription factor 1 (TTF-1) determine the activity of the bovine thyroglobulin gene upstream enhancer element

A thyroid-specific enhancer element located upstream from the bovine thyroglobulin gene had been shown to contain three contiguous regions that are protected by thyroid transcription factor 1 (TTF-1) in footprinting experiments in vitro. The functional relevance of the individual TTF-1 binding sites was investigated in a transient assay in primary cultured thyrocytes. Using reporter constructs containing synthetic oligonucleotides overlapping the protected sequences we were able to show that only two out of the three TTF-1 binding sites exhibit transcription enhancing activity. Within the context of the complete enhancer sequence, the central 'inactive' TTF-1 site could be deleted whithout any consequence on the activity of the enhancer in the assay, whereas the presence of both terminal 'active' TTF-1 sites had previously been shown to be strictly required for enhancer function. Our results thus show that the activity of the bovine thyroglobulin upstream enhancer relies on the presence of a pair of TTF-1 binding sites separated by about 30 bp. These results also emphasize the need to assess experimentally the functional relevance of TTF-1 binding sites identified in footprinting experiments.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe