Unmethylated thyroglobulin promoter may be repressed by methylation of flanking DNA sequences.

The thyroglobulin gene, like many other tissue-specific genes, appears to be specifically less methylated in the differentiated cell type where it is transcribed. The thyroglobulin gene promoter elements themselves are highly CG-deficient and do not contain any HpaII/MspI sites. In this study, using DNA constructs that were methylated in vitro with HpaII or MspI methylases, we show that DNA methylation of vector sequences is sufficient to repress the activity of the thyroglobulin gene promoter in transient transfection experiments. Reporter-gene expression from a plasmid containing only the proximal thyroglobulin gene promoter is sensitive to DNA methylation even in fully differentiated thyrocytes. Transcription from methylated plasmids containing the thyroglobulin gene enhancer and proximal promoter is also clearly reduced when the transfected cells are maintained under less-differentiated conditions. These results indicate that DNA methylation can influence, from a distance, the activity of an unmodified promoter. Our results also agree with the view that loss of DNA methylation does not constitute a prerequisite for thyroglobulin gene expression in differentiated thyrocytes, where the thyroglobulin gene enhancer and promoter are activated. However, the production of thyroglobulin transcripts could be severely impaired when this activation is not maximal, as is the case in less-differentiated cells or when the enhancer element is lacking. We suggest that DNA methylation helps to maintain the thyroglobulin gene in an inactive state unless all of the conditions required for its expression are fulfilled, and that the thyroid-specific demethylation events are a consequence of the activation state of the gene

Expression of TMEM47 gene during mouse embryogenesis

info:eu-repo/semantics/nonPublishe

TM4SF10 gene sequencing in XLMR patients identifies common polymorphisms but no disease-associated mutation

BACKGROUND: The TM4SF10 gene encodes a putative four-transmembrane domains protein of unknown function termed Brain Cell Membrane Protein 1 (BCMP1), and is abundantly expressed in the brain. This gene is located on the short arm of human chromosome X at p21.1. The hypothesis that mutations in the TM4SF10 gene are associated with impaired brain function was investigated by sequencing the gene in individuals with hereditary X-linked mental retardation (XLMR). METHODS: The coding region (543 bp) of TM4SF10, including intronic junctions, and the long 3' untranslated region (3 233 bp), that has been conserved during evolution, were sequenced in 16 male XLMR patients from 14 unrelated families with definite, or suggestive, linkage to the TM4SF10 gene locus, and in 5 normal males. RESULTS: Five sequence changes were identified but none was found to be associated with the disease. Two of these changes correspond to previously known SNPs, while three other were novel SNPs in the TM4SF10 gene. CONCLUSION: We have investigated the majority of the known MRX families linked to the TM4SF10 gene region. In the absence of mutations detected, our study indicates that alterations of TM4SF10 are not a frequent cause of XLMR.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

High-Content Microscopy Identifies New Neurite Outgrowth Regulators

Neurons, with their long axons and elaborate dendritic arbour, establish the complex circuitry that is essential for the proper functioning of the nervous system. Whereas a catalogue of structural, molecular, and functional differences between axons and dendrites is accumulating, the mechanisms involved in early events of neuronal differentiation, such as neurite initiation and elongation, are less well understood, mainly because the key molecules involved remain elusive. Here we describe the establishment and application of a microscopy-based approach designed to identify novel proteins involved in neurite initiation and/or elongation. We identified 21 proteins that affected neurite outgrowth when ectopically expressed in cells. Complementary time-lapse microscopy allowed us to discriminate between early and late effector proteins. Localization experiments with GFP-tagged proteins in fixed and living cells revealed a further 14 proteins that associated with neurite tips either early or late during neurite outgrowth. Coexpression experiments of the new effector proteins provide a first glimpse on a possible functional relationship of these proteins during neurite outgrowth. Altogether, we demonstrate the potential of the systematic microscope-based screening approaches described here to tackle the complex biological process of neurite outgrowth regulation