Browsing by Author "Conceição, N."
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- Comparative promoter analysis and its application to the identification of candidate regulatory factors of cartilage-expressed genesPublication . Conceição, N.; Cox, C. J.; Simões, B.; Viegas, M.; Cancela, LeonorChondrocyte gene regulation is important for the generation and maintenance of cartilage tissues. Analysis of the transcriptional regulation of cartilage-specific genes, encoding both collagenous and noncollagenous proteins, provides a useful strategy to identify transcription factors (TFs) that control chondrocyte specification and differentiation. Our work aims at the identification of candidate TFs important for cartilage maintenance and development through an in silico approach. In order to better define the transcriptional regulatory networks that affect chondrogenesis in zebrafish we propose a combination of comparative promoter analysis and transcription factor binding site analysis using a TRANSFAC position weight matrix search to identify cis-regulatory transcription factor binding motifs in a set of cartilage characteristic genes. With this methodology we have successfully identified several transcription factors known to be important for chondrogenesis thus validating our in silico approach.
- Evolutionary conservation of TFIIH subunits: Implications for the use of zebrafish as a model to study TFIIH function and regulationPublication . Silva, IAL; Cox, C. J.; Leite, Ricardo; Cancela, Leonor; Conceição, N.Transcriptional factor IIH (TFIIH) is involved in cell cycle regulation, nucleotide excision repair, and gene transcription. Mutations in three of its subunits, XPB, XPD, and TTDA, lead to human recessive genetic disorders such as trichothiodystrophy and xeroderma pigmentosum, the latter of which is sometimes associated with Cockayne's syndrome. In the present study, we investigate the sequence conservation of TFIIH subunits among several teleost fish species and compare their characteristics and putative regulation by transcription factors to those of human and zebrafish. We report the following findings: (i) comparisons among protein sequences revealed a high sequence identity for each TFIIH subunit analysed; (ii) among transcription factors identified as putative regulators, OCT1 and AP1 have the highest binding-site frequencies in the promoters of TFIIH genes, and (iii) TFIIH genes have alternatively spliced isoforms. Finally, we compared the protein primary structure in human and zebrafish of XPD and XPB – twoimportant ATP-dependent helicases that catalyse the unwinding of the DNA duplex at promoters during transcription – highlighting the conservation of domain regions such as the helicase domains. Our study suggests that zebrafish, a widely used model for many human diseases, could also act as an important model to study the function of TFIIH complex in repair and transcription regulation in humans.
- Gla-rich protein, a new player in tissue calcification?Publication . Cancela, Leonor; Conceição, N.; Laizé, VincentA novel g-carboxyglutamate (Gla)-containing protein, named Gla-rich protein (GRP) after its high content in Gla residues or upper zone of growth plate and cartilage matrix associated protein after its preferential expression by cartilage chondrocyte, was recently identified in sturgeon, mice, and humans through independent studies. GRP is the most densely g-carboxylated protein identified to date and its structure has been remarkably conserved throughout vertebrate evolution but is apparently absent from bird genomes. Several transcript and genomic variants affecting key protein features or regulatory elements were described and 2 paralogs were identified in the teleost fish genome. In the skeleton, most relevant levels of GRP gene expression were observed in cartilaginous tissues and associated with chondrocytes, suggesting a role in chondrogenesis. But GRP expression was also detected in bone cells, indicative of a more widespread role for the protein throughout skeletal formation. Although the molecular function of GRP is yet unknown, the high content of Gla residues and its accumulation at sites of pathological calcification in different human pathologies affecting skin or the vascular system and in breast cancer tumors suggest that GRP may function as a modulator of calcium availability. Because of its association with fibrillar collagens, GRP could also be involved in the organization and/or stabilization of cartilage matrix. Although transgenic mice did not reveal obvious phenotypic alterations in skeletal development or structure, zebrafish morphants lack craniofacial cartilage and exhibit limited calcification, suggesting a role for GRP during skeletal development, but additional functional data are required to understand its function.
- Identification of a promoter element within the zebrafish Collagen X¿1 gene responsive to Runx2 isoforms Osf2/Cbfa1 and til-1 but not to pebp2aA2Publication . Simões, B.; Conceição, N.; S B Viegas, Carla; Pinto, Jorge; Gavaia, Paulo J.; Kelsh, R. N.; Cancela, LeonorType X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xa1 gene (colXa1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2.
- Matrix Gla protein expression: a complex process involving the use of alternative promoters, multiple splicing events and microRNAsPublication . Cancela, Leonor; Laizé, Vincent; Conceição, N.; Tiago, Daniel; Maia, Ana-Teresa; Bensimon-Brito, A.; Gavaia, Paulo J.Matrix Gla protein (MGP) is a secreted vitamin K-dependent protein (VKD) located in the extracellular matrix and capable of binding calcium through its -carboxyglutamate residues. Although identified in 1983, transcriptional and post-transcriptional mechanisms regulating its expression remain unclear.
- Matrix Gla protein gene expression and protein accumulation co-localize with cartilage distribution during development of the teleost fish Sparus aurataPublication . Pinto, Jorge; Conceição, N.; Gavaia, Paulo J.; Cancela, LeonorMatrix Gla protein (MGP) is a member of the family of extracellular mineral-binding Gla proteins, expressed in several tissues with high accumulation in bone and cartilage. Although the precise molecular mechanism of action of this protein remains unknown, all available evidence indicates that MGP plays a role as an inhibitor of mineralization. We investigated the sites of gene expression and protein accumulation of MGP throughout development of the bony fish Sparus aurata, by in situ hybridization, Northern and RT-PCR Southern hybridization, and immunohistochemistry. The results obtained were compared with the patterns of developmental appearance of cartilaginous and mineralized structures in this species, identified by histological techniques and by detection of mRNA presence and protein accumulation of osteocalcin (Bone Gla protein), a marker for osteoblasts known to accumulate in bone mineralized extracellular matrix.
- Proteínas gla do osso e cartilagem: importância dos anfíbios e peixes como modelos biológicos para elucidação da sua função e evolução - gla proteins in bone and cartilage: the importance of fish and amphibian models to understand their function and evolutionPublication . Cancela, Leonor; Laizé, Vincent; Simes, D; Gavaia, Paulo J.; Conceição, N.; S B Viegas, Carla; Pombinho, A. R.; Marques, S.; Fidalgo, J.; Braga, D.Gla proteins, as the name indicates, undergo a post-translation modification where specific glutamic acid residues are γ-carboxylated through the action of the ubiquitous enzyme γ-carboxylase and using vitamin K as cofactor. Therefore, these proteins are also called vitamin K dependent proteins or VKD [1, 2]. We can assign VKDs to essentially four different groups: 1) those involved in blood coagulation (such as prothrombin and various coagulation factors; the first group to be discovered), 2) those involved in tissue mineralization (bone and matrix Gla proteins), 3) a nerve growth factor (gas6), and 4) those of unknown function (the latest group to be discovered). The carboxylase enzyme is present in fly and worm but not in yeast, indicating that γ-carboxylation is likely a feature appearing in multicellular eukaryotes.
- Zebrafish as a model to study craniofacial phenotypes related to human MEF2CmutationsPublication . Adrião, Andreia Lúcia Gonçalves; Conceição, N.; Gavaia, Paulo J.; Cancela, LeonorBackground: MEF2C is a transcription factor with important roles in differentiation and developmental processes, in particular in endochondral bone development. An association between human MEF2C gene mutations and particular phenotypes affecting craniofacial formation has been shown. Recent studies have shown that the zebrafish palate is under a genetic control similar to that controlling amniotic palatal skeleton, demonstrating the utility of this organism to study craniofacial development. Zebrafish has two mef2c genes and different lines of mutants are available. Therefore we have used zebrafish in an attempt to correlate specific MEF2C mutations with the abnormal human craniofacial phenotypes.