title: Central role of betaine-homocysteine S-methyltransferase 3 in chondral ossification and evidence for sub-functionalization in neoteleost fish.
abstract: BACKGROUND
  To better understand the complex mechanisms of bone formation it is fundamental that genes central to signaling/regulatory pathways and matrix formation are identified. Cell systems were used to analyze genes differentially expressed during extracellular matrix mineralization and bhmt3, coding for a betaine-homocysteine S-methyltransferase, was shown to be down-regulated in mineralizing gilthead seabream cells.
  
  
  METHODS
  Levels and sites of bhmt3 expression were determined by qPCR and in situ hybridization throughout seabream development and in adult tissues. Transcriptional regulation of bhmt3 was assessed from the activity of promoter constructs controlling luciferase gene expression. Molecular phylogeny of vertebrate BHMT was determined from maximum likelihood analysis of available sequences.
  
  
  RESULTS
  bhmt3 transcript is abundant in calcified tissues and localized in cartilaginous structures undergoing endo/perichondral ossification. Promoter activity is regulated by transcription factors involved in bone and cartilage development, further demonstrating the central role of Bhmt3 in chondrogenesis and/or osteogenesis. Molecular phylogeny revealed the explosive diversity of bhmt genes in neoteleost fish, while tissue distribution of bhmt genes in seabream suggested that neoteleostean Bhmt may have undergone several steps of sub-functionalization.
  
  
  CONCLUSIONS
  Data on bhmt3 gene expression and promoter activity evidences a novel function for betaine-homocysteine S-methyltransferase in bone and cartilage development, while phylogenetic analysis provides new insights into the evolution of vertebrate BHMTs and suggests that multiple gene duplication events occurred in neoteleost fish lineage.
  
  
  CONCLUSIONS
  High and specific expression of Bhmt3 in gilthead seabream calcified tissues suggests that bone-specific betaine-homocysteine S-methyltransferases could represent a suitable marker of chondral ossification.
authors.affiliation: Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal; PhD Program in Biomedical Sciences, Department of Biomedical Sciences and Medicine (DCBM), University of Algarve, Faro, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Rosa
authors.affiliation: Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Tiago
authors.affiliation: Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal.
authors.forename: Cátia L
authors.initials: CL
authors.lastname: Marques
authors.affiliation: Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal.
authors.forename: Parameswaran
authors.initials: P
authors.lastname: Vijayakumar
authors.affiliation: Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal.
authors.forename: Luis
authors.initials: L
authors.lastname: Fonseca
authors.affiliation: Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal; Department of Biomedical Sciences and Medicine (DCBM), University of Algarve, Faro, Portugal.
authors.forename: M Leonor
authors.initials: ML
authors.lastname: Cancela
authors.affiliation: Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal. Electronic address: vlaize@ualg.pt.
authors.forename: Vincent
authors.initials: V
authors.lastname: Laizé
mesh_terms: D000818:Animals; D050940:Betaine-Homocysteine S-Methyltransferase / Q000235:genetics / Q000378:metabolism*; D002356:Cartilage / Q000201:enzymology*; D002460:Cell Line; D020219:Chondrogenesis; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D010012:Osteogenesis; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D021541:Sea Bream / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D029941:Fish Proteins; D050940:Betaine-Homocysteine S-Methyltransferase
keywords: Betaine–homocysteine S-methyltransferase; Bone formation; Gene expression; Gilthead seabream Sparus aurata; In vitro mineralization; Molecular evolution; Taxonomic distribution; Transcriptional regulation