Left ventricular mass (LVM) and cardiac gene expression are complex traits regulated by factors both intrinsic and extrinsic to the heart. To dissect the major determinants of LVM, we combined expression quantitative trait locus1 and quantitative trait transcript2 (QTT) analyses of the cardiac transcriptome in the rat. Using these methods and in vitro functional assays, we identified osteoglycin (Ogn) as a major candidate regulator of rat LVM, with increased Ogn protein expression associated with elevated LVM. We also applied genome-wide QTT analysis to the human heart and observed that, out of ~22,000 transcripts, OGN transcript abundance had the © 2008 Nature Publishing Group Correspondence should be addressed to T.J.A. (t.aitman@csc.mrc.ac.uk) or S.A.C. (stuart.cook@imperial.ac.uk).. 11 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS The study was designed by S.A.C., E.P. and T.J.A.; S.A.C. obtained funding, supervised the study and coordinated the collaborations; R.S. performed PCR-based experiments and genotyping; H.L. and M.K.K. generated rat microarray data; B.S. and Y.M.P. generated human microarray data; M.B. generated immunofluorescence confocal micrographs; R.S. and H.L. performed cell culture and cloning experiments; R.S., B.S. and M.B. performed immunoblotting; P.J.M. and R.S. performed in vivo analyses in Ogn knockout mice; E.S.T., L.M.C., M.D.W. and G.W.C. provided and genotyped the Ogn knockout mice; N.H. and J.F. carried out sequence analysis of Ogn; T.W.K., V.K. and M.P. provided telemetric blood pressure data; P.P.P. provided human tissues for protein studies; S.K.P., D.J.P. and C.K. provided the human cardiac MRI data; E.P. designed, interpreted and supervised all statistical analyses; E.P., I.G. and R.S. performed statistical and bioinformatic analyses and were aided by J.M.; and E.P. and S.A.C. wrote the manuscript.Note: Supplementary information is available on the Nature Genetics website.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions Europe PMC Funders GroupAuthor Manuscript Nat Genet. Author manuscript; available in PMC 2009 September 11. Elevated indexed LVM is a major cause of morbidity and mortality and is regulated, in part, by hemodynamic indices3. However, only a small proportion of LVM variation is determined by hemodynamic effects4, and it has been proposed that genetic influences may also be important5,6. Many studies have shown that gene expression is heritable and that the genetic control of transcription affects physiological traits and disease phenotypes1,7. We have shown that gene transcription is highly heritable in the rat heart8, which led us to hypothesize that the genetic control of cardiac gene expression may be important in regulating LVM. Here, we used an integrated approach combining correlation of expression quantitative trait loci (eQTL)1 and genome-wide expression profiles with physiological traits, previously designated as quantitative trait transcript (QTT) analysis2, to ide...

Bovine cornea contains three unique keratan sulfate proteoglycans (KSPGs), of which two (lumican and keratocan) have been characterized using molecular cloning. The gene for the third protein (KSPG25) has not been identified. This study examined the relationship between the KSPG25 protein and the gene for osteoglycin, a 12-kDa bone glycoprotein. The N-terminal amino acid sequence of KSPG25 occurs in osteoglycin cDNA cloned from bovine cornea. The osteoglycin amino acid sequence makes up the C-terminal 47% of the deduced sequence of the KSPG25 protein. Antibodies to osteoglycin reacted with intact corneal KSPG, with KSPG25 protein, and with a 36-kDa protein, distinct from lumican and keratocan. KSPG25-related proteins, not modified with keratan sulfate, were also detected in several connective tissues. Northern blot analysis showed mRNA transcripts of 2.4, 2.5, and 2.6 kilobases in numerous tissues with the 2.4-kilobase transcript enriched in ocular tissues. Ribonuclease protection analysis detected several protected KSPG25 mRNA fragments, suggesting alternate splicing of KSPG25 transcripts. We conclude that the full-length translation product of the gene producing osteoglycin is a corneal keratan sulfate proteoglycan, also present in many non-corneal tissues without keratan sulfate chains. The multiple size protein products of this gene appear to result from in situ proteolytic processing and/or alternative splicing of mRNA. The name mimecan is proposed for this gene and its products.The corneal stroma of vertebrate organisms contains a unique class of molecules, corneal keratan sulfate proteoglycans (KSPGs), 1 consisting of several related proteins each bearing keratan sulfate. The specialized nature of corneal proteoglycans was recognized almost 60 years ago with the initial description of keratan sulfate, the most abundant glycosaminoglycan in cornea (1). Corneal keratan sulfate is a highly sulfated, linear polymer of N-acetyllactosamine, linked to asparagine residues in the KSPG core proteins (2). The unusual abundance of keratan sulfate in the cornea and studies of heritable metabolic diseases suggest that the KSPG molecules are essential in maintenance of corneal transparency (3, 4). Understanding the role of the KSPGs in corneal transparency, their interactions with cells and other matrix molecules, and the tissue-specific nature of their biosynthesis requires complete knowledge of their structure. Although we have excellent information regarding the carbohydrate components of the KSPGs, the core proteins as representatives of the primary KSPG gene products are still not fully understood.Research from our laboratory has shown that keratan sulfate is attached to three unique proteins in bovine cornea (5, 6). The cDNAs for two of these proteins, keratocan and lumican (originally designated 37A and 37B), have been cloned and sequenced (7,8). A third 25-kDa KSPG core protein, KSPG25, was recognized as unique from lumican and keratocan, but its primary sequence is not yet known. The deduced amino acid sequence...

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [35 S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor ␤, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.The corneal stroma is a disc of connective tissue that constitutes about 90% of the mammalian cornea. This tissue consists of a unique transparent extracellular matrix populated by keratocytes, flattened mesenchymal cells responsible for production and maintenance of this matrix. In healing corneal wounds keratocytes become activated, begin mitosis, and migrate to the wound location, where they secrete nontransparent scar components (1, 2). Cells in the healing wound are characterized by secretion of pro-inflammatory cytokines such as interleukin-1␣ and proteolytic enzymes involved in tissue remodeling, collagenase, gelatinase, and stromelysin (3, 4). This remodeling (fibroblastic) phenotype is simulated in vitro when keratocytes are cultured in medium containing fetal bovine serum and subcultured by trypsinization (5).The extracellular matrix of the normal corneal stroma is characterized by a unique class of molecules known as the corneal keratan sulfate proteoglycans (KSPG). 1 These consist of three structurally related proteins modified with N-linked keratan sulfate chains (6). The three proteins (lumican, keratocan, and mimecan) are found in a number of connective tissues but are expressed at much higher levels in the cornea compared with noncorneal tissues (7-9). In noncorneal tissues, these proteins are not modified with keratan sulfate (10). The high level of expression of these three proteins combined with a specialized glycosylation is a property unique to keratocytes and constitutes an essential feature of the role of the keratocyte in maintenance of corneal transparency. This conclusion is supported by the recent demonstration that mice bearing null mutations in the lumican gene lose corneal transparency, whereas mice with a similar...

Corneal stromal GAGs bind, and thus could alter the availability or conformation of, many proteins that may influence keratocyte and nerve growth cone behavior in the cornea.

Densely methylated DNA sequence islands, designated DMIs, have been observed in two Chinese hamster cell chromosomal replication origins by using a PCR-based chemical method of detection. One of the origins, on5S1, is located within or adjacent to the coding sequence for ribosomal protein S14 on chromosome 2q, and the other, ori-P, is -17 kbp downstream of the dhfr (dihydrofolic acid reductase) locus on chromosome 2p. The DMI in oris14 is 127 bp long, and the DMI in ori-is 516 bp long. Both DMIs are bilaterally methylated (i.e., all dCs are modified to 5-methyl dC) only in cells that are replicating their DNA. When cell growth and DNA replication are arrested, methylation of CpA, CpT, and CpC dinucleotides is lost and the sequence islands display only a subset of their originally methylated CpG dinucleotides. Several possible roles for DMImediated regulation of mammalian chromosomal origins are considered.To investigate the relationship between DNA cytosine methylation and site-specific point mutations in mammalian genes, we analyzed the positions of cytosine methylation in genomic DNA encoding the cloned Chinese hamster ovary (CHO) cell gene for ribosomal protein S14 (RPS14) and compared the methylation sites detected with the position of a recurrent transition mutation affecting the gene's fifth exon (39). During the course of that study, we noted an unusual methylation pattern within a short stretch of the DNA sequence at the 3' end of the gene. Although cytosine methylation at the 5' end and middle of CHO RPS14 occurs exclusively at CpG dinucleotides, in exponentially growing cells actively engaged in DNA replication, we found a unique chromosome segment at the 3' end of the gene in which every dC residue was methylated. Within this densely methylated island (DMI), both DNA strands were fully methylated, and 5-methyl cytosines were detected in CpA, CpT, and CpC as well as CpG dinucleotides.In the accompanying paper, we report that the 3' end of the CHO RPS14 locus also encodes an early-S-phase origin of bidirectional chromosomal DNA replication (OBR) designated oiS14 (40