Summary C andida albicans biofilms are composed of highly adherent and densely arranged cells with properties distinct from those of free‐floating (planktonic) cells. These biofilms are a significant medical problem because they commonly form on implanted medical devices, are drug resistant and are difficult to remove. C . albicans biofilms are not static structures; rather they are dynamic and develop over time. Here we characterize gene expression in biofilms during their development, and by comparing them to multiple planktonic reference states, we identify patterns of gene expression relevant to biofilm formation. In particular, we document time‐dependent changes in genes involved in adhesion and metabolism, both of which are at the core of biofilm development. Additionally, we identify three new regulators of biofilm formation, Flo8, Gal4, and Rfx2, which play distinct roles during biofilm development over time. Flo8 is required for biofilm formation at all time points, and Gal4 and Rfx2 are needed for proper biofilm formation at intermediate time points.

Osteoarthritis (OA) is a complex multifactorial disease with a strong genetic component. Several studies have suggested or identified epigenetic events that may play a role in OA progression and the gene expression changes observed in diseased cartilage. The aim of this review is to inform about current research in epigenetics and epigenetics in OA. Epigenetic mechanisms include DNA methylation, histone modifications, and microRNAs. Collectively, these enable the cell to respond quickly to environmental changes and can be inherited during cell division. However, aberrant epigenetic modifications are associated with a number of pathological conditions, including OA. Advancements in epigenetic research suggests that global analysis of such modifications in OA are now possible, however, with the exception of microRNAs, it will be a significant challenge to demonstrate how such events impact on the disease.

GDF5 is involved in synovial joint development, maintenance and repair, and the rs143383 C/T single nucleotide polymorphism (SNP) located in the 5'UTR of GDF5 is associated, at the genome-wide significance level, with osteoarthritis susceptibility, and with other musculoskeletal phenotypes including height, congenital hip dysplasia and Achilles tendinopathy. There is a significant reduction in the expression of the disease-associated T allele relative to the C allele in synovial joint tissues, an effect influenced by a second SNP (rs143384, C/T) also within the 5'UTR. The differential allelic expression (DAE) imbalance of the C and T alleles of rs143383 varies intra- and inter-individually, suggesting that DAE may be modulated epigenetically. The C alleles of both SNPs form CpG dinucleotides that are potentially amenable to regulation by methylation. Here, we have examined whether DNA methylation regulates GDF5 expression and the allelic imbalance caused by rs143383. We observed methylation of the GDF5 promoter and 5'UTR in cell lines and joint tissues, with demethylation correlating with increased GDF5 expression. The CpG sites created by the C alleles at rs143383 and rs143384 were variably methylated, and treatment of a heterozygous cell line with a demethylating agent further increased the allelic expression imbalance between the C and T alleles. This demonstrates that the genetic effect of the rs143383 SNP on GDF5 expression is modulated epigenetically by DNA methylation. The variability in DAE of rs143383 is therefore partly accounted for by differences in DNA methylation that could influence the penetrance of this allele in susceptibility to common musculoskeletal diseases.

Desbuquois dysplasia (DBQD) is a severe condition characterized by short stature, joint laxity, and advanced carpal ossification. Based on the presence of additional hand anomalies, we have previously distinguished DBQD type 1 and identified CANT1 (calcium activated nucleotidase 1) mutations as responsible for DBQD type 1. We report here the identification of five distinct homozygous xylosyltransferase 1 (XYLT1) mutations in seven DBQD type 2 subjects from six consanguineous families. Among the five mutations, four were expected to result in loss of function and a drastic reduction of XYLT1 cDNA level was demonstrated in two cultured individual fibroblasts. Because xylosyltransferase 1 (XT-I) catalyzes the very first step in proteoglycan (PG) biosynthesis, we further demonstrated in the two individual fibroblasts a significant reduction of cellular PG content. Our findings of XYLT1 mutations in DBQD type 2 further support a common physiological basis involving PG synthesis in the multiple dislocation group of disorders. This observation sheds light on the key role of the XT-I during the ossification process.

Osteoarthritis is a degenerative joint disease characterized by a progressive and irreversible loss of the articular cartilage, due in main part to the cleavage of type II collagen within the matrix by the enzyme matrix metalloproteinase (MMP)13. Here, we examined the methylation status of MMP13 promoter and report the demethylation of specific CpG dinucleotides within its promoter in osteoarthritic compared to normal cartilage, which correlates with increased MMP13 expression. Of the promoter CpG sites examined, the -104 CpG was consistently demethylated following treatment of human articular chondrocytes with 10 μM DNA-methyltransferase inhibitor 5-aza-2'-deoxycytidine, again correlating with increased MMP13 expression. Methylation of the -104 CpG site resulted in reduced promoter activity in the chondrosarcoma cell line SW1353 as shown by CpG-free luciferase reporter. Using electrophoretic mobility shift assays, we identified CREB as the regulating factor able to only bind to the MMP13 promoter when the -104 CpG is demethylated, and confirmed this binding by chromatin immunoprecipitation. Finally, we demonstrated that CREB induces MMP13 expression only following treatment of SW1353 with 0.5 μM Ca(2+) ionophore A23187. In summary, the -104 CpG is demethylated in osteoarthritic cartilage, correlating with the elevated MMP13 expression and cartilage destruction, providing a highly novel link between epigenetic status and arthritic disease.

GDF5 encodes an extracellular signalling molecule that is essential for normal skeletal development. The rs144383 C to T SNP located in the 5ʹUTR of this gene is functional and has a pleiotropic effect on the musculoskeletal system, being a risk factor for knee-osteoarthritis (OA), congenital hip dysplasia, lumbar disc degeneration and Achilles tendon pathology. rs143383 exerts a joint-wide effect on GDF5 expression, with expression of the OA-associated T allele being significantly reduced relative to the C allele, termed allelic expression imbalance. We have previously reported that the GDF5 locus is subject to DNA methylation and that allelic imbalance of rs143383 is mediated by SP1, SP3 and DEAF1 transcriptional repressors. In this study, we have assayed GDF5 methylation in normal and osteoarthritic cartilage, and investigated the effect of methylation on the allelic imbalance of rs143383. We observed demethylation of the GDF5 5ʹUTR in OA knee cartilage relative to both OA (p = 0.009) and non-OA (p = 0.001) hip cartilage, with the most significant demethylation observed at the highly conserved +37 CpG site located 4 bp upstream of rs143383. Methylation modulates the level and direction of allelic imbalance of rs143383, with methylation of the +37 CpG dinucleotide within the SP1/SP3 binding site having an allele-specific effect on SP1 and SP3 binding. Furthermore, methylation attenuated the repressive effects of SP1, SP3 and DEAF1 on GDF5 promoter activity. This data suggest that the differential methylation of the +37 CpG site between osteoarthritic hip and knee cartilage may be responsible for the knee-specific effect of rs143383 on OA susceptibility.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-014-1447-z) contains supplementary material, which is available to authorized users.

Heparan sulfate proteoglycans (HSPGs), strategically located at the cell-tissue-organ interface, regulate major biological processes, including cell proliferation, migration, and adhesion. These vital functions are compromised in tumors, due, in part, to alterations in heparan sulfate (HS) expression and structure. How these modifications occur is largely unknown. Here, we investigated whether epigenetic abnormalities involving aberrant DNA methylation affect HS biosynthetic enzymes in cancer cells. Analysis of the methylation status of glycosyltransferase and sulfotransferase genes in H-HEMC-SS chondrosarcoma cells showed a typical hypermethylation profile of 3-OST sulfotransferase genes. Exposure of chondrosarcoma cells to 5-aza-2'-deoxycytidine (5-Aza-dc), a DNA-methyltransferase inhibitor, up-regulated expression of 3-OST1, 3-OST2, and 3-OST3A mRNAs, indicating that aberrant methylation affects transcription of these genes. Furthermore, HS expression was restored on 5-Aza-dc treatment or reintroduction of 3-OST expression, as shown by indirect immunofluorescence microscopy and/or analysis of HS chains by anion-exchange and gel-filtration chromatography. Notably, 5-Aza-dc treatment of HEMC cells or expression of 3-OST3A cDNA reduced their proliferative and invading properties and augmented adhesion of chondrosarcoma cells. These results provide the first evidence for specific epigenetic regulation of 3-OST genes resulting in altered HSPG sulfation and point to a defect of HS-3-O-sulfation as a factor in cancer progression.

Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of ␤1,4-galactosyltransferase 7 (␤4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human ␤4GalT7, we conducted a phylogenetic analysis of the ␤1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila ␤4GalT7-UDP as template. Two evolutionary conserved motifs, 163 DVD 165 and 221 FWGWGREDDE 230 , are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type ␤4GalT7 and selected mutants identified Trp 224 as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp 228 acting as general base in the reaction catalyzed by human ␤4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human ␤4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human ␤4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics.