Genome-wide DNA methylation profile implicates potential cartilage regeneration at the late stage of knee osteoarthritis.

OBJECTIVE: The aim of this work was to characterize the genome-wide DNA methylation profile of cartilage from three regions of tibial plateau isolated from patients with primary knee osteoarthritis (OA), providing the first DNA methylation study that reflects OA progression. METHODS: The unique model system was used to section three regions of tibial plateau: the outer lateral tibial plateau (oLT), the inner lateral tibial plateau (iLT) and the inner medial tibial plateau (iMT) regions which represented the early, intermediate and late stages of OA, respectively. Genome-wide DNA methylation profile was examined using Illumina Infinium HumanMethylation450 BeadChip array. Comparisons of the iLT/oLT and iMT/oLT groups were carried out to identify differentially methylated (DM) probes (DMPs) associated with OA progression. DM genes were analyzed to identify the gene ontologies (GO), pathways, upstream regulators and networks. RESULTS: No significant DMPs were identified in iLT/oLT group, while 519 DMPs were identified in iMT/oLT group. Over half of them (68.2%) were hypo-methylated and enriched in enhancers and OpenSea. Upstream regulator analysis identified many microRNAs. DM genes were enriched in transcription factors, especially homeobox genes and in Wnt/ß-catenin signaling pathway. These genes also showed changes in expression when analyzed with expression profiles generated from previous studies. CONCLUSION: Our data suggested the changes in DNA methylation occurred at the late stage of OA. Pathways and networks enriched in identified DM genes highlighted potential etiologic mechanism and implicated the potential cartilage regeneration in the late stage of knee OA.

Activation of influenza virus RNA polymerase by the 5' and 3' terminal duplex of genomic RNA.

The current model for influenza virus mRNA transcription involves the sequential interaction of the viral polymerase with the 5'- and 3'-ends of vRNA, with each RNA-protein interaction triggering a polymerase function necessary for cap-primed transcription. Here we show that the order in which this ternary complex is assembled is in fact important. Polymerase bound simultaneously to a pre-annealed duplex of the 5'- and 3'-ends of vRNA had greatly increased levels of primer binding and endonuclease activities compared to a sequentially assembled complex. Increased primer binding was due to the activation of a high affinity binding site with a preference for primer length RNAs. This correlated with enhanced levels of cap-primed transcription. Polymerase that was bound initially to just 5' vRNA had low primer binding activity, but was endonucleolytically active. Neither activity was significantly increased by the subsequent addition of 3' vRNA, and this sequentially assembled complex had correspondingly low mRNA transcription activity. Nevertheless, both routes of assembly led to complexes that were highly competent for dinucleotide ApG-primed transcription. Therefore, polymerase complexes assembled on pre-annealed 5' and 3' terminal viral RNA sequences have distinct properties from those assembled by sequential loading of polymerase onto the 5'-end followed by the 3'-end. This suggests a mechanism by which the virus couples transcription initiation and termination during mRNA transcription.

Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase.

The first 11 nt at the 5' end of influenza virus genomic RNA were shown to be both necessary and sufficient for specific binding by the influenza virus polymerase. A novel in vitro transcription assay, in which the polymerase was bound to paramagnetic beads via a biotinylated 5'-vRNA oligonucleotide, was used to study the activities of different forms of the polymerase. Complexes composed of co-expressed PB1/PB2/PA proteins and a sub-complex composed of PB1/PA bound to the 5'-vRNA oligonucleotide, whereas PB1 expressed alone did not. The enriched 5'-vRNA/PB1/PB2/PA complex was highly active for ApG and globin mRNA primed transcription on a model 3'-vRNA template. RNA synthesis in the absence of added primers produced products with 5'-terminal tri- or diphosphate groups, indicating that genuine unprimed initiation of transcription also occurred. No transcriptase activity was detected for the PB1/PA complex. These results demonstrate a role for PA in the enhancement of 5' end binding activity of PB1, a role for PB2 in the assembly of a polymerase complex able to perform both cap-dependent and -independent synthesis and that NP is not required for the initiation of replicative transcription.

Palmitoyl acyltransferase, Zdhhc13, facilitates bone mass acquisition by regulating postnatal epiphyseal development and endochondral ossification: a mouse model.

ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis.

Genetic determinants of warfarin dosing in the Han-Chinese population.

UNLABELLED: Warfarin, a widely prescribed oral anticoagulant, is used for the prevention of thromboembolism. Polymorphisms in CYP2C9 and VKORC1 have been shown to be associated with warfarin dose requirements. However, it is likely that other genes could also affect warfarin dose. AIMS: In this study, we aimed to identify additional genes influencing warfarin dosing in the Han-Chinese population. MATERIALS & METHODS: In this study, we screened for SNPs in 13 genes (VKORC1, CYP2C9, CYP2C18, PROC, APOE, EPHX1, CALU, GGCX, ORM1, ORM2, factor II, factor VII and CYP4F2) and tested their associations with warfarin dosing with univariate and multiple regression analysis. RESULTS: Polymorphisms in the VKORC1 gene have the strongest effects on warfarin dose, followed by CYP2C9*3. In addition, our results showed that CYP2C18, PROC and EPHX1 have small but significant associations with warfarin dose. In multiple regression analysis, PROC and EPHX1 explained 3% of the dose variation. The incorporation of these two genes into warfarin dosing algorithms could improve the accuracy of prediction in the Han-Chinese population.

A novel functional VKORC1 promoter polymorphism is associated with inter-individual and inter-ethnic differences in warfarin sensitivity.

Warfarin, a commonly prescribed anticoagulant, exhibited large inter-individual and inter-ethnic differences in the dose required for its anticoagulation effect. Asian populations, including Chinese, require a much lower maintenance dose than Caucasians, for which the mechanisms still remain unknown. We determined DNA sequence variants in CYP2C9 and VKORC1 in 16 Chinese patients having warfarin sensitivity (< or = 1.5 mg/day, n = 11) or resistance (> or = 6.0 mg/day, n = 5), 104 randomly selected Chinese patients receiving warfarin, 95 normal Chinese controls and 92 normal Caucasians. We identified three CYP2C9 variants, CYP2C9*3, T299A and P382L, in four warfarin-sensitive patients. A novel VKORC1 promoter polymorphism (-1639 G > A) presented in the homozygous form (genotype AA) was found in all warfarin-sensitive patients. The resistant patients were either AG or GG. Among the 104 randomly selected Chinese patients receiving warfarin, AA genotype also had lower dose than the AG/GG genotype (P < 0.0001). Frequencies of AA, AG and GG genotypes were comparable in Chinese patients receiving warfarin (79.7, 17.6 and 2.7%) and normal Chinese controls (82, 18 and 0%), but differed significantly from Caucasians (14, 47 and 39%) (P < 0.0001). The promoter polymorphism abolished the E-box consensus sequences and dual luciferase assay revealed that VOKRC1 promoter with the G allele had a 44% increase of activity when compared with the A allele. The differences in allele frequencies of A/G allele and its levels of VKORC1 promoter activity may underscore the inter-individual differences in warfarin dosage as well as inter-ethnic differences between Chinese and Caucasians.