**5.1 DNA methylation**

 DNA methylation involves the addition of a methyl group to a DNA at CpG dinucleotide, to convert cytosine to 5-methylcytosine. CpG islands are usually clustered near the promoter in about 30% of the gene. Methylation of these sequences results in silencing of these genes, and vice versa, hypo-methylation results in expression of the respective genes. DNA methylation factors are established and modified according to the environmental factors by three DNA methyltransferases (DNMT1, DNMT3a and DNMT3b). Earlier studies using chick embryos indicate the possible role of methylation in gene expression of type I and type II collagen in chondrocyte differentiation and dedifferentiation [50]. In our studies on chick chondrocytes in culture, we noticed a strong correlation of chondrocyte morphology to DNA methylation status as shown in **Figure 2**. The chondrocytes when treated with DNMT inhibitor 5-aza-2'deoxycytidine exhibit fibroblastic morphology and express type I and type X collagen with an upregulation of alkaline phosphatase enzyme [51]. Two CpG sites within the type X collagen promoter appear to be demethylated during MSC differentiation into chondrocyte morphology [52]. Recently, it was demonstrated that Wnt signalling caused both repressive chromatin mark (H3K27me3) and DNA methylation over the SOX9 promoter and that Wnt-induced irreversible silencing of Sox9 gene requires DNA methylation of this locus that is specifically countered by FGF signalling [53]. FGF blocks the recruitment of DNMT3a to the SOX-9 promoter by inducing the interaction and phosphorylation of DNMT3a by extracellular kinases ERK 1and ERK 2. Similarly, a number of studies indicated the control of Runx2 promoter activation by methylation. The number of MMP promoters show decreased methylation at single CpG island in OA cartilage as compared to normal.

### **5.2 Histone modifications**

Gene regulation is also controlled through the close packaging of eukaryotic DNA into nucleosomes. Nucleosomes are thought to be repressive for

#### **Figure 2.**

*The effect of culture conditions on the morphology of chondrocytes: When chick chondrocytes from caudal region sternum were grown in the presence of demethylation drug 5aza-2'deoxyctydine (5azadC), (A) the chondrocytes assume more flattened fibroblastic morphology and show no staining with alcian blue (stain specific for sulphate PG). However, the control chondrocyte without any treatment showed extensive ECM staining (B).* 

transcription; but through the post-translational modification of histones such as acetylation, phosphorylation, methylation and ubiquitination, this inhibition can be regulated.

Acetylation is mediated through acetyltransferase (HAT) and occurs on specific lysine residues on the N-terminal tails of histones, loosening the histones: DNA interactions, thus employing the access of transcriptional factors to the DNA. Deacetylation is of two types, one that requires Zn-catalysed deacetylation (HDAC) and the sirtuin deacetylase that requires NAD+, and removes these acetyl groups resulting in hypo-acetylation. Numerous transcriptional activators or repressors recruit HDAC and HAT activity.

Histone methylation is important for the formation of active and inactive genomic regions and is associated with transcription activation and silencing. Methylation of histone tails of lysine and arginine residues is catalysed by histone methyltransferase (HMT) and protein arginine methyltransferase (PRMT) which can add one or more methyl groups to regulate transcription [54]. Although histone methylation is more dynamic than DNA methylation, some specific histone methylation is tightly regulated and maintained through DNA replication. HDAC can block cytokine-induced PG release and cartilage resorption in cartilage explant model indicating that HDAC activity is important for the catabolic activity of chondrocytes [55, 56].

#### **5.3 Micro RNA**

MiRNA is a small 20–23 base pair-long cytoplasmic RNA that regulates post-transcriptional gene expression through binding to target mRNA. This interaction of miRNA with the target mRNA results in degradation of mRNA, thus suppression of translation. The first studied miRNA in cartilage was miR-140, which was first identified as cartilage restricted in developing zebrafish [57]. In humans, the expression of miR-140 increases during chondrogenesis and is more abundant in articular cartilage, but its expression is reduced in OA [58]. It has also been reported that the expression of miR-140 is regulated by the cartilage-specific master transcriptional factor Sox-9 in zebrafish and mammalian cells [59].
