ESET histone methyltransferase is essential to hypertrophic differentiation of growth plate chondrocytes and formation of epiphyseal plates

Abstract

The ESET (also called SETDB1) protein contains an N-terminal tudor domain that mediates protein-protein interactions and a C-terminal SET domain that catalyzes methylation of histone H3 at lysine 9. We report here that ESET protein is transiently upregulated in prehypertrophic chondrocytes in newborn mice. To investigate the in vivo effects of ESET on chondrocyte differentiation, we generated conditional knockout mice to specifically eliminate the catalytic SET domain of ESET protein only in mesenchymal cells. Such deletion of the ESET gene caused acceleration of chondrocyte hypertrophy in both embryos and young animals, depleting chondrocytes that are otherwise available to form epiphyseal plates for endochondral bone growth. ESET-deficient mice are thus characterized by defective long bone growth and trabecular bone formation. To understand the underlying mechanism for ESET regulation of chondrocytes, we carried out co-expression experiments and found that ESET associates with histone deacetylase 4 to bind and inhibit the activity of Runx2, a hypertrophy-promoting transcription factor. Repression of Runx2-mediated gene transactivation by ESET is dependent on its H3-K9 methyltransferase activity as well as its associated histone deacetylase activity. In addition, knockout of ESET is associated with repression of Indian hedgehog gene in pre- and early hypertrophic chondrocytes. Together, these results provide clear evidence that ESET controls hypertrophic differentiation of growth plate chondrocytes and endochondral ossification during embryogenesis and postnatal development.

Differentially Expressed MicroRNAs in Chondrocytes from Distinct Regions of Developing Human Cartilage

“…For immunofluorescent antibody (Ab) staining, de-paraffinized sections were treated with 1% hyaluronidase (Sigma) for 30 min at 37°C. Sections were rinsed with 1× PBS and blocked with 10% goat serum for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: 1) a rabbit polyclonal “anti-IIA” antibody (Ab) that recognizes the exon 2-encoded cysteine-rich domain of the amino propeptide of type II procollagen (1/400 dilution) [43]; 2) a rat polyclonal Ab against the triple helical domain of type II collagen (1/100 dilution) [44]; 3) a rabbit polyclonal Ab against type I collagen (1/300 dilution) (abcam®); and 4) a rabbit polyclonal Ab against the hypertrophic chondrocyte marker, type X collagen (1/1000 dilution) [45], [46]. Each antibody was diluted in 2% goat serum.…”

The Role of MicroRNAs and Their Targets in Osteoarthritis

“…It is now well established that epigenetic regulation by methyltransferase enzymes is important for chondrocyte differentiation and may play a role in OA pathology [156-158]. MicroRNA-370 and miR-373 target hydroxymethyltransferase-2 (SHMT-2) and methyl-CpG-binding protein-2 (MECP-2) [159].…”

O-GlcNAc Modification of the runt-Related Transcription Factor 2 (Runx2) Links Osteogenesis and Nutrient Metabolism in Bone Marrow Mesenchymal Stem Cells

“…In chondrocytes the functional interaction between the H3K9 histone methyltransferase ESET (i.e. SETDB1), Runx2, and HDAC4 represses Runx2 responsive genetic programs that regulate terminal chondrocyte differentiation during endochondral ossification, and mesenchymal ESET-deficient mice display premature chondrocyte hypertrophy, lower levels of trabecular bone formation, and defective expression of mineralization markers in long bones (81). Although it has yet to be determined whether direct methylation of Runx2 affects its function, inhibition of SAM methyltransferases in C2C12 stromal cells attenuated BMP2-induced activation of Runx2 (78).…”