Set7/9 Impacts COL2A1 Expression Through Binding and Repression of SirT1 Histone Deacetylation

Abstract

Type II collagen is a key cartilaginous extracellular protein required for normal endochondral development and cartilage homeostasis. COL2A1 gene expression is positively regulated by the NAD-dependent protein deacetylase Sirtuin 1 (SirT1), through its ability to bind chromatin regions of the COL2A1 promoter and enhancer. Although SirT1/Sox9 binding on the enhancer site of COL2A1 was previously demonstrated, little is known about its functional role on the gene promoter site. Here, we examined the mechanism by which promoter-associated SirT1 governs COL2A1 expression. Human chondrocytes were encapsulated in three-dimensional (3D) alginate beads where they exhibited upregulated COL2A1 mRNA expression and increased levels of SirT1 occupancy on the promoter and enhancer regions, when compared to monolayer controls. Chromatin immunoprecipitation (ChIP) analyses of 3D cultures showed augmented levels of the DNA-binding transcription factor SP1, and the histone methyltransferase Set7/9, on the COL2A1 promoter site. ChIP reChIP assays revealed that SirT1 and Set7/9 form a protein complex on the COL2A1 promoter region of 3D-cultured chondrocytes, which also demonstrated elevated trimethylated lysine 4 on histone 3 (3MeH3K4), a hallmark of Set7/9 methyltransferase activity. Advanced passaging of chondrocytes yielded a decrease in 3MeH3K4 and Set7/9 levels on the COL2A1 promoter and reduced COL2A1 expression, suggesting that the SirT1/Set7/9 complex is preferentially formed on the COL2A1 promoter and required for gene activation. Interestingly, despite SirT1 occupancy, its deacetylation targets (ie, H3K9/14 and H4K16) were found acetylated on the COL2A1 promoter of 3D-cultured chondrocytes. A possible explanation for this phenotype is the enrichment of the histone acetyltransferases P300 and GCN5 on the COL2A1 promoter of3 D-cultured chondrocytes. Our study indicates that Set7/9 prevents the histone deacetylase activity of SirT1, potentiating euchromatin formation on the promoter site of COL2A1 and resulting in morphology-dependent COL2A1 gene transactivation.

Acetylation reduces SOX 9 nuclear entry and ACAN gene transactivation in human chondrocytes

“…(2014). Briefly, first‐passage (P1) cultured human chondrocytes were mixed with a 1.25% sodium alginate solution to obtain a final concentration of 1 × 10 6 cells mL −1 .…”

“…Protein, mRNA and chromatin were isolated from alginate bead cultures as previously described by Oppenheimer et al . (2014). …”

“…Previous studies uncovered a key role for the nicotinamide adenine dinucleotide (NAD)‐dependent class III protein deacetylase SIRTUIN 1 (SIRT1) in cartilage homeostasis and gene expression (Dvir‐Ginzberg et al ., 2008; Oppenheimer et al ., 2014) While SIRT1 was shown to induce transcriptional repression in many cell types (Carafa et al ., 2012), the unexpected, contrasting discovery was made that it strikingly potentiated SOX9 gene targets (i.e. ACAN and COL2A1 ) in human chondrocytes (Dvir‐Ginzberg et al ., 2008; Dvir‐Ginzberg & Steinmeyer, 2013; Oppenheimer et al ., 2014).…”

“…ACAN and COL2A1 ) in human chondrocytes (Dvir‐Ginzberg et al ., 2008; Dvir‐Ginzberg & Steinmeyer, 2013; Oppenheimer et al ., 2014). In vitro studies showed that SIRT1 bound to and deacetylated SOX9, but did not affect SOX9 binding to the COL2A1 enhancer, as observed by chromatin immunoprecipitation assays of chondrocytes overexpressing wild‐type SIRT1 and a dominant negative H355Y SIRT1 mutant (Dvir‐Ginzberg et al ., 2008).…”

“…Further studies suggested that most of SIRT1 action on COL2A1 expression occurred through recruitment of the histone methyl‐transferase SET7/9 and other histone acetyl transferases (i.e. P300 and GCN5) to the promoter and enhancer sites of the gene (Dvir‐Ginzberg et al ., 2008; Oppenheimer et al ., 2014). On the other hand, the mechanisms whereby SIRT1 may regulate ACAN expression are yet unknown.…”