Set7/9 represses SirT1 to induce collagen type II expression in 3D cultured human chodrocytes

Abstract

Objective: Collagen type II (Col-II) plays a significant role in skeletal development and cartilage homeostasis and its loss with age is associated with degenerative joint diseases such as osteoarthritis (OA). Previously findings establish that the protein deacetylase SirT1 is required for enhanced expression of Col-IIa1, a major cartilage structural component of Col-II. Here we profile the mechanism by which SirT1 activates promoter-driven Col-IIa1 expression. Methods: Chondrocytes derived from human osteoarthritic knee joints were encapsulated in three-dimensional alginate hydrogel microbeads (3D) and compared to paired monolayer (2D, passage 2) cultures derived from the same donor tissue. 3D-cultured cells displayed augmented expression of Col-IIa1 and were subject to serial chromatin immunoprecipitation (ChIP) and ChIP reChIP analyses to characterize epigenetic variations as compared to 2D cultures. Results: ChIP analyses exhibited enrichment for SirT1 and the histone methyl transferase Set7/9, which formed a complex in 3D cultures wherein Col-IIa1 was augmented in expression. Consistent with enriched Set7/9 levels on the Col-IIa1 promoter, ChIP data revealed enriched levels of trimethylation on histone 3 lysine 4 (3MeH3K4). Acetylated H3K9/14 and H4K16 on Col-IIa1 promoters of 3D-cultured chondrocytes were also elevated indicating that SirT1 is enzymatically repressed by Set7/9 on the transactivated Col-IIa1 promoter. Elevated marks of the Histone acetyl transferases GCN5 and P300 also supported the local enrichment of their acetylated histone marks (i.e. H3K9/ 14,H4K5 and H4K16). Conclusions: The data support that Set7/9 is capable of locally repressing SirT1 enzymatic activity on the promoter of Col-IIa1, resulting in its augmented expression dependent on cell morphology.