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THU0033 SET7/9 and SIRT1 induce promoter-driven collagen 2ALPHA1 expression in 3D cultured human chodrocytes
  1. H. Oppenheimer1,
  2. H. Meir1,
  3. A. Haze2,
  4. L. Kandel2,
  5. M. Leibergall2,
  6. M. Dvir-Ginzberg1
  1. 1Hebrew University of Jerusalem
  2. 2Hadassah Mount Scopus Hospital, Jerusalem, Israel

Abstract

Background Osteoarthritis (OA) is characterized by disrupted balance between extracellular matrix (ECM) synthesis and breakdown, leading to loss of articular cartilage (AC) and severe joint pain-dysfunction. Collagen type IIα1 (Col2α1) is a predominant cartilage-type ECM component that forms densely packed fibers which provide tensile force to the tissue, enabling it to withstand mechanical compression. Previous published findings establish that the protein deacetylase SirT1 is a positive regulator of Col2α1 expression. SirT1 achieves this by recruiting PGC1a and Sox9 to the enhancer region of Col2α1. Additional data in SirT1 overexpressing chondrocytes show that SirT1 is enriched on the promoter region of actively transcribed Col2α1 and reduced during OA development.

Objectives Here we propose to explore the SirT1-mediated promoter-driven mechanism of Col2α1 expression in human chondrocytes.

Methods Isolated human OA-derived (passage 2) chondrocytes were encapsulated in three dimensional (3D) alginate hydrogel microbeads and systematically compared to their equivalent monolayer (2D) cultures. Col2α1 expression was examined via confocal microscopy immunofluorescence, quantitative PCR (qPCR) and immunoblot analyses. SirT1 protein and RNA levels were analyzed via immunoblot and qPCR analyses, respectively. Chromatin immunoprecipitation (ChIP) for SirT1, SirT1-associated chromatin modifying enzymes (CMEs) and histone modifications were carried out on the Col2α1 promoter site. Sequential ChIP was executed to characterize SirT1 complex formation with CMEs on the Col2α1-promoter of actively transcribed Col2α1.

Results 3D-cultured human chondrocytes possessed augmented expression of Col2α1 as compared to their equivalent 2D cultures. ChIP analyses for the promoter of Col2α1 exhibited enrichment for SirT1 and the histone methyl transferase SET7/9 in 3D cultured chondrocytes vs. 2D cultures. Sequential-ChIP assays revealed that SirT1 and SET7/9 form a protein complex on the promoter region of Col2α1 and show elevated marks of Histone 3 Lysine 4 trimethylation (3MeH3K4), where Col2a1 is actively transcribed. Contrary to the enrichment SirT1 on the Col2a1 promoter site, ChIP assays revealed enhanced acetylation of its histone target H4K16 during Col2a1 expression.

Conclusions (i) 3D/2D experimental platforms provide a valuable tool for deciphering epigenetic mechanisms of gene regulation towards a better understanding of processes leading to tissue degeneration and disease, as in OA. (ii) Our preliminary data show that SirT1 and SET7/9 co-regulate COL2α1 expression through their enhanced association on its promoter region. Increased marks of histone acetylation especially in known SirT1 deacetylation targets (i.e. H4K16) may be a result the inhibitory effect of SET7/9 on the SirT1 deacetylase activity, consistent with previous reports. Expectedly, these investigations will provide mechanistic insight of cartilage ECM production, towards identifying novel epigenetic drug targets for the treatment of OA in susceptible individuals.

Disclosure of Interest None Declared

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