Laboratory of Cartilage Biology, Institute of Dental Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
J Bone Miner Res. 2014 Feb;29(2):348-60. doi: 10.1002/jbmr.2052.
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.
Ⅱ型胶原是一种关键的软骨细胞外基质蛋白，对于正常的软骨内发育和软骨稳态至关重要。COL2A1 基因的表达受到 NAD 依赖性蛋白去乙酰化酶 Sirtuin 1（SirT1）的正向调控，SirT1 能够结合 COL2A1 启动子和增强子区域的染色质。尽管之前已经证明了 SirT1/Sox9 结合在 COL2A1 增强子位点上，但关于其在基因启动子位点上的功能作用知之甚少。在这里，我们研究了启动子相关的 SirT1 调控 COL2A1 表达的机制。将人软骨细胞包埋在三维（3D）藻酸盐珠中，与单层对照相比，它们的 COL2A1 mRNA 表达上调，并且 SirT1 在启动子和增强子区域的占有率增加。3D 培养物的染色质免疫沉淀（ChIP）分析显示，DNA 结合转录因子 SP1 和组蛋白甲基转移酶 Set7/9 的水平在 COL2A1 启动子位点上增加。ChIP 再 ChIP 实验表明，SirT1 和 Set7/9 在 3D 培养的软骨细胞的 COL2A1 启动子区域形成蛋白质复合物，该复合物还显示组蛋白 3 赖氨酸 4 三甲基化（3MeH3K4）水平升高，这是 Set7/9 甲基转移酶活性的标志。软骨细胞的高级传代导致 COL2A1 启动子上的 3MeH3K4 和 Set7/9 水平降低，COL2A1 表达减少，表明 SirT1/Set7/9 复合物优先形成在 COL2A1 启动子上，并需要基因激活。有趣的是，尽管 SirT1 占据，但在 3D 培养的软骨细胞的 COL2A1 启动子上发现其去乙酰化靶标（即 H3K9/14 和 H4K16）乙酰化。这种表型的一个可能解释是组蛋白乙酰转移酶 P300 和 GCN5 在 3D 培养的软骨细胞的 COL2A1 启动子上富集。我们的研究表明，Set7/9 阻止了 SirT1 的组蛋白去乙酰化酶活性，增强了 COL2A1 启动子上的常染色质形成，从而导致形态依赖性 COL2A1 基因反式激活。
