Determine the effects of Hdac3 deletion on H3K27ac profiles in murine chondrocytes[RNA-Seq]

Histone deacetylase inhibitors are efficacious epigenetic-based therapies for some cancers and neurological disorders; however, these drugs inhibit multiple Hdacs and have detrimental effects on the pre- and post-natal skeleton. To better understand how Hdac inhibitors affect the skeleton, we focused on understanding the role of one of their targets, Hdac3, in endochondral bone formation by deleting it in immature murine chondrocyte micro masses with Adeno-Cre. Hdac3-deficient chondrocytes expressed higher levels of pro-inflammatory and matrix degrading genes (e.g., Il-6, Mmp3, Mmp13, Saa3) and lower levels of genes related to the extracellular matrix production, bone development and ossification (e.g., Acan, Col2a1, Ihh, Col10a1). Histone acetylation was increased in and around genes with elevated expression. Overall design: High Throughput RNA sequencing and Chromatin immunopreciptation sequencing experiments were performed in chondrocyte cultures. Differential analysis was conducted on ChIP-seq and RNA-seq data to identify H3K27Ac profile for up and down regulated genes in Hdac3-deficient murine chondrocytes.

组蛋白去乙酰化酶抑制剂（Histone deacetylase inhibitors）是针对部分癌症及神经系统疾病的有效表观遗传学疗法；然而此类药物会抑制多种HDAC（组蛋白去乙酰化酶），并对产前及产后骨骼造成不良影响。为更深入阐明HDAC抑制剂对骨骼的作用机制，本研究聚焦于其靶标之一HDAC3在软骨内骨形成中的功能，通过腺病毒-Cre（Adeno-Cre）在未成熟小鼠软骨细胞微团培养物中敲除该基因展开相关研究。实验结果显示，HDAC3缺陷型软骨细胞中，促炎与基质降解相关基因（如Il-6、Mmp3、Mmp13、Saa3）的表达水平显著升高，而与细胞外基质生成、骨骼发育及骨化相关的基因（如Acan、Col2a1、Ihh、Col10a1）表达水平显著降低。在表达上调的基因及其周边区域，组蛋白乙酰化水平有所升高。整体实验设计：本研究在软骨细胞培养物中开展了高通量RNA测序（RNA-seq）及染色质免疫沉淀测序（Chromatin immunoprecipitation sequencing，ChIP-seq）实验；对ChIP-seq与RNA-seq数据进行差异分析，以鉴定HDAC3缺陷型小鼠软骨细胞中差异表达基因的H3K27Ac（组蛋白H3赖氨酸27乙酰化）修饰谱。