Control of gene expression by epigenetic regulators is fundamental to tissue development and homeostasis. Loss-of-function (LOF) studies using siRNAs for epigenetic regulators require that RNA interference rapidly reduces the cellular levels of the corresponding mRNAs and/or proteins. The most abundant chromatin structural proteins (i.e., the core histones H2A, H2B, H3 and H4) have relatively long half-lives and do not turn over rapidly, although their mRNAs are labile. The question arises whether epigenetic regulatory enzymes (e.g., Ezh2) or proteins that interact with histones via selective modifications (e.g., Cbx1 to Cbx8, Brd4) are stable or unstable. Therefore, we performed classical α-amanitin and cycloheximide inhibition assays that block, respectively, mRNA transcription and protein translation in mouse MC3T3 osteoblasts, ATDC5 chondrocytes and C2C12 myoblasts. We find that mRNA levels of Cbx proteins and Ezh2 were significantly depleted after 24 hrs, while their corresponding proteins remained relatively stable. As positive control, the half-life of the labile cyclin D1 protein was found to be less than 1 hr. Our study suggests that histone code readers and writers are relatively stable chromatin-related proteins, which is consistent with their long-term activities in maintaining chromatin organization and phenotype identity. These findings have conceptual ramifications for the interpretation of RNAi experiments that reduce the mRNA but not protein levels of epiregulatory proteins. We propose that siRNAs for at least some epigenetic regulatory proteins may exert their biological effects by blocking translation and new protein synthesis rather than by decreasing pre-existing protein pools.

表观遗传调节剂对基因表达的控制是组织发育和体内平衡的基础。使用siRNA作为表观遗传调节剂的功能丧失（LOF）研究要求RNA干扰迅速降低相应mRNA和/或蛋白质的细胞水平。最丰富的染色质结构蛋白（即核心组蛋白H2A，H2B，H3和H4）具有相对较长的半衰期，尽管其mRNA不稳定，但不能快速翻转。问题是表观遗传调节酶（例如Ezh2）或通过选择性修饰与组蛋白相互作用的蛋白质（例如Cbx1至Cbx8，Brd4）是稳定的还是不稳定的。因此，我们进行了经典的α-amanitin和cycloheximide抑制试验，分别阻断了小鼠MC3T3成骨细胞中的mRNA转录和蛋白质翻译，ATDC5软骨细胞和C2C12成肌细胞。我们发现24小时后，Cbx蛋白和Ezh2的mRNA水平显着减少，而它们相应的蛋白保持相对稳定。作为阳性对照，发现不稳定的细胞周期蛋白D1蛋白的半衰期少于1小时。我们的研究表明，组蛋白代码的阅读者和作者是相对稳定的染色质相关蛋白，这与其在维持染色质组织和表型同一性方面的长期活动是一致的。这些发现对RNAi实验的解释具有概念性的影响，该实验可减少上调蛋白的mRNA但不降低蛋白水平。