In eukaryotes, nucleosomes form a barrier to DNA templated reactions and must be dynamically disrupted to provide access to the genome. During nucleosome (re)assembly, histones can be replaced by new histones, erasing post-translational modifications. Measuring histone turnover in mammalian cells has mostly relied on inducible overexpression of histones, which may influence and distort natural histone deposition rates. We have previously used recombination-induced tag exchange (RITE) to study histone dynamics in budding yeast. RITE is a method to follow protein turnover by genetic switching of epitope tags using Cre recombinase and does not rely on inducible overexpression. Here, we applied RITE to study the dynamics of the replication-independent histone variant H3.3 in human cells. Epitope tag-switching could be readily detected upon induction of Cre-recombinase, enabling the monitoring old and new H3.3 in the same pool of cells. However, the rate of tag-switching was lower than in yeast cells. Analysis of histone H3.3 incorporation by chromatin immunoprecipitation did not recapitulate previously reported aspects of H3.3 dynamics such as high turnover rates in active promoters and enhancers. We hypothesize that asynchronous Cre-mediated DNA recombination in the cell population leads to a low time resolution of the H3.3-RITE system in human cells. We conclude that RITE enables the detection of old and new proteins in human cells and that the time-scale of tag-switching prevents the capture of high turnover events in a population of cells. Instead, RITE might be more suited for tracking long-lived histone proteins in human cells.

在真核生物中，核小体形成了DNA模板反应的屏障，必须动态破坏以提供对基因组的访问。在核小体（重新）组装过程中，可以用新的组蛋白代替组蛋白，从而消除翻译后修饰。测量哺乳动物细胞中的组蛋白更新主要依赖于组蛋白的诱导型过表达，这可能会影响和扭曲天然组蛋白的沉积速率。我们以前使用重组诱导的标签交换（RITE）来研究发芽酵母中的组蛋白动力学。RITE是通过使用Cre重组酶的抗原决定簇标签进行遗传转换来跟踪蛋白质更新的方法，并且不依赖于诱导型过表达。在这里，我们应用RITE研究了人类细胞中非复制依赖性组蛋白变体H3.3的动力学。诱导Cre重组酶后可以很容易地检测到抗原决定簇标签转换，从而可以在同一细胞池中监测新旧H3.3。但是，标签转换率低于酵母细胞。通过染色质免疫沉淀对组蛋白H3.3掺入的分析没有概括先前报道的H3.3动力学方面，例如活性启动子和增强子的高周转率。我们假设细胞群中的异步Cre介导的DNA重组导致人类细胞中H3.3-RITE系统的低时间分辨率。我们得出的结论是，RITE能够检测人类细胞中的旧蛋白质和新蛋白质，并且标签切换的时标会阻止捕获细胞群中的高周转事件。代替，