Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, 41 Medlars Drive, Bldg 41/Rm B1300, Bethesda, MD, 20892, USA.
Epigenetics Chromatin. 2024 Jun 2;17(1):19. doi: 10.1186/s13072-024-00543-9.
Over the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function.
We report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes.
These data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.
在过去的几十年中，生化和荧光标记物的使用阐明了原本不可能的机制和细胞学过程。某些核蛋白的挑战性性质包括丰度低、抗体识别差和瞬态动力学。解决这些问题的一种方法是在目标蛋白上添加肽或更大的蛋白标签，以提高富集、纯化和可视化效果。然而，许多这些研究都是基于这样的假设，即标记蛋白可以完全再现天然蛋白的功能。
我们报告说，当 C 端 TAP 标记的着丝粒蛋白 A 组蛋白变体被引入时，它会经历改变的着丝粒蛋白结合，在翻译后修饰（PTMs）方面存在差异，利用与天然 CENP-A 不同的组蛋白伴侣，并且可以在人类细胞中部分取代天然 CENP-A。此外，与天然 CENP-A 核小体相比，这些标记的含有 CENP-A 的核小体在早期 G1 期时的着丝粒掺入减少，与连接组蛋白 H1.5 的结合较差。
这些数据表明，表达标记的组蛋白变体 CENP-A 可能会导致非天然途径的意外利用，从而改变组蛋白变体的生物学功能。
