Yalagapati Surya Prakash, Ahmadli Ulkar, Sinha Aditya, Kalidass Manikandan, Dabravolski Siarhei, Zuo Sheng, Yadala Ramakrishna, Rutten Twan, Talbert Paul, Berr Alexandre, Lermontova Inna
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) OT Gatersleben, Corrensstr 3, 06466 Seeland, Germany.
Department of Biotechnology Engineering, Braude Academic College of Engineering, Snunit 51, P.O. Box 78, Karmiel 2161002, Israel.
Nucleic Acids Res. 2025 Feb 8;53(4). doi: 10.1093/nar/gkae1242.
In eukaryotes, accurate chromosome segregation during cell division relies on the centromeric histone H3 variant, CENH3. Our previous work identified KINETOCHORE NULL2 (αKNL2) as a plant CENH3 assembly factor, which contains a centromere-targeting motif, CENPC-k, analogous to the CENPC motif found in CENP-C. We also demonstrated that αKNL2 can bind DNA in vitro in a sequence-independent manner, without the involvement of its CENPC-k motif. In this study, we show that the CENPC-k and CENPC motifs alone are insufficient for centromere targeting in Nicotiana benthamiana and Arabidopsis thaliana. In silico analysis identified adjacent DNA-binding regions near the CENPC-k and CENPC motifs, suggesting their role in centromeric DNA interaction. We further demonstrated that protein fragments containing these motifs effectively target centromeres. Deletion of these DNA-binding domains reduced the centromeric localization of αKNL2-C, while fusing CENPC-k to the non-specific DNA-binding domain of histone-like nucleoid structuring protein from Escherichia coli successfully targeted it to centromeres. Our findings suggest that the centromeric targeting of αKNL2 and CENP-C proteins relies on the CENPC-k/CENPC motifs, and that their sequence-independent DNA-binding activity enhances their centromere anchoring. These insights into the mechanisms of αKNL2 and CENP-C targeting may facilitate the engineering of kinetochore structures by directing chromatin-modifying proteins to centromeres.
在真核生物中，细胞分裂期间准确的染色体分离依赖于着丝粒组蛋白H3变体CENH3。我们之前的研究确定了动粒缺失蛋白2（αKNL2）是一种植物CENH3组装因子，它含有一个着丝粒靶向基序CENPC-k，类似于在CENP-C中发现的CENPC基序。我们还证明，αKNL2能够在体外以不依赖序列的方式结合DNA，且不涉及其CENPC-k基序。在本研究中，我们表明，单独的CENPC-k和CENPC基序不足以在本氏烟草和拟南芥中靶向着丝粒。计算机分析确定了CENPC-k和CENPC基序附近相邻的DNA结合区域，表明它们在着丝粒DNA相互作用中的作用。我们进一步证明，含有这些基序的蛋白质片段能够有效地靶向着丝粒。删除这些DNA结合结构域会降低αKNL2-C在着丝粒的定位，而将CENPC-k与大肠杆菌类组蛋白核仁结构蛋白的非特异性DNA结合结构域融合，则成功地将其靶向着丝粒。我们的研究结果表明，αKNL2和CENP-C蛋白的着丝粒靶向依赖于CENPC-k/CENPC基序，且它们不依赖序列的DNA结合活性增强了它们对着丝粒的锚定作用。这些对αKNL2和CENP-C靶向机制的见解可能有助于通过将染色质修饰蛋白引导至着丝粒来构建动粒结构。
