Sandmann Michael, Talbert Paul, Demidov Dmitri, Kuhlmann Markus, Rutten Twan, Conrad Udo, Lermontova Inna
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, D-06466 Stadt Seeland, Germany.
Howard Hughes Medical Institute, Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109.
Plant Cell. 2017 Jan;29(1):144-155. doi: 10.1105/tpc.16.00720. Epub 2017 Jan 6.
KINETOCHORE NULL2 (KNL2) is involved in recognition of centromeres and in centromeric localization of the centromere-specific histone cenH3. Our study revealed a cenH3 nucleosome binding CENPC-k motif at the C terminus of KNL2, which is conserved among a wide spectrum of eukaryotes. Centromeric localization of KNL2 is abolished by deletion of the CENPC-k motif and by mutating single conserved amino acids, but can be restored by insertion of the corresponding motif of Arabidopsis CENP-C. We showed by electrophoretic mobility shift assay that the C terminus of KNL2 binds DNA sequence-independently and interacts with the centromeric transcripts in vitro. Chromatin immunoprecipitation with anti-KNL2 antibodies indicated that in vivo KNL2 is preferentially associated with the centromeric repeat Complete deletion of the CENPC-k motif did not influence its ability to interact with DNA in vitro. Therefore, we suggest that KNL2 recognizes centromeric nucleosomes, similar to CENP-C, via the CENPC-k motif and binds adjoining DNA.
动粒缺失蛋白2（KNL2）参与着丝粒的识别以及着丝粒特异性组蛋白cenH3在着丝粒的定位。我们的研究揭示了KNL2 C末端存在一个与cenH3核小体结合的CENPC-k基序，该基序在广泛的真核生物中是保守的。删除CENPC-k基序和突变单个保守氨基酸会消除KNL2在着丝粒的定位，但通过插入拟南芥CENP-C的相应基序可以恢复。我们通过电泳迁移率变动分析表明，KNL2的C末端不依赖于DNA序列结合，并在体外与着丝粒转录本相互作用。用抗KNL2抗体进行的染色质免疫沉淀表明，在体内KNL2优先与着丝粒重复序列相关联。完全删除CENPC-k基序并不影响其在体外与DNA相互作用的能力。因此，我们认为KNL2类似于CENP-C，通过CENPC-k基序识别着丝粒核小体并结合相邻的DNA。
