Targeting of Arabidopsis KNL2 to Centromeres Depends on the Conserved CENPC-k Motif in Its C Terminus OPEN Michael Sandmann, a Paul Talbert, b Dmitri Demidov, a Markus Kuhlmann, a Twan Rutten, a Udo Conrad, a and Inna Lermontova a,1 a Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, D-06466 Stadt Seeland, Germany b Howard Hughes Medical Institute, Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 ORCID IDs: 0000-0001-6893-8431 (M.S.); 0000-0003-3104-0825 (M.K.); 0000-0001-5891-6503 (T.R.); 0000-0002-9416-3470 (U.C.); 0000-0003-3386-2590 (I.L.) 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 Arabidopsis thaliana 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 pAL1. 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. INTRODUCTION Centromeres are the chromosomal positions at which the ki- netochore complex assembles. The kinetochore complex is required for correct sister chromatid segregation, sister chro- matid cohesion, chromosome movement, and cell cycle regu- lation (Henikoff and Dalal, 2005). Normal kinetochore establishment depends on the centromeric histone H3 variant cenH3 originally described as human CENP-A (Earnshaw and Rothfield, 1985). CenH3 is an essential centro- meric mark and substitutes for H3 histones at centromeric sites. The centromeric chromatin consists of blocks of H3 containing nucleosomes intermingled with cenH3 chromatin (Blower et al., 2002). In most plants and animals, centromeres are located within regions containing large numbers of tandem repeats of short se- quences. CenH3-containing chromatin occupies only part of these repeats (Blower et al., 2002). In Arabidopsis thaliana, ;15% of the 178-bp centromeric repeat pAL1 is associated with Arabidopsis cenH3 (Nagaki et al., 2003). Thus, in most organisms, except Saccharomyces cerevisiae (Meluh et al., 1998), cenH3 localization is not solely determined by centromeric DNA sequences, but is rather regulated epigeneti- cally. Some studies have proposed that cenH3 plays the role of epigenetic mark, since ectopically incorporated cenH3 (Lo et al., 2001; Nasuda et al., 2005), as well as tethering of cenH3 to noncentromeric loci (Mendiburo et al., 2011; Teo et al., 2013), leads to de novo kinetochore assembly at positions free of centromeric repeats. Incorporation of cenH3 is anticorrelated with other epigenetic marks such as DNA methylation and some histone modifications (Zhang et al., 2008). It was shown for Arabidopsis that the centromeric transcripts and corresponding small interfering RNAs might be involved in methylation of centromeric/pericentromeric DNA (Teixeira and Colot, 2010). Single-stranded centromeric transcripts can bind centromeric chro- matin and may serve as a structural template to help in recruiting kinetochore proteins (Wong et al., 2007; Du et al., 2010). The cenH3 deposition pathway includes three steps: (1) ini- tiation, i.e., generation of the epigenetic context for cenH3 in- corporation; (2) deposition; and (3) maintenance (reviewed in De Rop et al., 2012). (1) In mammals, the Mis18 complex, which includes Mis18a, Mis18b, and Mis18BP1 (KNL2) proteins, plays a role in the ini- tiation step and replenishment of cenH3. The human Mis18 complex is transiently present at centromeres after mitotic exit prior to cenH3 loading, and downregulation of Mis18 complex components results in a reduced amount of cenH3 at centromeres (Fujita et al., 2007). Fission yeast (Schizosaccharomyces pombe) Mis18 is also a part of the cenH3 assembly machinery (Hayashi et al., 2004). The Caenorhabditis elegans KNL2 and cenH3 co- localize at centromeres throughout the cell cycle and coordinate kinetochore assembly and chromosome segregation (Maddox et al., 2007). Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. Knockout of Arabidopsis KNL2 leads to anaphase bridges, reduced fertility, reduced levels of DNA methylation, and reduced transcription of the genes encoding cenH3 and histone modification proteins such as SUVH4 and SUVH9 (Lermontova et al., 2013). Establishment of the epigenetic context for cenH3 loading requires other proteins. In mammalian cells, the kinetochore protein CENP-C recruits epigenetic factors such as DNA 1 Address correspondence to lermonto@ipk-gatersleben.de. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Inna Lermontova (lermonto@ipk-gatersleben.de). OPEN Articles can be viewed without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.16.00720 The Plant Cell, Vol. 29: 144–155, January 2017, www.plantcell.org ã 2017 American Society of Plant Biologists. All rights reserved.