Centromeres serve as platforms for the assembly of kinetochores and are 
essential for nuclear division. Here we identified Neurospora crassa centromeric 
DNA by chromatin immunoprecipitation followed by high-throughput sequencing 
(ChIP-seq) of DNA associated with tagged versions of the centromere foundation 
proteins CenH3 (CENP-A) and CEN-C (CENP-C) and the kinetochore protein CEN-T 
(CENP-T). On each chromosome we found an ∼150- to 300-kbp region of enrichment 
for all three proteins. These regions correspond to intervals predicted to be 
centromeric DNA by genetic mapping and DNA sequence analyses. By ChIP-seq we 
found extensive colocalization of CenH3, CEN-C, CEN-T, and histone H3K9 
trimethylation (H3K9me3). In contrast, H3K4me2, which has been found at the 
cores of plant, fission yeast, Drosophila, and mammalian centromeres, was not 
enriched in Neurospora centromeric DNA. DNA methylation was most pronounced at 
the periphery of centromeric DNA. Mutation of dim-5, which encodes an H3K9 
methyltransferase responsible for nearly all H3K9me3, resulted in altered 
distribution of CenH3-green fluorescent protein (GFP). Similarly, CenH3-GFP 
distribution was altered in the absence of HP1, the chromodomain protein that 
binds to H3K9me3. We conclude that eukaryotes with regional centromeres make use 
of different strategies for maintenance of CenH3 at centromeres, and we suggest 
a model in which centromere proteins nucleate at the core but require DIM-5 and 
HP1 for spreading.