Heterochromatin Is Required for Normal Distribution of Neurospora crassa CenH3

Abstract

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.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01285-10.

ACKNOWLEDGMENTS

We thank Lanelle Connolly for technical support, Mark Dasenko, Steve Drake, Matthew Peterson, and Scott Givan at the OSU CGRB core facility for assistance with Illumina sequencing, and Noah Fahlgren and Henry Priest for helpful discussions and for sharing code. Shinji Honda and Eric Selker kindly provided pZero plasmids, as well as hpo and dim-5 mutant strains. Yi Zhou and Jason Stajich gave us access to RepeatMasker files. We are grateful to James C. Carrington for his support and encouragement of this work.

This work was supported by the American Cancer Society (RSG-08-030-01-CCG to M.F.), start-up funds from the OSU Computational and Genome Biology Initiative, and funds supporting the OSU CGRB.

We have no conflicting interests.