Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco

Kiyotaka Nagaki, Fukashi Shibata, Asaka Kanatani, Kazunari Kashihara, Minoru Murata

    Research output: Contribution to journalArticlepeer-review

    9 Citations (Scopus)

    Abstract

    The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres.

    Original languageEnglish
    Pages (from-to)771-779
    Number of pages9
    JournalPlant Cell Reports
    Volume31
    Issue number4
    DOIs
    Publication statusPublished - Apr 2012

    Keywords

    • CENH3
    • Centromere-specific histone H3
    • Centromeric DNA sequence
    • Chromatin immunoprecipitation and ChIP
    • HaloTag

    ASJC Scopus subject areas

    • Agronomy and Crop Science
    • Plant Science

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