Abstract
SummaryIn vertebrate cells, centromeres are specified epigenetically through the deposition of the centromere-specific histone CENP-A. Following CENP-A deposition, additional proteins are assembled on centromeric chromatin. However, it remains unknown whether additional epigenetic features of centromeric chromatin are required for kinetochore assembly. Here, we used ChIP-seq analysis to examine centromere-specific histone modifications at chicken centromeres, which lack highly repetitive sequences. We found that H4K20 monomethylation (H4K20me1) is enriched at centromeres. Immunofluorescence and biochemical analyses revealed that H4K20me1 is present at all centromeres in chicken and human cells. Based on immunoprecipitation data, H4K20me1 occurs primarily on the histone H4 that is assembled as part of the CENP-A nucleosome following deposition of CENP-A into centromeres. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces the level of H4K20me1 at centromeres and results in kinetochore assembly defects. We conclude that H4K20me1 modification of CENP-A nucleosomes contributes to functional kinetochore assembly.
Highlights
Centromeres are essential genomic regions that direct faithful chromosome segregation
H4K20 Monomethylation Is Detected at Centromere Regions in DT40 and HeLa Cells Based on Chromatin immunoprecipitation (ChIP) Analyses We began by performing ChIP-seq analyses in chicken DT40 cells using specific monoclonal antibodies against a range of core histone modifications, including H3K4me1/me2/me3, H3K9me1/me2/me3, H3K27me1/me2/me3, H3K36me1/me2/ me3, and H4K20me1/me2/me3 (Figure S1 available online). Most of these histone modifications did not display any significant accumulation at centromeres assembled on nonrepetitive sequences (Figures S1A and S1C), some of them were detected at repetitive centromeres, presumably because of the recognition of the associated heterochromatin (Figures S1B and S1D)
H4K20me3, an established marker for pericentromeric heterochromatin (Jørgensen et al, 2013), or H4K20me2 was detected at repetitive centromeres in chicken cells (Figure S1D), but not at centromeres containing nonrepetitive unique sequences, such as centromere Z, which lacks heterochromatin (Shang et al, 2010) (Figure S1C)
Summary
Centromeres are essential genomic regions that direct faithful chromosome segregation. The centromere-specific histone H3 variant CENP-A is a critical epigenetic marker for centromere specification (Allshire and Karpen, 2008; Guse et al, 2011; Hori et al, 2013; Mendiburo et al, 2011; Perpelescu and Fukagawa, 2011), but whether additional epigenetic features are required for centromere specification and/or kinetochore assembly remains a key unanswered question. It is unclear whether histone modifications (Ruthenburg et al, 2007) are required for distinct functions at centromeres. A recent study has further confirmed that the functioning kinetochore of chicken cells contains $50 kb of DNA (Ribeiro et al, 2014)
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