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Chromosome Res. 2015 Dec;23(4):753-66. doi: 10.1007/s10577-015-9486-4. Epub 2015 Sep 5.

Distribution of histone H4 modifications as revealed by a panel of specific monoclonal antibodies.

Author information

1
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
2
Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan.
3
CREST, JST, Kawaguchi, Saitama, 332-0012, Japan.
4
Department of Advanced Medical Initiatives, Faculty of Medicine, Kyushu University, Fukuoka, 812-8582, Japan.
5
RIKEN Center for Life Science Technologies, Yokohama, 230-0045, Japan.
6
RIKEN Structural Biology Laboratory, Yokohama, 230-0045, Japan.
7
School of Life Sciences, Hokkaido University, Sapporo, 001-0021, Japan.
8
MAB Institute Inc, Sapporo, 001-0021, Japan.
9
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan. hkimura@bio.titech.ac.jp.
10
Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan. hkimura@bio.titech.ac.jp.
11
CREST, JST, Kawaguchi, Saitama, 332-0012, Japan. hkimura@bio.titech.ac.jp.

Abstract

Post-translational histone modifications play a critical role in genome functions such as epigenetic gene regulation and genome maintenance. The tail of the histone H4 N-terminus contains several amino acids that can be acetylated and methylated. Some of these modifications are known to undergo drastic changes during the cell cycle. In this study, we generated a panel of mouse monoclonal antibodies against histone H4 modifications, including acetylation at K5, K8, K12, and K16, and different levels of methylation at K20. Their specificity was evaluated by ELISA and immunoblotting using synthetic peptide and recombinant proteins that harbor specific modifications or amino acid substitutions. Immunofluorescence confirmed the characteristic distributions of target modifications. An H4K5 acetylation (H4K5ac)-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated. This unique feature allowed us to detect newly assembled H4, which is diacetylated at K5 and K12, and distinguish it from hyperacetylated H4, where K5 and K8 are both acetylated. Chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) revealed that acetylation of both H4K8 and H4K16 were enriched around transcription start sites. These extensively characterized and highly specific antibodies will be useful for future epigenetics and epigenome studies.

KEYWORDS:

Chromatin; Epigenetics; Histone modification; Monoclonal antibody

PMID:
26343042
PMCID:
PMC4666908
DOI:
10.1007/s10577-015-9486-4
[Indexed for MEDLINE]
Free PMC Article

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