Abstract
This chapter describes a method used to analyze the behavior of histone modifications in S phase in Arabidopsis using a whole-mount immunostaining technique. Previous studies have demonstrated that dramatic changes in local chromatin structure are required for the initiation and progression of DNA replication, and that histone modifications play an essential role in the determination of chromatin structure in S phase. Since euchromatic and heterochromatic regions are replicated in distinct S-phase stages, it is important to identify histone modifications at each stage. Here, we introduce a protocol for whole-mount immunostaining combined with 5-ethynyl-2′-deoxyuridine (EdU) staining, which enables the visualization of spatial patterns in histone modifications in the early and late S-phase nuclei of Arabidopsis roots.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schuermann D, Fritsch O, Lucht JM, Hohn B (2009) Replication stress leads to genome instabilities in Arabidopsis DNA polymerase δ mutants. Plant Cell 21:2700–2714
Domenichini S, Benhamed M, de Jaeger G et al (2012) Evidence for a role of Arabidopsis CDT1 proteins in gametophyte development and maintenance of genome integrity. Plant Cell 24:2779–2791
Concia L, Brooks AM, Wheeler E et al (2018) Genome-wide analysis of the Arabidopsis replication timing program. Plant Physiol 176:2166–2185
Belsky JA, Macalpine HK, Lubelsky Y et al (2015) Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly. Genes Dev 29:212–224
Rivera C, Gurard-Levin ZA, Almouzni G, Loyola A (2014) Histone lysine methylation and chromatin replication. Biochim Biophys Acta Gene Regul Mech 1839:1433–1439
Sugimoto N, Fujita M (2017) Molecular mechanism for chromatin regulation during MCM loading in mammalian cells. In: Advances in experimental medicine and biology. Springer, Heidelberg, pp 61–78
Lee TJ, Pascuzzi PE, Settlage SB et al (2010) Arabidopsis thaliana chromosome 4 replicates in two phases that correlate with chromatin state. PLoS Genet 6:1–18
Jiang D, Berger F (2017) DNA replication–coupled histone modification maintains Polycomb gene silencing in plants. Science 357:1146–1149
Hayashi K, Hasegawa J, Matsunaga S (2013) The boundary of the meristematic and elongation zones in roots: endoreduplication precedes rapid cell expansion. Sci Rep 3:1–8
Acknowledgments
This work was supported by MEXT KAKENHI Grant Numbers 17H06470 and 17H06477 to M.U., and JSPS KAKENHI Grant Number 17K15415 to H.T.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Takatsuka, H., Umeda, M. (2021). Whole-Mount Immunostaining for the Identification of Histone Modifications in the S-Phase Nuclei of Arabidopsis Roots. In: Coutts, A.S., Weston, L. (eds) Cell Cycle Oscillators . Methods in Molecular Biology, vol 2329. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1538-6_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1538-6_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1537-9
Online ISBN: 978-1-0716-1538-6
eBook Packages: Springer Protocols