Chromatin Characterization in Xenopus laevis Cell-Free Egg Extracts and Embryos

Wei Lin Wang, Takashi Onikubo, David Shechter

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Xenopus laevis development is marked by accelerated cell division solely supported by the proteins maternally deposited in the egg. Oocytes mature to eggs with concomitant transcriptional silencing. The unique maternal chromatin state contributing to this silencing and subsequent zygotic activation is likely established by histone posttranslational modifications and histone variants. Therefore, tools for understanding the nature and function of maternal and embryonic histones are essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Here we describe protocols for isolating pronuclear sperm chromatin from Xenopus egg extracts and hydroxyapatite-based histone purification from this chromatin. The histones purified through this method can be directly assembled into chromatin through in vitro assembly reactions, providing a unique opportunity to biochemically dissect the effect of histone variants, histone modifications, and other factors in chromatin replication and assembly. We also describe how to isolate chromatin from staged embryos and analyze the proteins to reveal dynamic developmental histone modifications. Finally, we present protocols to measure chromatin assembly in extracts, including supercoiling and micrococcal nuclease assays. Using these approaches, analysis of maternal and zygotic histone posttranslational modifications concomitant with cell-cycle and developmental transitions can be tested.

Original languageEnglish (US)
JournalCold Spring Harbor Protocols
Volume2019
Issue number2
DOIs
StatePublished - Feb 1 2019

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Histone Code
Xenopus laevis
Histones
Chromatin
Ovum
Embryonic Structures
Chromatin Assembly and Disassembly
Mothers
Post Translational Protein Processing
Micrococcal Nuclease
Durapatite
Xenopus
Cell Division
Eggs
Oocytes
Spermatozoa
Cell Cycle
Cells
Proteins
Transcription

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Chromatin Characterization in Xenopus laevis Cell-Free Egg Extracts and Embryos. / Wang, Wei Lin; Onikubo, Takashi; Shechter, David.

In: Cold Spring Harbor Protocols, Vol. 2019, No. 2, 01.02.2019.

Research output: Contribution to journalArticle

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