XB-ART-54613Cold Spring Harb Protoc February 1, 2019; 2019 (2): pdb.prot099879.
Chromatin Characterization in Xenopus laevis Cell-Free Egg Extracts and Embryos.
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.
PubMed ID: 29475998
PMC ID: PMC6107429
Article link: Cold Spring Harb Protoc
Grant support: R01 GM108646 NIGMS NIH HHS