TY - JOUR
T1 - Chromatin characterization in xenopus laevis cell-free egg extracts and embryos
AU - Wang, Wei Lin
AU - Onikubo, Takashi
AU - Shechter, David
N1 - Funding Information:
This work was supported by The American Cancer Society-Robbie Sue Mudd Kidney Cancer Research Scholar Grant (124891-RSG-13-396-01-DMC) and National Institutes of Health (NIH) grant R01GM108646 (both to D.S.).
Publisher Copyright:
© 2019 Cold Spring Harbor Laboratory Press.
PY - 2019/2/1
Y1 - 2019/2/1
N2 - 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 hydroxy-apatite-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.
AB - 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 hydroxy-apatite-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.
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U2 - 10.1101/pdb.prot099879
DO - 10.1101/pdb.prot099879
M3 - Article
C2 - 29475998
AN - SCOPUS:85060955915
SN - 1559-6095
VL - 2019
SP - 133
EP - 142
JO - Cold Spring Harbor Protocols
JF - Cold Spring Harbor Protocols
IS - 2
ER -