Chromatin Assembly of DNA Templates Microinjected Into Xenopus Oocytes

Danièle Roche, Geneviève Almouzni, and Jean-Pierre Quivy

Summary

The packaging of deoxyribonucleic acid (DNA) into chromatin within the eukaryotic nucleus can affect processes such as DNA replication, transcription, recombination, and repair. Therefore, studies aimed at understanding at the molecular level how these processes are operating have to take into account the chromatin context. We present a method to assemble DNA into chromatin by nuclear microinjection into Xenopus oocytes. This method allows in vivo chromatin formation in a nuclear environment. We provide the experimental procedures for oocyte preparation, DNA injection, and analysis of the assembled chromatin.

Key Words: Microinjection; oocytes; chromatin assembly; nucleosomes. 1. Introduction

In the nucleus of eukaryotic cells, DNA is packaged into chromatin, a nucleopro-tein complex consisting of a basic repeating unit known as the nucleosome. A single nucleosome contains two turns of DNA wrapped around a core histone octamer comprised of the histones H2A, H2B, H3, and H4 (1). Nucleosomes represent the first level of compaction in chromatin, the dynamics of which will influence access to enzymes involved in DNA metabolism (2). In addition to these basic components, linker histones and a variety of nonhistone proteins are incorporated to achieve complete genome organization within a higher-order chromatin structure (3).

Thus, studies aimed at understanding mechanisms such as transcription, replication, repair, or recombination at the molecular level have to incorporate the nucleo-somes and chromatin components. Although chromatin can be reconstituted using pure histones (4,5), the nucleosomal templates generated in this way do not necessarily possess some physiological characteristics of native chromatin, such as the spacing of the nucleosomes, the diversity of histone posttranslational modifications, or the presence of nonhistone-associated proteins.

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

Cdna Injection Into Xenopus Oocyte

Fig. 1. Simplified scheme of the experimental strategy. Above: single-stranded circular DNA (ssDNA) molecules with a-32P-dCTP are injected into the nucleus (germinal vesicle, dashed circle) of a stage VI oocyte. The animal and vegetal poles are indicated. After 3 h at 18°C, the DNA is extracted and purified. Below: following its injection, the circular naked ssDNA undergoes a complementary strand synthesis (dashed arrow). During this synthesis, a-32P-dCTP is incorporated in the synthesized DNA (32P), and nucleosomes (N in circles) are deposited concomitantly. After 3 h, the reaction is complete and yields a radioactively labeled double-stranded closed circular DNA molecule (dsDNA) on which nucleosomes are assembled (about 1 nucleosome for 185 bp).

Fig. 1. Simplified scheme of the experimental strategy. Above: single-stranded circular DNA (ssDNA) molecules with a-32P-dCTP are injected into the nucleus (germinal vesicle, dashed circle) of a stage VI oocyte. The animal and vegetal poles are indicated. After 3 h at 18°C, the DNA is extracted and purified. Below: following its injection, the circular naked ssDNA undergoes a complementary strand synthesis (dashed arrow). During this synthesis, a-32P-dCTP is incorporated in the synthesized DNA (32P), and nucleosomes (N in circles) are deposited concomitantly. After 3 h, the reaction is complete and yields a radioactively labeled double-stranded closed circular DNA molecule (dsDNA) on which nucleosomes are assembled (about 1 nucleosome for 185 bp).

Efficient chromatin assembly can be reproduced in vitro in crude extracts derived from Xenopus oocytes or eggs (6-9), Drosophila embryos (10-12), or human cells (13-16). However, these extracts are usually not simultaneously competent for transcription, repair, and replication. The injection of DNA templates in Xenopus oocytes as a "living test tube" (17) provides a convenient and highly efficient means to assemble in vivo the DNA into chromatin in a nuclear compartment (germinal vesicle) proficient for transcription and DNA synthesis and repair (18-23). Thus, the impact of the chromatin structure on these processes can be studied directly, provided that the DNA template contains adequate features such as sequence control elements, reporter genes, and so on. In this chapter, we describe this powerful approach for chromatin assembly and discuss potential applications.

The experimental strategy is summarized in Fig. 1. Following injection of a circular single-stranded DNA (ssDNA; M13 derivative) into the nucleus (germinal vesicle) of a stage VI oocyte, complementary-strand DNA synthesis occurs, leading to a closed circular double-stranded DNA (dsDNA) (21). Coupled to this process, nucleosomes are efficiently assembled onto the DNA template. If radioactive deoxycitidine-5'-triphosphate (a-32P) (a-32P-dCTP) is coinjected with DNA, it will be incorporated during synthesis, enabling the concomitant labeling of the DNA assembled into chromatin.

After 3 h, the efficiency of nucleosome assembly can be assessed by purification of injected DNA. Two assays can be performed: (1) a supercoiling assay and (2) a micro-

supercoiling assay

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