Cell-free transcription in Xenopus egg extract

John K Barrows,David T Long

doi:10.1074/jbc.ra119.011350

Abstract

Soluble extracts prepared from Xenopus eggs have been used extensively to study various aspects of cellular and developmental biology. During early egg development, transcription of the zygotic genome is suppressed. As a result, traditional extracts derived from unfertilized and early stage eggs possess little or no intrinsic transcriptional activity. In this study, we show that Xenopus nucleoplasmic extract (NPE) supports robust transcription of a chromatinized plasmid substrate. Although prepared from eggs in a transcriptionally inactive state, the process of making NPE resembles some aspects of egg fertilization and early embryo development that lead to transcriptional activation. With this system, we observed that promoter-dependent recruitment of transcription factors and RNA polymerase II leads to conventional patterns of divergent transcription and pre-mRNA processing, including intron splicing and 3' cleavage and polyadenylation. We also show that histone density controls transcription factor binding and RNA polymerase II activity, validating a mechanism proposed to regulate genome activation during development. Together, these results establish a new cell-free system to study the regulation, initiation, and processing of mRNA transcripts.

Highlights

Soluble extracts prepared from Xenopus eggs have been used extensively to study various aspects of cellular and developmental biology
To determine the relative efficiency of transcription in nucleoplasmic extract (NPE), we compared its activity with other Xenopus egg extracts shown to have limited transcriptional activity, including HSS and CSF
The CMV promoter was previously shown to support transcription in Xenopus oocytes [18] and cultured somatic cell lysate [32], indicating that it is recognized by Xenopus transcription machinery

Summary

Introduction

Soluble extracts prepared from Xenopus eggs have been used extensively to study various aspects of cellular and developmental biology, including nuclear formation [3,4,5], DNA replication and repair (6 –9), cellular and checkpoint signaling (10 –13), mitosis (14 –16), and apoptosis [17] These extracts have been found to possess little or no intrinsic transcriptional activity [18], limiting study of a fundamental biological process with this model system. The single-cell embryo undergoes multiple rounds of rapid DNA synthesis and cellular division to form a fluid-filled sphere of cells called a blastula At this point in embryo development, the genome transitions to a transcriptionally active state through a process referred to as the midblastula transition (MBT)2 [20]

Results

Discussion

Conclusion