Cell-free transcription in Xenopus egg extract

Abstract

Edited by John M. Denu 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.

BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract

“…In the embryos of rapidly developing species, transcription activity is regulated by the density of maternally-supplied histones ( 39 , 40 ). We previously showed that a similar mechanism also controls transcription in NPE ( 26 ), which supports nucleosome formation ( Supplementary Figure S3A ) ( 41 ). To test whether BRCA1’s ability to suppress transcription was linked to histone density, we compared transcription in mock- and BRCA1-depleted extracts incubated with 2.5 ng/ul pActin and increasing concentrations of a ‘carrier’ plasmid with no sequence homology.…”

“…Despite extensive study, the context and functional role that BRCA1 plays in transcription regulation remains poorly understood. We recently showed that nucleoplasmic extract (NPE) readily promotes transcription of plasmid-borne gene constructs ( 26 ), unlike traditional egg extracts where transcription is suppressed ( 27 ). Using the NPE system, we sought to investigate BRCA1’s role as a transcription regulator.…”

Studying chromosome biology with single-molecule resolution in Xenopus laevis egg extracts

“…Equally, reconstitution involves purification of multiple proteins and fine-tuning of reaction conditions, whilst extracts contain a complete complement of proteins required for the reactions discussed here, including previously uncharacterised proteins. This justifies development of new single-molecule approaches in extracts to give a detailed insight into pathways like non-NHEJ DNA repair mechanisms and transcription [50]. Further study of the intersection between chromosome replication, repair and organisation in egg extracts will be interesting [26,33].…”

The TFIIH Complex is Required to Establish and Maintain Mitotic Chromosome Structure

“…This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted November 7, 2021. ; https://doi.org/10.1101/2021.11.06.467569 doi: bioRxiv preprint 2015; Barrows and Long, 2019). Interestingly, in vitro chromatid reconstitution in this system specifically requires the use of an egg-specific histone (Shintomi et al, 2015;Shechter et al, 2009), histone H2A.X-F (also known as H2A.X.3), and it was suggested that this variant is a better substrate of the FACT complex chaperone than is the canonical histone H2A, which is also present in egg extracts (Shintomi et al, 2015;Wang et al, 2014).…”

“…To examine possible relationships between transcription-associated activities and mitotic chromosome condensation, we used cell-free Xenopus M phase egg extracts, a system in which condensin function is well characterized and general transcription is silenced (Kinoshita et al, 2015;Hirano and Mitchison, 1994;Barrows and Long, 2019;Chen et al, 2019;Amodeo et al, 2015). As previously demonstrated (Hirano and Mitchison, 1993;Shintomi et al, 2015), sperm nuclei incubated with a high-speed supernatant (HSS) of egg extracts first swell during the exchange of protamines for maternal histones, assemble into a diffuse "cloud" of chromatin, and gradually form clusters of condensed single-chromatid structures (Figure 1A).…”