Barrows JK and Long DT (2019), Cell-free transcription in Xenopus egg extract.

XB-ART-56472

J Biol Chem 2019 Dec 20;29451:19645-19654. doi: 10.1074/jbc.RA119.011350.

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Cell-free transcription in Xenopus egg extract.

Barrows JK , Long DT .

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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.

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Species referenced: Xenopus laevis
Genes referenced: actb

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	Figure 1. Nucleoplasmic extract (NPE) supports robust transcription of plasmid substrates. (A) pCMV schematic. Relative location of promoter and GFP are indicated. (B) Different concentrations of pCMV were incubated in NPE, HSS, or CSF extract in the presence of [Î±32P] UTP. Samples were withdrawn at 180 minutes, resolved by agarose gel electrophoresis, and visualized by autoradiography. (C) Total UTP incorporation from (B) was quantified and graphed. (D) pActin schematic showing the 5â€™ and 3â€™ regions cloned from Xenopus actb. (E) pActin was incubated in NPE, HSS, or CSF and UTP incorporation was analyzed in parallel to (B) to allow a direct comparison. (F) Total UTP incorporation from (E) was quantified and graphed relative to peak intensity in (B). (G) Total protein from each extract was resolved by SDS-polyacrylamide gel electrophoresis and visualized with Coomassie stain or by Western blot using the indicated antibodies.
	Figure 2. NPE supports regulated and promoter-dependent transcription. (A) pActin schematic. Sequence elements are shown relative to the transcription start site (+1). âControlâ and âPromoterâ primer pair locations are indicated. (B) pActin was incubated at 10 ng/ÂµL in NPE supplemented with buffer or Î±amanitin. RNA was isolated at the indicated time points and quantified by RT-qPCR. (C) Different concentrations of pActin or ÎPromoter plasmid were incubated in NPE for 120 minutes. RNA was isolated and quantified by RT-qPCR using the Promoter primers. (D) Transcription from (C) was normalized based on starting plasmid concentration. (E) pActin or ÎPromoter plasmid were incubated in NPE at 25 ng/ÂµL. At 30 minutes, DNA-bound protein was analyzed by ChIP with the indicated antibodies. (F) At 120 minutes, RNA was isolated from the reactions in (E) and quantified by RT-qPCR using the Promoter primers. Error bars represent +/- one standard deviation. See Figure S2 for experimental replicates.
	Figure 3. Histone occupancy regulates transcriptional activity in NPE. (A) pActin was incubated in NPE at the indicated concentrations for 30 minutes. DNA-bound protein was then analyzed by ChIP using histone H3 antibodies. (B) pActin was incubated in NPE at the indicated concentrations for 60 minutes. Next, reaction samples were diluted in MNase buffer and treated with 100 U MNase at 37Â°C for the indicated time. DNA was then isolated and resolved by agarose gel electrophoresis. Input DNA (IN) and topological isoforms of the resolved plasmids are indicated: open circular (OC), supercoiled (SC), and linear. (C) The total intensity of all three full-length plasmid molecules identified in (B) was quantified and graphed. (D) Mock-depleted (ÎMock) and histone-depleted (ÎH) NPE were analyzed by Western blot with the indicated antibodies. (E) pActin was incubated in ÎMock or ÎH extract at 10 ng/ÂµL. At 30 minutes, DNA-bound protein was analyzed by ChIP using the indicated antibodies. (F) RNA was isolated from the reactions in (E) at 120 minutes and quantified by RT-qPCR using the Promoter primers. Error bars represent +/- one standard deviation.
	Figure 4. Analysis of whole-plasmid transcription and pre-mRNA processing in NPE. (A) pActin was incubated at 25 ng/ÂµL in NPE for 120 minutes. RNA was then purified and analyzed by paired-end RNA sequencing. The total of all mapped reads were graphed for both the forward (+) and reverse (âˆ’) orientations. (B) The Î”Promoter plasmid was incubated in NPE and analyzed by RNA-seq as described in (A). (C) Reads from (A) and (B) were overlaid onto the same graph for direct comparison. A diagram of pActin showing the relative position of major sequence elements is shown above. See text for description of arrowheads. (D-F) pActin was incubated in NPE at the indicated concentrations for 120 minutes. RNA was then isolated and analyzed by RT-qPCR to determine the efficiency of (D) elongation, (E) splicing, and (F) cleavage and polyadenylation. Elongation was graphed as a percentage of amplification with Elongation primers versus Promoter primers. Splicing and cleavage and polyadenylation were graphed as a percentage of amplification with Unspliced and Uncleaved primers, respectively. Error bars represent +/- one standard deviation.

References [+] :

Amodeo, Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition. 2015, Pubmed, Xenbase