Alasdair J. E. Gordon

Transcriptional Infidelity Promotes Heritable Phenotypic Change in a Bistable Gene Network

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

Heritable Change Caused by Transient Transcription Errors

Transmission of cellular identity relies on the faithful transfer of information from the mother to the daughter cell. This process includes accurate replication of the DNA, but also the correct propagation of regulatory programs responsible for cellular identity. Errors in DNA replication (mutations) and protein conformation (prions) can trigger stable phenotypic changes and cause human disease, yet the ability of transient transcriptional errors to produce heritable phenotypic change (‘epimutations’) remains an open question. Here, we demonstrate that transcriptional errors made specifically in the mRNA encoding a transcription factor can promote heritable phenotypic change by reprogramming a transcriptional network, without altering DNA. We have harnessed the classical bistable switch in the lac operon, a memory-module, to capture the consequences of transient transcription errors in living Escherichia coli cells. We engineered an error-prone transcription sequence (A9 run) in the gene encoding the lac repressor and show that this ‘slippery’ sequence directly increases epigenetic switching, not mutation in the cell population. Therefore, one altered transcript within a multi-generational series of many error-free transcripts can cause long-term phenotypic consequences. Thus, like DNA mutations, transcriptional epimutations can instigate heritable changes that increase phenotypic diversity, which drives both evolution and disease.

Epigenetic switches: can infidelity govern fate in microbes?

Unicellular organisms are constantly subject to sudden changes in environment. Here, we describe recent progress in understanding how epigenetic mechanisms can generate differentiation within genetically identical single cells of a clonal population. Such intrinsic phenotypic heterogeneity within a population may be considered as a bet-hedging strategy in fluctuating environments. One aspect we highlight is how transient errors in information transfer, be it errors in transcription or translation, or alternatives in protein folding, can influence the quantity and the quality of the resulting proteins, and therefore, contribute to genetic noise within individual cells. These stochastic events can provide the impetus for heritable phenotypic change in bistable epigenetic regulatory networks that are susceptible to noise and proteins capable of dominant variant conformations.

Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation

To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the “Kibo” module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134xa0days flight, 72xa0mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.3-fold) in thymidine kinase deficient (TK−) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK− mutation frequency upon exposure to a subsequent 2xa0Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores.

Lost in transcription: transient errors in information transfer

Errors in information transfer from DNA to RNA to protein are inevitable. Here, we focus on errors that occur in nascent transcripts during transcription, epimutations. Recent approaches using novel cDNA library preparation and next-generation sequencing begin to directly determine the rate of epimutation and allow analysis of the epimutational spectrum of transcription errors, the type and sequence context of the errors produced in a transcript by an RNA polymerase. The phenotypic consequences of transcription errors have been assessed using both forward and reverse epimutation systems. These studies reveal that transient transcription errors can produce a modification of cell phenotype, partial phenotypic suppression of a mutant allele, and a heritable change in cell phenotype, epigenetic switching in a bistable gene network.

DksA involvement in transcription fidelity buffers stochastic epigenetic change

DksA is an auxiliary transcription factor that interacts with RNA polymerase and influences gene expression. Depending on the promoter, DksA can be a positive or negative regulator of transcription initiation. Moreover, DksA has a substantial effect on transcription elongation where it prevents the collision of transcription and replication machineries, plays a key role in maintaining transcription elongation when translation and transcription are uncoupled and has been shown to be involved in transcription fidelity. Here, we assessed the role of DksA in transcription fidelity by monitoring stochastic epigenetic switching in the lac operon (with and without an error-prone transcription slippage sequence), partial phenotypic suppression of a lacZ nonsense allele, as well as monitoring the number of lacI mRNA transcripts produced in the presence and absence of DksA via an operon fusion and single molecule fluorescent in situ hybridization studies. We present data showing that DksA acts to maintain transcription fidelity in vivo and the role of DksA seems to be distinct from that of the GreA and GreB transcription fidelity factors.

Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity

Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage DNA, RNA, protein and lipids. The MutT protein in Escherichia coli removes 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-GTP) from the nucleotide pools precluding incorporation into DNA and RNA. While 8-oxo-dGTP incorporation into DNA is mutagenic, it is not clear if 8-oxo-GTP incorporation into RNA can have phenotypic consequences for the cell. We use a bistable epigenetic switch sensitive to transcription errors in the Escherichia coli lacI transcript to monitor transient RNA errors. We do not observe any increase in epigenetic switching in mutT cells. We revisit the original observation of partial phenotypic suppression of a lacZamber allele in a mutT background that was attributed to RNA errors. We find that Lac+ revertants can completely account for the increase in β-galactosidase levels in mutT lacZamber cultures, without invoking participation of transient transcription errors. Moreover, we observe a fluctuation type of distribution of β-galactosidase appearance in a growing culture, consistent with Lac+ DNA revertant events. We conclude that the absence of MutT produces a DNA mutator but does not equally create an RNA mutator.

Transcription infidelity and genome integrity: the parallax view

ABSTRACT It was recently shown that removal of GreA, a transcription fidelity factor, enhances DNA break repair. This counterintuitive result, arising from unresolved backtracked RNA polymerase impeding DNA resection and thereby facilitating RecA-loading, leads to an interesting corollary: error-free full-length transcripts and broken chromosomes. Therefore, transcription fidelity may compromise genomic integrity.

How Acts of Infidelity Promote DNA Break Repair: Collision and Collusion Between DNA Repair and Transcription

Transcription is a fundamental cellular process and the first step in gene regulation. Although RNA polymerase (RNAP) is highly processive, in growing cells the progression of transcription can be hindered by obstacles on the DNA template, such as damaged DNA. The authors recent findings highlight a trade‐off between transcription fidelity and DNA break repair. While a lot of work has focused on the interaction between transcription and nucleotide excision repair, less is known about how transcription influences the repair of DNA breaks. The authors suggest that when the cell experiences stress from DNA breaks, the control of RNAP processivity affects the balance between preserving transcription integrity and DNA repair. Here, how the conflict between transcription and DNA double‐strand break (DSB) repair threatens the integrity of both RNA and DNA are discussed. In reviewing this field, the authors speculate on cellular paradigms where this equilibrium is well sustained, and instances where the maintenance of transcription fidelity is favored over genome stability.