Zinc finger artificial transcription factor–based nearest inactive analogue/nearest active analogue strategy used for the identification of plant genes controlling homologous recombination

Summary In previous work, we selected a particular transcription factor, designated VP16‐HRU, from a pool of zinc finger artificial transcription factors (ZF‐ATFs) used for genome interrogation. When expressed in Arabidopsis thaliana under control of the ribosomal protein S5A promoter, the RPS5A::VP...

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Bibliographic Details
Published in:Plant biotechnology journal 2013-12, Vol.11 (9), p.1069-1079
Main Authors: Jia, Qi, Verk, Marcel C., Pinas, Johan E., Lindhout, Beatrice I., Hooykaas, Paul J.J., Zaal, Bert J.
Format: Article
Language:English
Subjects:
Arabidopsis - genetics
Arabidopsis thaliana
DNA damage
Ectopic expression
Epigenetics
Gene Expression
Gene Expression Profiling
Genes
Genes, Synthetic
genome interrogation
Genome, Plant - genetics
Genomes
High-Throughput Nucleotide Sequencing
Homologous recombination
Homologous Recombination - genetics
Interrogation
Kinases
mutagenesis
Mutation
next‐generation sequencing
Phenotype
Phenotypes
Plants, Genetically Modified
Promoter Regions, Genetic - genetics
Radiation
Sequence Analysis, DNA
somatic intrachromosomal homologous recombination
Transcription factors
Transcription Factors - genetics
transcriptome analysis
VP16 protein
Zinc
zinc finger artificial transcription factor
Zinc finger proteins
Zinc Fingers
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Summary:Summary In previous work, we selected a particular transcription factor, designated VP16‐HRU, from a pool of zinc finger artificial transcription factors (ZF‐ATFs) used for genome interrogation. When expressed in Arabidopsis thaliana under control of the ribosomal protein S5A promoter, the RPS5A::VP16‐HRU construct led to a 200‐ to 300‐fold increase in the frequency of somatic intrachromosomal homologous recombination (iHR). Because the expression of each ZF‐ATF leads to a large number of transcriptional changes, we designed a strategy employing a collection of structurally similar ZF‐ATFs to filter out the transcriptional changes relevant to the phenotype by deep sequencing. In that manner, 30 transcripts were found to be consistently induced in plants with enhanced homologous recombination (HR). For 25 of the cognate genes, their effect on the HR process was assessed using cDNA/gDNA expression constructs. For three genes, ectopic expression indeed led to enhanced iHR frequencies, albeit much lower than the frequency observed when a HR‐inducing ZF‐ATF was present. Altogether, our data demonstrate that despite the large number of transcriptional changes brought about by individual ZF‐ATFs, causal changes can be identified. In our case, the picture emerged that a natural regulatory switch for iHR does not exist but that ZF‐ATFs‐like VP16‐HRU act as an ectopic master switch, orchestrating the timely expression of a set of plant genes that each by themselves only have modest effects, but when acting together support an extremely high iHR frequency.
ISSN:1467-7644
1467-7652
DOI:10.1111/pbi.12101