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Abstract Bacteria contain conserved mechanisms to control the intracellular levels of metal ions. Metalloregulatory transcription factors bind metal cations and play a central role in regulating gene expression of metal transporters. Often, these transcription factors regulate transcription by binding to a specific DNA sequence in the promoter region of target genes. Understanding the preferred DNA‐binding sequence for transcriptional regulators can help uncover novel gene targets and provide insight into the biological role of the transcription factor in the host organism. Here, we identify consensus DNA‐binding sequences and subsequent transcription regulatory networks for two metalloregulators from the ferric uptake regulator (FUR) and diphtheria toxin repressor (DtxR) superfamilies inThermus thermophilusHB8. By homology search, we classify the DtxR homolog as a manganese‐specific, MntR (TtMntR), and the FUR homolog as a peroxide‐sensing, PerR (TtPerR). Both transcription factors repress separate ZIP transporter genes in vivo, andTtPerR acts as a bifunctional transcription regulator by activating the expression of ferric and hemin transport systems. We showTtPerR andTtMntR bind DNA in the presence of manganese in vitro and in vivo; however,TtPerR is unable to bind DNA in the presence of iron, likely due to iron‐mediated histidine oxidation. Unlike canonical PerR homologs,TtPerR does not appear to contribute to peroxide detoxification. Instead, theTtPerR regulon and DNA binding sequence are more reminiscent of Fur or Mur homologs. Collectively, these results highlight the similarities and differences between two metalloregulatory superfamilies and underscore the interplay of manganese and iron in transcription factor regulation.
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Direct regulation of shikimate, early phenylpropanoid, and stilbenoid pathways by Subgroup 2 R2R3‐MYBs in grapevine
SUMMARY The stilbenoid pathway is responsible for the production of resveratrol in grapevine (Vitis viniferaL.). A few transcription factors (TFs) have been identified as regulators of this pathway but the extent of this control has not been deeply studied. Here we show how DNA affinity purification sequencing (DAP‐Seq) allows for the genome‐wide TF‐binding site interrogation in grape. We obtained 5190 and 4443 binding events assigned to 4041 and 3626 genes for MYB14 and MYB15, respectively (approximately 40% of peaks located within −10 kb of transcription start sites). DAP‐Seq of MYB14/MYB15 was combined with aggregate gene co‐expression networks (GCNs) built from more than 1400 transcriptomic datasets from leaves, fruits, and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15, and MYB13, a third uncharacterized member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS) targets, these regulators bind to 30 of 47STSfamily genes. Moreover, all three MYBs bind to severalPAL,C4H, and4CLgenes, in addition to shikimate pathway genes, theWRKY03stilbenoid co‐regulator and resveratrol‐modifying gene candidates among which ROMT2‐3 were validated enzymatically. A high proportion of DAP‐Seq bound genes were induced in the activated transcriptomes of transientMYB15‐overexpressing grapevine leaves, validating our methodological approach for delimiting TF targets. Overall, Subgroup 2 R2R3‐MYBs appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. The integration of DAP‐Seq and reciprocal GCNs offers a rapid framework for gene function characterization using genome‐wide approaches in the context of non‐model plant species and stands up as a valid first approach for identifying gene regulatory networks of specialized metabolism.
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- Award ID(s):
- 1916804
- PAR ID:
- 10367759
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 110
- Issue:
- 2
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 529-547
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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