Can AtTZF1 act as a transcriptional activator or repressor in plants?

Abstract

In animals, Tandem CCCH Zinc Finger (TZF) proteins can affect gene expression at both transcriptional and post-transcriptional levels. In Arabidopsis thaliana, AtTZF1 is a member of the TZF family characterized by a plant-unique tandem zinc finger motif. AtTZF1 can bind both DNA and RNA in vitro, and it can traffic between the nucleus and cytoplasmic foci. However, no in vivo DNA/RNA targets have been identified so far, and little is known about the molecular mechanisms underlying AtTZF1's profound effects on plant growth, development, and stress responses. In order to determine whether AtTZF1 can function as a transcription factor, transactivation assays were conducted. Results indicated that AtTZF1 fusion proteins could not exert obvious transcriptional activity in a maize protoplast transient expression system. However, this conclusion might be biased due to poor nuclear localization of AtTZF1 fusion proteins in the assay system.

Figure 1

GAL4^DBD-AtTZF1 transactivation assay in yeast. (A) ClustalW sequence alignment of 53 amino acid minimal activation EELR regions in the Oslic transcription factor, At1g75340 (OsLIC homolog) and AtTZF1. AtTZF1 EELR shows partial conservation. (B) Yeast cells were first selected on -Trp single dropout plates for the positive transformation of GAL4^DBD-AtTZF1 plasmid. (C) Neither GAL4^DBD nor Gal4^DBD-AtTZF1 showed any activation of His or Ade synthesis genes and thus did not grow on -Trp-His-Ade triple dropout plates. Only yeast transformed with positive control GAL4^DBD-SNF1 and GAL4^AD-SNF4 activated transcription and grew on selection media.

Figure 2

GAL4^DBD transactivation assays conducted in maize protoplasts. (A) AtTZF1 does not exhibit trancriptional activation activity. GFP-GAL4^DBD-AtTZF1 does not activate min35S-Gal4^BS:firefly luciferase reporter. GFP-AtTZF1 without the GAL4^DBD was included as a negative control and maize transcriptional activator Gal4^DBD-C1^cterm-GFP [13] was included as a positive control. (B) AtTZF1 does not exhibit transcriptional repression activity. GFP-Gal4^DBD-AtTZF1 does not repress CaMV35S-Gal4^BS:firefly luciferase reporter. (C) Schematic representation of reporter constructs. Reporter min35S-Gal4^BS:firefly luciferase was used for activation assays and CaMV35S-Gal4^BS:firefly luciferase was used for repression assays. CaMV35S:renilla luciferase was used as an internal control. Readings were taken using Promega Dual-Luciferase^® reporter assay system. Readings are normalized using the ratios of firefly/Renilla luciferase activities and presented as fold expressions over the blank control containing only reporter plasmids without effectors.