The yeast protein Xtc1 functions as a direct transcriptional repressor

Abstract

The yeast protein Xtc1 was identified as a protein that binds directly and specifically to the activation domains of acidic activators such as E2F-1, Gal4 and VP16. Additionally, it was shown to co-purify with the RNA polymerase II holoenzyme complex and it was suggested that Xtc1 functions as a regulator of transcription that modulates the response of RNA polymerase II to transcriptional activators. We have further analyzed the transcription function of Xtc1 and show that its fusion to a heterologous DNA binding domain can repress transcription of a reporter gene in vivo in an Srb10/11-dependent manner. We suggest that the presence of Xtc1 in the RNA polymerase II holoenzyme could help to recruit an Srb10-active form of the holoenzyme to target promoters. This same protein has also been implicated in mitochondrial DNA recombination, maintenance and repair. Determination of the subcellular localization using a GFP-Xtc1 fusion shows that it localizes to both the nucleus and the mitochondria in vivo, which is consistent with Xtc1 having a function in both cellular compartments.

Figure 1

The reporters used for β-galactosidase assays.

Figure 2

Repression by Xtc1 in vivo. β-galactosidase assays for all experiments were performed from at least three independent transformants assayed in triplicate and the standard error was between 10 and 20%. (A) β-galactosidase assays of transcriptional repression by Gal4–Xtc1 on reporters GG9, SS38-3 or SS36. (B) β-galactosidase assays of repression by Lex–Xtc1. The CYC1 reporter containing LexA operator sites is on plasmid JK1621 (2µ, URA3).

Figure 3

Mapping the repression domain. β-galactosidase assays were performed using various deletions of Xtc1 fused to the Gal4 DNA binding domain. (A) The mapping experiments were carried out using the reporter GG9. A schematic representation of the fusion protein Gal4–Xtc1 is shown above the graph. The mapped repression domain is shown in gray and the primary amino acid sequence is shown below the graph. (B) Transcriptional activation by the construct ΔN100 in the context of the reporter RY171.

Figure 4

Western analysis of the expression of the Gal4 DNA binding domain fusion constructs. Western blot analysis was performed with 500 µg of yeast extracts using a Gal4 DNA binding domain antibody. All extracts were made from strain GGY1_tH::GG9 transformed with the appropriate fusion construct, except the extract ΔN100, which was made from strain GGY1_tH::RY171.

Figure 5

GFP–Xtc1 localizes to both the nucleus and the mitochondria. For fluorescence microscopy, yeast wild-type strain W3031B was transformed with either pGFP-C-Xtc1 (A) or pGFP-N-Xtc1 (B). Nuclear and mitochondrial DNA were visualized by staining with 2.5 µg/ml DAPI. The arrows show co-localization of the fusion of GFP to Xtc1 with the mitochondria (A) or with both the nucleus and the mitochondria (B) visualized by DAPI staining of the respective genomes. The nucleus and the mitochondria are represented with N and M, respectively.