Figure 1.
A schematic representation of the Hap4 and Yap1 proteins.
The Saccharomyces cerevisiae Hap4 and Yap1 proteins are schematically represented relative to the two Hansenula polymorpha HpHap4-A and HpHap4-B ones. The main motifs are indicated in grey (N-terminal Hap4), black (bZIP of BR motif; the BR motif is the DNA binding part of the bZIP motif) and striped (CRD or cysteine rich domain).
Figure 2.
A simple phylogenetic tree of the hemiascomycete yeasts.
Only a limited number of yeast species are presented which span the complete clade of Hemiascomycete yeasts from S. cerevisiae to Y. lipolytica, S. pombe being the outgroup. H. polymorpha is placed on the tree at the same position as P. angusta which is the anamorphic species. The position of the « Whole Genome Duplication » (WGD) is indicated. The tree is taken from [48].
Table 1.
Strains and plasmids used in this study.
Table 2.
List of H. polymorpha genes and corresponding primers used for qRT-PCR.
Figure 3.
Analysis of transcriptomic data of the ScΔhap4 strains.
The analysed data are the normalized log2 ratios (intensity in WT strain versus intensity in the mutant strains) obtained from the microarray experiments (four biological replicates per condition) in the ScΔhap4 genetic background. The WT strain is defined as ScΔhap4 plus the empty plasmid pBFG1 and the three mutant strains as ScΔhap4plus pBFG1 containing the ScHap4, the HpHAP4-A or the HpHAP4-B genes. A: Heat map clustergram. This figure reveals the changes in gene expression between experiments. The experiments and the genes are clustered in a tree from their Pearson correlation coefficient values. Gene expression level is represented on a heat map by colour level (green when overexpressed and red when underexpressed compared to the WT strain). B: Presentation of the experiments in the 3D space of the principal components. The two axes of the figure are the three first principal components determined by Principal Component Analysis (PCA). They explain, respectively, 36%, 27% and 16% of the variance.
Table 3.
Genes up-regulated by HpHAP4-B in the Δhap4 background.
Figure 4.
Functional comparison between ScYAP1 and the HpHAP4-B genes.
Part A: Comparison of the new bZIP type motif identified in HpHap4-B with the YAP family motif. Q234, Q239, A241 and F/Y242 are four basic regions (DNA-binding sites) characteristic residues of the YAP protein family which are rarely or never observed in other bZIP proteins [34]. Part B: Heterologous complementation of the growth deficiency of S. cerevisiae Δyap1 in the presence of H202 by the HpHAP4-B gene. The gene is expressed on a multicopy plasmid (pBFG1, see Methods) and the growth monitored on minimal medium (W0) containing 0.5 mM of H202. 1: S. cerevisiae Δyap1 strain (with or without H202). 2:Δyap1 strain with empty plasmid BFG1. 3: Δyap1 with BFG1 plasmid containing HpHap4-B. 4: Δyap1 with BFG1 plasmid containing HpHap4-B with deleted bZIP domain (see Methods). Tests 2, 3 and 4 were performed on medium containing H202. Strains were grown on minimal glucose medium supplemented with the necessary aminoacids at 28°C for 5 days.
Figure 5.
Gel shift experiment with a synthetic ARE sequence.
Probe alone (lane 1), S. cerevisiae Δyap1 strain (lane 2), ScΔyap1 with empty plasmid pBFG1 (lane 3), ScΔyap1 with pBFG1 plasmid carrying ScHAP4 (lane 4), pBFG1-ScYAP1(lane 5), pBFG1-HpHAP4-A (lane 6), pBFG1-HpHAP4-B (lane 7) pBFG1-HpHAP4-B-bZIP (devoid of Hphap4-B BR region; lane 8).
Figure 6.
Principal Component Analysis of transcriptome data of the ScΔyap1 strains.
The analysed data are the normalized median signals obtained in two mutants and in two different growth conditions (with or without H2O2), compared to the WT by microarray experiments in a ScΔyap1 genetic background. The WT is defined as ScΔyap1 plus the empty plasmid pBFG1, and the mutants as ScΔyap1 plus pBFG1 carrying either the ScYAP1 or the HpHAP4-B gene. For each condition, four independent experiments were performed. The two axes of the figure are the second and the third first principal components and explain 7.2% of the variance.
Table 4.
Gene ontology categories of the genes regulated by HpHap4-B and ScYap1.
Figure 7.
Growth of different H. polymorpha strains on media containing either antimycin A, SHAM or hydrogen peroxide.
A: H. polymorpha HpΔhap4-A and double knock-out strains, but not HpΔhap4-B deletion mutant, are sensitive to antimycin A on glucose. B: Growth on xylose plus SHAM or H202. 1: HpNCYC495leu1_1 strain. 2: HpNCYC495leu1_1 with pYT1 (empty plasmid, see Material and Methods). 3: HpΔhap4-A. 4: HpΔhap4-B. 5: HpΔhap4-A HpΔhap4-B (double knock-out strain).
Table 5.
qRT-PCR of HpHAP4-A and HpHAP4-B regulation of gene expression in H. polymorpha.
Figure 8.
Phylogenetic tree of fungi in relation with the presence/absence of HpHap4-B-type of proteins.
The phylogenetic tree was adapted from [49]. Species in which proteins such as HpHap4-B (containing both the N- Hap4 and the BR motifs) could be identified are boxed. Aligned with each species, the presence of proteins of the Yap1 type (Yap1 BR motif), HpHap4-B type (BR plus N-ter motifs) and HAP4-A type (N-ter motif) of protein is indicated next to each species. The position of H. polymorpha, which is not on the tree, is indicated, underlining that this yeast belongs to the clade where C. albicans and D. hansenii are localized. * These species were not correctly annotated; for P. nodorum, the BR and N-ter motif are overlapping and degenerate while for U. maydis, the protein annotated as Yap1 turned out to contain also an N-ter motif and belongs therefore to the HpHap4-B family of proteins.
Figure 9.
Identification of a “Relic sequence” in S. cerevisiae.
Upper part (Hap4) is the N-ter Hap4 motif with small extensions (the motif itself spans aminoacids 60 to 76). Bottom part (Yap7) is a small sequence in the Yap7 protein which is 68% similar to the upper sequence. Yap7 is a member of the bZIP family of S. cerevisiae transactivators. Its function is not precisely known [50]. Refer to Fig 1 for the various motifs of these proteins.