Article Figures & Data
Figures
- FIG. 1.
Regulation of proline utilization in A. nidulans. (A) Overview of proline cluster regulation. The prnD-B intergenic region is shown. prnD encodes the proline oxidase, and prnB encodes the specific proline transporter (12, 14). The pathway-specific transcription factor PrnA is essential for proline induction of both genes. In the absence of preferential carbon (glucose) and nitrogen (ammonium) sources and the presence of proline, PrnA and the GATA factor AreA bind to their cognate sites in the intergenic region, resulting in the expression of prnD and prnB. Repression requires both glucose activation of the negative regulator CreA and ammonium inactivation of AreA. Full repression occurs only in the simultaneous presence of glucose and ammonium. Repression acts directly on prnB expression; prnD repression is indirect and results from inducer exclusion. (B) Effect of adaB and gcnE deletion on prnB and prnD transcription. Strains (Table 1) were pregrown in liquid minimal medium under noninducing conditions (5 mM urea-0.1% fructose) with the appropriate supplements, harvested, divided into aliquots, and further incubated for 2 h under the conditions indicated. Noninducing (NI), 5 mM urea and 0.1% fructose; inducing (I), induced by 20 mM proline; inducing-repressing (IR), 20 mM proline and repression by 1% glucose and 20 mM ammonium-l(+)-tartrate. Expression levels (bottom) were quantified using phosphorimaging and ImageQuant software analysis. Normalized signals were obtained by comparison of specific signals with actin gene (acnA) expression signals. The induced levels in the adaB+ and gcnE+ strains are given in every case the arbitrary value of 100; filled columns represent prnB, and open columns represent prnD expression.
- FIG. 2.
Nucleosome positioning in the prnD-prnB intergenic region in adaB+ and adaBΔ strains. (A) Indirect end-labeling MNase I analysis of the prnD-prnB promoter region using probe SC1. This probe reveals nucleosomes +2 to −4. MNase analysis of nucleosomes +3 and +4 is not shown as the patterns obtained for the mutant strains are identical to the pattern of the wild type. Growth conditions were identical to those described in the legend of Fig. 1: NI, noninducing; I, inducing; IR, inducing-repressing. Numbers beside the autoradiograms correspond to the positions of the main cuts relative to the prnD ATG. These were calculated from molecular size markers run in every gel. Asterisks indicate the positions of the relevant changes observed. Triangles indicate increasing concentrations of MNase I. nDNA, naked DNA. To the left of the adaBΔ NI lanes, a schematic representation of the nucleosome structure is shown. The wild-type patterns under all conditions have been previously published (12); we include in the same gel for comparison only the pattern obtained under inducing-repressing conditions, the only one where an adaB deletion pattern differs from that of the wild type. (B) Schematic representation of nucleosome positioning (based on the results shown in panel A and data from reference 12). All eight nucleosomes of the intergenic region are drawn. Arrows indicate MNase I digests. Their thicknesses indicate the relative intensities of the bands in the autoradiogram shown in panel A and in other autoradiograms (data not shown) covering nucleosomes +3 and +4. Dashed arrows indicate weakly cut sites. Under noninducing and inducing conditions, the nucleosome patterns are identical in the adaB+ and adaBΔ strains. White ovals represent fully positioned nucleosomes, while partially positioned nucleosomes are shown by diagonally hatched ovals. The interpretation and significance of this partial positioning are discussed in the text (see also references 12 and 25). Symbols: white lozenges, CreA-binding sites 3.1 and 3.2, which are mutated in the prnd20 and prnd22 strains and result in derepression (see text); gray ovals, AreA-binding sites 13 and 14, shown to be the physiologically relevant AreA binding sites (15); white triangles, high-affinity PrnA binding sites 2 and 3 (14); black triangle, TATA box (15). The fragment amplified in the ChIP analysis (fragment a) is shown as a solid line below nucleosome +2. nDNA, pattern obtained with naked DNA. To the right of the scheme we indicate the strain(s) of the corresponding nucleosome pattern.
- FIG. 3.
ChIP assay comparing acetylated H3K9 and -K14 in nucleosome +2 of the prnD-prnB intergenic region between adaB+ and adaBΔ (A) and between gcnE+ and gcnEΔ (B) strains (see Table 1 for genotype details). The ratio between total H3 (C-terminal [C-term] epitope) and H3-acetyl (recognizing H3 K9/K14) in the adaB+ wild-type strain grown under noninducing conditions (NI) was set to 1. Error bars indicate the standard deviation of four experiments (two biological and two technical repetitions per condition). Growth conditions: NI, noninducing; I, inducing; IR, simultaneously inducing-repressing.
Tables
- TABLE 1.
A. nidulans strains used throughout this work
Strain Genotype Reference or sourcea gcnE + pyrG89 argB2 pantoB100 riboB2 yA2 This work gcnEΔ pyrG89 argB2 pantoB100 riboB2 yA2 gcnEΔ::pyrG+ This work adaB+ biA1 FGSC strain A26 adaBΔ biA1 argB::trpCΔB adaBΔ::argB+ This work MH 9233 wA3 biA1 argB::trpCΔB pyroA4 riboB2 26 ↵ a FGSC, Fungal Genetics Stock Center (http://www.fgsc.net ).
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Photographs obtained from the adaBΔ strain.
Zipped PPT file, 1.4MB.
- Supplemental file 1
-
Photographs obtained from the adaBΔ strain.
