Chromatin Rearrangements in the prnD-prnB Bidirectional Promoter: Dependence on Transcription Factors

Abstract

The prnD-prnB intergenic region regulates the divergent transcription of the genes encoding proline oxidase and the major proline transporter. Eight nucleosomes are positioned in this region. Upon induction, the positioning of these nucleosomes is lost. This process depends on the specific transcriptional activator PrnA but not on the general GATA factor AreA. Induction of prnB but not prnD can be elicited by amino acid starvation. A specific nucleosomal pattern in the prnB proximal region is associated with this process. Under conditions of induction by proline, metabolite repression depends on the presence of both repressing carbon (glucose) and nitrogen (ammonium) sources. Under these repressing conditions, partial nucleosomal positioning is observed. This depends on the CreA repressor's binding to two specific cis-acting sites. Three conditions (induction by the defective PrnA80 protein, induction by amino acid starvation, and induction in the presence of an activated CreA) result in similar low transcriptional activation. Each results in a different nucleosome pattern, which argues strongly for a specific effect of each signal on nucleosome positioning. Experiments with trichostatin A suggest that both default nucleosome positioning and partial positioning under induced-repressed conditions depend on deacetylated histones.In simple eukaryotes, some genes are transcribed divergently from a common bidirectional promoter. Well-studied examples are the GAL1-GAL10 promoter of Saccharomyces cerevisioe (reviewed in references 10 and 35) and the niiA-niaD promoter of Aspergillus nidulans (37, 41, 52). Here we analyzed the nucleosome rearrangements of the bidirectional prnD-prnB intergenic region. This is a 1.7-kb region located between the gene coding for proline oxidase (prnD) and the one coding for the major, specific proline transporter (prnB) (29, 50). These genes are located in the prn gene cluster in the right arm of chromosome VII (Fig. 1). The regulation of prnD and prnB involves a multiplicity of metabolic signals. The pathway-specific transcription factor PrnA is essential for proline induction of both genes (11,21,22,46). The prnD-prnB intergenic region is a genuine bidirectional promoter, as mutations in the two PrnA binding sites present in this region affect the transcription of both prnD and prnB (21; I. García, D. Gómez, and C. Scazzocchio, unpublished results).Transcription of prnB but not of prnD can also be induced by amino acid starvation. This effect depends on the integrity of a canonical GCN4 binding site in the proximity of the prnB TATA box (56).Repression of both prnB and prnD occurs only when both carbon (glucose) and nitrogen-repressing (ammonium) sources are present simultaneously (2, 5, 6, 21, 22). Repression acts directly on prnB expression, while repression of prnD is indirect and results from inducer exclusion (5, 16, 22). Repression necessitates both the activation of the negative regulator CreA and the inactivation of the GATA factor AreA. A model to account for this pattern of re...

Nucleosome Positioning and Histone H3 Acetylation Are Independent Processes in the Aspergillus nidulans prnD-prnB Bidirectional Promoter

“…AreA in the proline cluster is required only in the presence of glucose, which is consistent with its proposed function, which is to counteract the repressive effect of the global carbon repressor CreA. A functional CreA protein and two CreA binding sites in the prnD-prnB bidirectional promoter were shown to be essential for nucleosome repositioning under repression conditions (12). A first indication that histone acetylation is involved in the CreA-mediated repression of the prnD-prnB bidirectional promoter process came from the observation that trichostatin A (TSA), an inhibitor of histone deacetylases, mimics the effect of a CreA loss-of-function mutation or of cis-acting CreA binding site mutations (termed prn d ).…”

“…Around 1.4 kb of the adaB 3Ј region was amplified with primers 3Ј forward (5Ј-CGAATCATCACGTCGAC TTGAATTTAGAGGCC-3Ј; the SalI site introduced by mutation of the HindIII restriction site, positioned 50 bp downstream of the empirically determined polyadenylation site, is underlined) and 3Ј reverse (5Ј-GCGAGTTGACTGAG CTCGAGAAGGATCACCCTC-3Ј 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.…”

“…We have recently described the relationship between the transcriptional activation of a number of Aspergillus nidulans promoters driving genes of primary metabolism and their chromatin architecture (3,12,25,(27)(28)(29). The prn cluster of A. nidulans comprises all the genes involved in proline utilization as a sole nitrogen and/or carbon source.…”

“…A first indication that histone acetylation is involved in the CreA-mediated repression of the prnD-prnB bidirectional promoter process came from the observation that trichostatin A (TSA), an inhibitor of histone deacetylases, mimics the effect of a CreA loss-of-function mutation or of cis-acting CreA binding site mutations (termed prn d ). Nucleosome positioning after repression and transcriptional repression is completely lost when CreA is nonfunctional and also partially lost when TSA is present in the culture medium (12).…”

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

“…The prnB gene has been extensively used as a model for transcriptional and post-transcriptional regulation of gene expression (15)(16)(17)(18)(19)(20).…”