Gcn5 Mutants Research Articles

Histone acetyltransferase GCN5 is essential for heat stress-responsive gene activation and thermotolerance in Arabidopsis.

Exposure to temperatures exceeding the normal optimum levels, or heat stress (HS), constitutes an environmental disruption for plants, resulting in severe growth and development retardation. Here we show that loss of function of the Arabidopsis histone acetyltransferase GCN5 results in serious defects in terms of thermotolerance, and considerably impairs the transcriptional activation of HS-responsive genes. Notably, expression of several key regulators such as the HS transcription factors HSFA2 and HSFA3, Multiprotein Bridging Factor 1c (MBF1c) and UV-HYPERSENSITIVE 6 (UVH6) is down-regulated in the gcn5 mutant under HS compared with the wild-type. Chromatin immunoprecipitation (ChIP) assays indicated that GCN5 protein is enriched at the promoter regions of HSFA3 and UVH6 genes, but not in HSFA2 and MBF1c, and that GCN5 facilitates H3K9 and H3K14 acetylation, which are associated with HSFA3 and UVH6 activation under HS. Moreover, constitutive expression of UVH6 in the gcn5 mutant partially restores heat tolerance. Taken together, our data indicate that GCN5 plays a key role in the preservation of thermotolerance via versatile regulation in Arabidopsis. In addition, expression of the wheat TaGCN5 gene re-establishes heat tolerance in Arabidopsis gcn5 mutant plants, suggesting that GCN5-mediated thermotolerance may be conserved between Arabidopsis and wheat.

Synergistic action of histone acetyltransferase GCN5 and receptor CLAVATA1 negatively affects ethylene responses in Arabidopsis thaliana.

GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5) is a histone acetyltransferase (HAT) and the catalytic subunit of several multicomponent HAT complexes that acetylate lysine residues of histone H3. Mutants in AtGCN5 display pleiotropic developmental defects including aberrant meristem function. Shoot apical meristem (SAM) maintenance is regulated by CLAVATA1 (CLV1), a receptor kinase that controls the size of the shoot and floral meristems. Upon activation through CLV3 binding, CLV1 signals to the transcription factor WUSCHEL (WUS), restricting WUS expression and thus the meristem size. We hypothesized that GCN5 and CLV1 act together to affect SAM function. Using genetic and molecular approaches, we generated and characterized clv gcn5 mutants. Surprisingly, the clv1-1 gcn5-1 double mutant exhibited constitutive ethylene responses, suggesting that GCN5 and CLV signaling act synergistically to inhibit ethylene responses in Arabidopsis. This genetic and molecular interaction was mediated by ETHYLENE INSENSITIVE 3/ EIN3-LIKE1 (EIN3/EIL1) transcription factors. Our data suggest that signals from the CLV transduction pathway reach the GCN5-containing complexes in the nucleus and alter the histone acetylation status of ethylene-responsive genes, thus translating the CLV information to transcriptional activity and uncovering a link between histone acetylation and SAM maintenance in the complex mode of ethylene signaling.

PH-Jump Induced Leucine Zipper Folding beyond the Diffusion Limit

The folding of a pH-sensitive leucine zipper, that is, a GCN4 mutant containing eight glutamic acid residues, has been investigated. A pH-jump induced by a caged proton (o-nitrobenzaldehyde, oNBA) is employed to initiate the process, and time-resolved IR spectroscopy of the amide I band is used to probe it. The experiment has been carefully designed to minimize the buffer capacity of the sample solution so that a large pH jump can be achieved, leading to a transition from a completely unfolded to a completely folded state with a single laser shot. In order to eliminate the otherwise rate-limiting diffusion-controlled step of the association of two peptides, they have been covalently linked. The results for the folding kinetics of the cross-linked peptide are compared with those of an unlinked peptide, which reveals a detailed picture of the folding mechanism. That is, folding occurs in two steps, one on an ∼1-2 μs time scale leading to a partially folded α-helix even in the monomeric case and a second one leading to the final coiled-coil structure on distinctively different time scales of ∼30 μs for the cross-linked peptide and ∼200 μs for the unlinked peptide. By varying the initial pH, it is found that the folding mechanism is consistent with a thermodynamic two-state model, despite the fact that a transient intermediate is observed in the kinetic experiment.

Acetylome Profiling Reveals Overlap in the Regulation of Diverse Processes by Sirtuins, Gcn5, and Esa1

Although histone acetylation and deacetylation machineries (HATs and HDACs) regulate important aspects of cell function by targeting histone tails, recent work highlights that non-histone protein acetylation is also pervasive in eukaryotes. Here, we use quantitative mass-spectrometry to define acetylations targeted by the sirtuin family, previously implicated in the regulation of non-histone protein acetylation. To identify HATs that promote acetylation of these sites, we also performed this analysis in gcn5 (SAGA) and esa1 (NuA4) mutants. We observed strong sequence specificity for the sirtuins and for each of these HATs. Although the Gcn5 and Esa1 consensus sequences are entirely distinct, the sirtuin consensus overlaps almost entirely with that of Gcn5, suggesting a strong coordination between these two regulatory enzymes. Furthermore, by examining global acetylation in an ada2 mutant, which dissociates Gcn5 from the SAGA complex, we found that a subset of Gcn5 targets did not depend on an intact SAGA complex for targeting. Our work provides a framework for understanding how HAT and HDAC enzymes collaborate to regulate critical cellular processes related to growth and division.

Oxidant-specific regulation of protein synthesis in Candida albicans

Eukaryotic cells typically respond to stress conditions by inhibiting global protein synthesis. The initiation phase is the main target of regulation and represents a key control point for eukaryotic gene expression. In Saccharomyces cerevisiae and mammalian cells this is achieved by phosphorylation of eukaryotic initiation factor 2 (eIF2α). We have examined how the fungal pathogen Candida albicans responds to oxidative stress conditions and show that oxidants including hydrogen peroxide, the heavy metal cadmium and the thiol oxidant diamide inhibit translation initiation. The inhibition in response to hydrogen peroxide and cadmium largely depends on phosphorylation of eIF2α since minimal inhibition is observed in a gcn2 mutant. In contrast, translation initiation is inhibited in a Gcn2-independent manner in response to diamide. Our data indicate that all three oxidants inhibit growth of C. albicans in a dose-dependent manner, however, loss of GCN2 does not improve growth in the presence of hydrogen peroxide or cadmium. Examination of translational activity indicates that these oxidants inhibit translation at a post-initiation phase which may account for the growth inhibition in a gcn2 mutant. As well as inhibiting global translation initiation, phosphorylation of eIF2α also enhances expression of the GCN4 mRNA in yeast via a well-known translational control mechanism. We show that C. albicans GCN4 is similarly induced in response to oxidative stress conditions and Gcn4 is specifically required for hydrogen peroxide tolerance. Thus, the response of C. albicans to oxidative stress is mediated by oxidant-specific regulation of translation initiation and we discuss our findings in comparison to other eukaryotes including the yeast S. cerevisiae.

Sublethal Cadmium Intoxication In Arabidopsis thaliana Impacts Translation at Multiple Levels

To study the impact of translational regulation during heavy metal poisoning, Arabidopsis thaliana cell cultures were submitted to sublethal cadmium stress. At the concentration used, cadmium had a minimal impact on the growth of the culture but induced an accumulation of high molecular weight polysomes without de novo production of new ribosomes together with a reduction of protein synthesis. In addition, cadmium stress induces phosphorylation of eukaryotic initiation factor 2α by GCN2 and, in planta, gcn2 mutants are more sensitive to cadmium stress, suggesting a role for this translational regulation mechanism in the response to cadmium stress. Microarray analysis of total and polysomal RNAs in control and cadmium-treated cells reveals a large class of genes for which a variation in total RNA abundance is not linked to a variation in polysomal loading, suggesting that transcription and translation are uncoupled and that these genes are not recruited at the initiation step of translation.

The histone acetyltransferase GCN5 affects the inflorescence meristem and stamen development in Arabidopsis

A central question in biology is to understand how gene expression is precisely regulated to give rise to a variety of forms during the process of development. Epigenetic effects such as DNA methylation or histone modification have been increasingly shown to play a critical role in regulation of genome function. GCN5 is a prototypical histone acetyltransferase that participates in regulating developmental gene expression in several metazoan species. In Arabidopsis thaliana, plants with T-DNA insertions in GCN5 (also known as HAG1) display a variety of pleiotropic effects including dwarfism, loss of apical dominance, and floral defects affecting fertility. We sought to determine when during early development floral abnormalities first arise. Using scanning electron microscopy, we demonstrate that gcn5-1/hag1-1 and gcn5-5/hag1-5 mutants display overproliferation of young buds and development of abnormal structures around the inflorescence meristem. gcn5 mutants also display defects in stamen number and arrangement at later stages. This analysis provides temporal and spatial information to aid in the identification of GCN5 target genes in the developing flower. Preliminary studies of putative targets using reverse transcriptase PCR suggest that the floral meristem identity gene LEAFY is among factors upregulated in gcn5-1 mutants.

Members of the GCN5 Histone Acetyltransferase Complex Regulate PLETHORA-Mediated Root Stem Cell Niche Maintenance and Transit Amplifying Cell Proliferation inArabidopsis

The PLETHORA (PLT) stem cell transcription factors form a developmentally instructive protein gradient in Arabidopsis thaliana roots. Histone acetylation is known to facilitate gene transcription and plays an important role in developmental processes. Here, we show that histone acetyltransferase GCN5 (for general control nonderepressible 5) attenuates the PLT gradient. Based on genetic evidence, we establish that GCN5 is essential for root stem cell niche maintenance and acts in the PLT pathway. The GCN5-associated factor ADA2b (for alteration/deficiency in activation 2b) is also positioned in the PLT pathway and regulates PLT expression, similar to GCN5. Both GCN5 and ADA2b mediate proliferation of the transit amplifying cells, but ADA2b does not affect stem cell niche maintenance. Overexpression of PLT2 rescues the stem cell niche defect of gcn5 mutants, indicating that GCN5 regulation of PLT expression is essential for maintenance of the root stem cell niche. We conclude that histone acetylation complexes play an important role in shaping a developmentally instructive gradient in the root.

Functional connection between histone acetyltransferase Gcn5p and methyltransferase Hmt1p

Histone acetylation and methylation are linked to a variety of nuclear activities, most notably transcriptional regulation. Both synergistic and antagonistic relationships between these two modifications have been reported in different systems. Here we show that the budding yeast histone H4 arginine 3 (R3) methyltransferase Hmt1p binds acetylated histones H3 and H4, and importantly, that acetylated H4 is a significantly better methylation substrate for Hmt1p. Kinetic studies show that acetylation at any of the four acetylatable lysine residues of histone H4 results in more efficient methylation. Among the four, K8 acetylation imposes the strongest effect on reducing K M, consistent with the observed acetylation-stimulated interaction. In vivo, hmt1Δ cells rescue the transcriptional defect caused by GCN5 deletion, indicating that one of the functions of Gcn5p is to neutralize the negative effect of Hmt1p. Mutating either K8 or R3 to alanine causes similar growth defects in selective histone and gcn5 mutant background, suggesting that these two residues function in the same pathway for optimal vegetative growth. Together, these results reveal a functional connection between histone acetylation, methylation, and two of the responsible enzymes, Gcn5p and Hmt1p.

Insights into prevention of human neural tube defects by folic acid arising from consideration of mouse mutants

Almost 30 years after the initial study by Richard W. Smithells and coworkers, it is still unknown how maternal periconceptional folic acid supplementation prevents human neural tube defects (NTDs). In this article, questions about human NTD prevention are considered in relation to three groups of mouse models: NTD mutants that respond to folate, NTD mutants and strains that do not respond to folate, and mutants involving folate-pathway genes. Of the 200 mouse NTD mutants, only a few have been tested with folate; half respond and half do not. Among responsive mutants, folic acid supplementation reduces exencephaly and/or spina bifida aperta frequency in the Sp(2H), Sp, Cd, Cited2, Cart1, and Gcn5 mutants. Prevention ranges from 35 to 85%. The responsive Sp(2H) (Pax3) mutant has abnormal folate metabolism, but the responsive Cited2 mutant does not. Neither folic nor folinic acid reduces NTD frequency in Axd, Grhl3, Fkbp8, Map3k4, or Nog mutants or in the curly tail or SELH/Bc strains. Spina bifida frequency is reduced in Axd by methionine and in curly tail by inositol. Exencephaly frequency is reduced in SELH/Bc by an alternative commercial ration. Mutations in folate-pathway genes do not cause NTDs, except for 30% exencephaly in folate-treated Folr1. Among folate-pathway mutants, neural tube closure is normal in Cbs, Folr2, Mthfd1, Mthfd2, Mthfr, and Shmt1 mutants. Embryos die by midgestation in Folr1, Mtr, Mtrr, and RFC1 mutants. The mouse models point to genetic heterogeneity in the ability to respond to folic acid and also to heterogeneity in genetic cause of NTDs that can be prevented by folic acid.

The Histone Chaperone Anti-silencing Function 1 Stimulates the Acetylation of Newly Synthesized Histone H3 in S-phase

Anti-silencing function 1 (Asf1) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. We have found that budding yeast lacking Asf1 has greatly reduced levels of histone H3 acetylated at lysine 9. Lysine 9 is acetylated on newly synthesized budding yeast histone H3 prior to its assembly onto newly replicated DNA. Accordingly, we found that the vast majority of H3 Lys-9 acetylation peaked in S-phase, and this S-phase peak of H3 lysine 9 acetylation was absent in yeast lacking Asf1. By contrast, deletion of ASF1 has no effect on the S-phase specific peak of H4 lysine 12 acetylation; another modification carried by newly synthesized histones prior to chromatin assembly. We show that Gcn5 is the histone acetyltransferase responsible for the S-phase-specific peak of H3 lysine 9 acetylation. Strikingly, overexpression of Asf1 leads to greatly increased levels of H3 on acetylation on lysine 56 and Gcn5-dependent acetylation on lysine 9. Analysis of a panel of Asf1 mutations that modulate the ability of Asf1 to bind to histones H3/H4 demonstrates that the histone binding activity of Asf1 is required for the acetylation of Lys-9 and Lys-56 on newly synthesized H3. These results demonstrate that Asf1 does not affect the stability of the newly synthesized histones per se, but instead histone binding by Asf1 promotes the efficient acetylation of specific residues of newly synthesized histone H3.

Global Translational Responses to Oxidative Stress Impact upon Multiple Levels of Protein Synthesis

Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H(2)O(2) in the yeast Saccharomyces cerevisiae. Our data show that H(2)O(2) causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the alpha-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H(2)O(2) in a gcn2 mutant. Polysome analysis indicated that H(2)O(2) causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome- and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H(2)O(2) stress. For some candidate mRNAs, we show that a low concentration of H(2)O(2) results in increased protein production. In contrast, a high concentration of H(2)O(2) promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress.

GCN5 Functions in Telomere Maintenance and Neural Development

We reported previously GCN5 deletion in the mouse leads to embryonic lethality due to increased apoptosis. New data show that this apoptosis is p53 dependent and may be triggered by telomere defects. Telomeres are shortened in cells from GCN5 mutant embryos, and these cells display increased numbers of chromosomal end associations and fusions. In the absence of both p53 and GCN5, an even greater incidence of telomere fusion is observed. These double mutant embryos also display defects in neural tube closure and brain development. Very similar defects in neural development are observed in mice homozygous for GCN5 HAT catalytic site mutations and in mice that carry a hypomorphic allele of GCN5. These data provide the first evidence that GCN5 may be required both for telomere maintenance and proper expression of genes required for neural development in the mouse.

A histone H3 Ser10 phosphorylation‐independent function of Snf1 and Reg1 proteins rescues a gcn5¬C mutant in HIS3 expression

Gcn5 serves as the catalytic subunit of the SAGA histone acetyltransferase (HAT) complex and is critical for HIS3 activation in Saccharomyces cerevisiae. A truncated Reg1 protein, Reg1(1–740), was found to be a dominant suppressor rescuing the HIS3 transcriptional defects of a gcn5 mutant (E173H). The function of Reg1(1–740) protein requires an intact cis-acting element for the Gcn4 transcriptional activator, and another SAGA component, Spt3, suggesting that the observed suppression was achieved via the normal SAGA regulatory pathway. Reg1 protein functionally and physically interacts with a histone H3 kinase, Snf1. Indeed, Snf1 plays an important role for normal and Reg1(1–740)-dependent HIS3 activation. However, substituting the phosphorylation residue in H3, i.e. Ser10, with alanine or glutamate neither attenuates nor augments the suppression phenotypes. These results argue against an essential role of H3 phosphorylation in HIS3 expression. Both Reg1(1–740) and overexpressed Snf1 rescue the E173H allele of gcn5 selectively, suggesting physical interactions between Gcn5 and these two proteins. In vivo co-purification experiments confirmed this notion, and importantly, in vitro data showed that Gcn5 can be phosphorylated by Snf1 protein. Together, these data suggest that Reg1 and Snf1 proteins activate HIS3 in a histone H3 phosphorylation-independent manner that may also involve a non-catalytic function or a novel HAT activity of the Gcn5 protein. This work was supported by NIH grant GM 62282

Molecular cloning of an arabidopsis homologue of GCN2, a protein kinase involved in co-ordinated response to amino acid starvation.

DNA homologous to the yeast ( Saccharomyces cerevisiae) protein kinase gene, GCN2, was amplified from arabidopsis [ Arabidopsis thaliana (L.) Heynh.] RNA and given the name AtGCN2. The AtGCN2 peptide sequence included adjacent protein kinase and histidyl tRNA synthetase-like domains and showed 45% sequence identity with the GCN2 peptide sequence in the protein kinase domain. AtGCN2 transcripts were detectable in RNA from roots, leaves, stems, buds, flowers, siliques and seedlings. GCN2 is required for yeast cells to respond to amino acid starvation. Expression of AtGCN2 in yeast gcn2 mutants complemented the mutation, enabling growth in the presence of sulfometuron methyl, an inhibitor of branched-chain amino acid biosynthesis, and 3-aminotriazole, an inhibitor of histidine biosynthesis.

Transcription factor GCN4 for control of amino acid biosynthesis also regulates the expression of the gene for lipoamide dehydrogenase

The yeast LPD1 gene encoding lipoamide dehydrogenase is subject to the general control of amino acid biosynthesis mediated by the GCN4 transcription factor. This is striking in that it demonstrates that GCN4-mediated regulation extends much farther upstream than simply to the direct pathways for amino acid and purine biosynthesis. In yeast, lipoamide dehydrogenase functions in at least three multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (which function in the entry of pyruvate into, and metabolism via, the citric acid cycle) and glycine decarboxylase. When wild-type cells were shifted from growth on amino acid-rich to amino acid-deficient medium, the expression of lipoamide dehydrogenase was induced approx. 2-fold. In a similar experiment no such induction was observed in isogenic gcn4 mutant cells. Northern analysis indicated that amino acid starvation affected levels of the LPD1 transcript. In the upstream region of LPD1 are three matches to the consensus for control mediated by GCN4. Directed mutagenesis of each site, and of all combinations of sites, suggests that only one site might be important for the general control response under the conditions tested. Gel-retardation analysis with GCN4 protein synthesized in vitro has indicated that GCN4 can bind in vitro to at least two of the consensus motifs.

UV-responsive Genes of Arabidopsis Revealed by Similarity to the Gcn4-mediated UV Response in Yeast

A UV response that involves the Ras proteins and AP-1 transcription factors has recently been described in mammals and yeast. To test whether an equivalent response exists in plants, we monitored the expression of Arabidopsis histidinol dehydrogenase gene (HDH), a homologue of the yeast HIS4 gene, which is strongly induced by UV light and is a target of the transcriptional activator Gcn4. We show that HDH mRNA levels increase specifically in response to UV-B light. Only small increases were detected upon exposure to other wavelengths. To isolate plant genes involved in this UV response, a gcn4 mutant was transfected with an Arabidopsis thaliana cDNA library. A new type of nucleotide diphosphate kinase (NDPK Ia) with a significant homology to the human tumor suppressor protein Nm23 rescued the gcn4 phenotype. NDPK Ia specifically binds to the HIS4 promoter in vitro and induces HIS4 transcription in yeast. In Arabidopsis, the NDPK Ia protein is located in the nucleus and cytosol. Expression studies in seedlings revealed that the level of NDPK Ia mRNA, like that of HDH, increases in response to UV-B light. It appears that NDPK Ia and HDH are components of a novel UV-responsive pathway in A. thaliana.

Nucleosome structure of the yeast CHA1 promoter: analysis of activation-dependent chromatin remodeling of an RNA-polymerase-II-transcribed gene in TBP and RNA pol II mutants defective in vivo in response to acidic activators.

The Saccharomyces cerevisiae CHA1 gene encodes the catabolic L-serine (L-threonine) dehydratase. We have previously shown that the transcriptional activator protein Cha4p mediates serine/threonine induction of CHA1 expression. We used accessibility to micrococcal nuclease and DNase I to determine the in vivo chromatin structure of the CHA1 chromosomal locus, both in the non-induced state and upon induction. Upon activation, a precisely positioned nucleosome (nuc-1) occluding the TATA box and the transcription start site is removed. A strain devoid of Cha4p showed no chromatin alteration under inducing conditions. Five yeast TBP mutants defective in different steps in activated transcription abolished CHA1 expression, but failed to affect induction-dependent chromatin rearrangement of the promoter region. Progressive truncations of the RNA polymerase II C-terminal domain caused a progressive reduction in CHA1 transcription, but no difference in chromatin remodeling. Analysis of swi1, swi3, snf5 and snf6, as well as gcn5, ada2 and ada3 mutants, suggested that neither the SWI/SNF complex nor the ADA/GCN5 complex is involved in efficient activation and/or remodeling of the CHA1 promoter. Interestingly, in a sir4 deletion strain, repression of CHA1 is partly lost and activator-independent remodeling of nuc-1 is observed. We propose a model for CHA1 activation based on promoter remodeling through interactions of Cha4p with chromatin components other than basal factors and associated proteins.

Gcn5p is involved in the acetylation of histone H3 in nucleosomes

Enzymatic extracts from a gcn5 mutant and wild-type strains of Saccharomyces cerevisiae were chromatographically fractionated and the histone acetyltransferase activities compared. When free histones were used as substrate, extracts from wild-type cells showed two peaks of activity on histone H3 but extracts from gcn5 mutant cells showed only one. With nucleosomes as substrate, the histone acetyltransferase activities present in extracts from the gcn5 mutant strain were not able to modify H3 whereas wild-type cell extracts acetylated intensely this histone. The activity that acetylated nucleosome-bound H3 behaved as a 170-kDa complex. We suggest that Gcn5p represents a catalytic subunit within a multiprotein complex containing proteins that confer on it the ability to acetylate H3 in nucleosomes. © 1997 Federation of European Biochemical Societies.

The Aspergillus niger GCN4 homologue, cpcA, is transcriptionally regulated and encodes an unusual leucine zipper.

The general control transcriptional regulator gene cpcA of Aspergillus niger was cloned by complementation of a Saccharomyces cerevisiae delta gcn4 mutant strain. The encoded protein conferred resistance to amino acid analogues when expressed in yeast. Disruption of cpcA in A. niger resulted in a strain which is sensitive towards 3-aminotriazole and fails to respond to amino acid starvation, cpcA encodes a transcript of approximately 2400 nucleotides in length that includes a 5' leader region of 900 nucleotides. The 5' leader region contains two small open reading frames, suggesting translational control of gene expression. Steady-state mRNA levels of cpcA increase by a factor of three upon amino acid starvation. The coding region of cpcA is interrupted by a 57 bp intron and the deduced amino acid sequence displays an approximately 30% overall identity to yeast GCN4p and Neurospora crassa cpc1p. Critical amino acid residues of the transcriptional activation domains of GCN4p are conserved in cpcAp. The basic DNA-binding domain shows up to 70% amino acid sequence identity to other basic zipper (bZIP)-type transcriptional activators. cpcAp binds specifically to a GCN4p recognition element in gel retardation experiments. The C-terminal dimerization domain encodes a leucine zipper with only a single leucine residue.