Biosci. Biotechnol. Biochem., 69 (4), 663–672, 2005 Review An Overview of the CCAAT-Box Binding Factor in Filamentous Fungi: Assembly, Nuclear Translocation, and Transcriptional Enhancement

Masashi KATO

Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Filamentous fungi are frequently used for the pro- of amylolytic have been extensively studied on duction of industrial , since they produce a the A. oryzae taaG2 . Using a hybrid model system variety of enzymes including polysaccharide-degrading of A. nidulans transformed by the A. oryzae taaG2 gene, enzymes. Among the many filamentous fungi, Aspergil- we found that three factors, CCAAT-box binding lus species, such as A. oryzae and A. niger, are known as protein, CreA, and AmyR, are involved in taaG2 gene strong producers of amylolytic enzymes. We have been expression and regulation (Fig. 1).1–9) studying on the regulatory mechanisms underlying the The CCAAT-box binding protein binds to a CCAAT expression of A. oryzae amylolytic genes. Based on sequence at approximately 300 in the taaG2 analyses using a hybrid model system of A. nidulans and is responsible for high-level expression of the taaG2 transformed by a gene encoding A. oryzae Taka-amyla- gene.2–4) CreA is a negative regulatory factor with a se A, the major amylase (taaG2), we have found that C2H2-type zinc finger motif that mediates carbon three factors, CCAAT-box binding protein, CreA, and catabolite repression. Analysis with recombinant CreA AmyR, are involved in taaG2 and protein indicated that four CreA binding sites in the regulation. In this review, the focus is on the CCAAT- taaG2 promoter region are involved in the repression of box binding protein of filamentous fungi. The assembly, taaG2 expression by glucose.1) AmyR is a specific nuclear translocation, and transcriptional enhancement transcriptional activator with a Zn(II)2 Cys6 binuclear mechanisms of the CCAAT-box binding protein are cluster DNA-binding motif. AmyR binds at approxi- discussed. mately 200 in the promoter region of taaG2. The binding site, designated SRE, is required for inducible Key words: Aspergillus; amylase; CCAAT-box; Hap expression.7–13) In this review, I have focused on a wide complex; NF-Y domain regulatory factor, which is a CCAAT-box binding protein in aspergilli.

Filamentous fungi are frequently used for the pro- duction of industrial enzymes because they produce a variety of enzymes including polysaccharide-degrading enzymes. They are also effective hosts for the produc- tion of foreign proteins due to their high secretion capability. Many filamentous fungi produce amylolytic enzymes. Among these, Aspergillus species, such as A. oryzae and A. niger, are known to be good producers. Hence, Aspergillus species are widely used in the Japanese fermentation industry, such as in sake brewing. Starch is a major storage polysaccharide in green plants and is composed of -1,4-glucan chains with -1,6 branches. Aspergillus species produce -amylase, glucoamylases, Fig. 1. Schematic Representation of the Taka-Amylase A Promoter and -glucosidases. These enzymes synergistically Region. degrade starch to produce glucose. -Amylases hydro- Taka-amylase A is a major amylase of A. oryzae. The CCAAT- lyze the -1,4 linkages of amylose, and glucoamylases box binding protein and AmyR bind to the CCAAT-box containing and -glucosidases exolytically hydrolyze the -1,4 region (313 to 300) and SRE, the starch responsive element (200 to 178), respectively. The general transcription factors and linkages from the nonreducing ends and release - RNA polymerase II are also shown. CreA, the carbon catabolite glucose and -glucose respectively. repressor, and its binding sites are omitted to avoid complexity. The Regulatory mechanisms that underlie the expression positions of the cis elements in the promoter are arbitrary.

To whom correspondence should be addressed. Fax: +81-52-789-4086; E-mail: [email protected] 664 M. KATO I. The CCAAT Sequence and CCAAT Bind- Hap2p, Hap3p, and Hap5p, are necessary for DNA ing Proteins binding, while Hap4p is involved in transcriptional activation.29,32,33) On the other hand, the mammalian The CCAAT sequence is one of the most common CCAAT-box binding factor, NF-Y, is composed of three cis-elements present in the promoter regions of numer- subunits, NF-YA (or CBF-B, corresponding to Hap2p), ous eukaryotic genes. A statistical analysis of over 500 NF-YB (or CBF-A, corresponding to Hap3p), and NF- promoters revealed that the CCAAT sequence is an YC (or CBF-C, corresponding to Hap5p). No Hap4p element present in approximately 30% of eukaryotic homolog has been identified in higher eukaryotes, promoters.14,15) The CCAAT sequence is present in the indicating that three subunits might be sufficient for forward or reverse orientation between 60 and 100 DNA binding and transcriptional activation. Indeed, the of the major transcription start sites. Several DNA N-terminal region of NF-YA and the C-terminal region binding proteins named for their CCAAT sequence of NF-YC were found to be responsible for transcrip- binding have been isolated and characterized: CTF/NF1 tional activation based on assessment by both in vitro (CCAAT /Nuclear Factor 1);16–19) transcription systems with recombinant proteins and C/EBP (CCAAT/ Binding Protein);20–25) and transfections of mammalian cells with the LexA and NF-Y/Hap (Nuclear Factor Y/Hem activation pro- GAL4 fusion genes. These activation domains are rich in tein).26–29) Factors in the CTF/NF1 and C/EBP groups glutamine and hydrophobic residues, and are not genuine CCAAT-box binding proteins because show amino acid sequence similarities to each other and they recognize the palindromic sequences in a manner to the glutamine-rich activation domain of transcription that is different from the widespread consensus derived factor Sp1.34–36) by statistical analysis. On the other hand, promoter compilation and site-selection analysis of the transcrip- II. CCAAT Binding Proteins of Filamentous tion factor of the NF-Y/Hap family revealed the binding Fungi sequence –C G/A G/A C C A A T C/G A/G C A/C–, in agreement with the consensus sequence deduced from The CCAAT sequence in filamentous fungi has been the statistical studies described above.30,31) The factor shown to modulate the expression of several genes, such belonging to the NF-Y/Hap family has been discovered as the A. nidulans acetamidase gene (amdS), the in a variety of organisms. These factors have been A. oryzae Taka-amylase A gene (taa), the A. nidulans designated Hap complex (, penicillin biosynthesis genes (ipnA and aatA), and the Shizosaccharomyces pombe, Kluveromyces lactis, Ara- Neurospora crassa NADP-specific glutamate dehydro- bidopsis thaliana, and Aspergillus species), CBF, or NF- genase gene (am).13,37) Y(Xenopus and mammals). These factors are summa- In filamentous fungi, the CCAAT-binding proteins, rized in Table 1. The Hap complex in S. cerevisiae was which are homologs of the yeast Hap complex, have also the first factor in this family to be identified as a heme- been characterized:13,37) AnCF (A. nidulans CCAAT- dependent transcriptional activator. This complex re- binding factor) for amdS by Hynes and his colleagues, sponds to two stimuli, heme and nonfermentable energy AnCP (A. nidulans CCAAT-binding protein) for taa by sources, and it comprises four subunits, Hap2p, Hap3p, our group, PENR1 for ipnA and aatA by Brakhage’s Hap4p, and Hap5p. Among the four subunits, three, group, and AAB for am by Kinsey’s group. The hapC

Table 1. CCAAT-Box Binding Proteins in the NF-Y/Hap Family

Name Subunits Examples of regulated genes References Filamentous fungi Aspergillus species Hap complex HapB/C/E taaG2, amdS, aatA, ipnA, 13, 37 (AnCP, AoCP, gatA, gdhA, fmdS, eglA, AnCF, PenR1) agdA Neurospora crassa AAB1 Hap2/3/5 am 41, 42 Trichoderma reesei Hap complex Hap2/3/5 cbh2, xyn1, xyn2 43, 44

Yeast Saccharomyces cerevisiae Hap complex Hap2/3/4/5 CYC1, CIT1, COX6, HEM1 79 CYT1, LPD1, ASN1, SOD2 Kluveromyces lactis Hap complex Hap2/3/4/5 KlDLD 80 Shizosaccharomyces pombe Hap complex Php2/3/5 CYC1 81

Plant Arabidopsis thaliana Hap complex Hap2/3/5 development genes 82, 86

Amphibian Xenopus laevis NF-Y NF-YA/B/C HSP70 83

Mammal mouse NF-Y NF-YA/B/C MHC II genes, Globin , 79, 84, 85 (CBF) (CBF-A/B/C) Collagen 1(I), Osteopontin, Albmin, PDGF , GPAT, CyclA, E2F1, RRR2, CCAAT-Box Binding Protein in Filamentous Fungi 665 gene, which is a homolog of the hap3 gene of induction of Taka-amylase A, as described in the first S. cerevisiae, has been isolated from A. nidulans by section of this paper. A nuclear protein designated Hynes and his colleagues. Disruption of the hapC gene AnCP, which was renamed the Hap complex, has been resulted in loss of binding of AnCP, AnCF, and PENR1 shown to bind in vitro to the CCAAT sequence in the to the CCAAT sequence, indicating that HapC is a taa promoter.4) This has also been confirmed by DNase I component of AnCP, AnCF, and PENR1.3,38,39) We footprinting analysis and electrophoretic mobility shift further confirmed this result by a supershift assay with assay (EMSA) using the taa promoter DNA as a probe.2) anti-HapC antibody.3) Furthermore, a DNA-complex has By means of EMSA with synthetic oligonucleotide successfully been reconstituted using recombinant HapC probes corresponding to the proximal regions containing together with the recombinant S. cerevisiae Hap2p and the CCAAT sequence of the taa gene, the minimum Hap5p subunits.3) The hapB and hapE genes, which are sequence comprising a 14 base-paired region including homologs of the hap2 and hap5 genes of S. cerevisiae, the CCAAT pentanucleotides, has been found to be have also been cloned from A. nidulans, and a DNA- necessary for formation of a stable complex with the binding complex has been reconstituted with recombi- Hap complex. In other words, the pentanucleotide nant HapC, HapE, and HapB by Hynes’ and Brakhage’s CCAAT sequence alone is not sufficient for binding of groups.40) All these data indicate that AnCP, AnCF, and the Hap complex to the taa promoter. PENR1 are identical, and are the A. nidulans counterpart The in vivo regulatory function of the Hap complex of the S. cerevisiae Hap complex. Hence, we designated has been confirmed by replacing the CCAAT sequence this factor the A. nidulans Hap complex. with CGTAA. This mutation abolishes the binding of the Hap complex to the sequence in vitro and reduces the taa III. Hap Complexes from Other Filamen- expression level to approximately 20% of that observed tous Fungi with the wild-type construct. Although the amylase activity of the strain transformed with the mutated taa We also cloned the A. oryzae counterparts for three gene is low, amylase activity is significantly induced by subunit genes, hapB, hapC, and hapE. These subunits starch and repressed by glucose, indicating that the exhibit high similarity (82% to 95%) in comparison with function of the Hap complex is not essential for starch those of A. nidulans.5,6) Furthermore, all A. oryzae genes induction of taa gene expression.2) This is different from can complement the corresponding deletion mutants of the function of the Hap complex described for the cbh2 A. nidulans, indicating that these are functionally inter- gene induction in T. reesei, since the cbh2-activating changeable with their A. nidulans counterparts. element, CAE, containing the CCAAT sequence is A CCAAT-binding complex involved in regulation of essential for inducible expression of the gene.43) the glutamate dehydrogenase gene (am) has been To clarify further the possibility that CCAAT-binding purified from N. crassa by Chen and Kinsey.41) Based factors different from the Hap complex might be on partial amino acid sequences, aab1, a homolog of the involved in enhancement of taa expression, we ex- HAP5 gene, has been cloned and characterized. Dis- pressed the authentic taa gene and the mutant taa gene ruption of the aab1 gene reduced the expression level of where the CCAAT sequence is replaced with CGTAA in the am gene to approximately 50% of the wild-type a hapC disruptant. The two types of transformants level, suggesting that AAB1 is a component of the produce only a little amylase activity, at almost the same CCAAT-binding complex.42) level, indicating that no factors are able to substitute for By antibody supershift experiments with anti-A. ni- the Hap complex in transcriptional enhancement of the dulans HapC antiserum prepared by us3) as well as taa gene.46) cross-competition experiments with CCAAT-containing The CCAAT sequence is also found in the promoter oligonucleotides derived from the A. nidulans amdS regions of other amylolytic genes, such as agdAs promoter, Kubicek and his colleagues have found that a encoding -glucosidases of A. nidulans, A. oryzae, and Hap-like complex binds to a CCAAT sequence in the A. niger. The distance between the CCAAT sequence promoter of the cellobiohydrolase gene (cbh2)in and SRE, the starch responsive element or one of the Trichoderma reesei.43) The T. reesei hap2, hap3, and AmyR binding sequences (CGGAAATT), is 14 bp and hap5 homologs have been cloned and their functionality is well conserved among three agdA genes, suggesting has been confirmed by complementing with the corre- that the Hap complex can also modulate expression of sponding A. nidulans hap mutants.44) We also detected a the agdA gene.9) CCAAT-binding activity in the cell extracts prepared With regard to the cellulolytic genes, the eglA gene from T. viride using the method we have developed.45) encoding the A. nidulans endo- -1,4-glucanase A is hapC-dependently expressed.5) To confirm this, we have IV. Regulation of the Amylolytic and Cellu- further examined the question both at the transcription lolytic Genes by the Hap Complex level by Northern analysis and at the enzyme level by activity staining. The A. nidulans eglA gene contains We have used A. nidulans as an intermediate host to two CCAAT sequences in the promoter region,47) but it analyze the molecular mechanisms underlying the starch remains to be determined in vivo whether one or both of 666 M. KATO the sequences are functional in enhancement of eglA acids respectively. Protein sequence alignment analysis gene expression. with the program CLASTALW indicated the presence of a histone-fold motif, which is required for heterodimer V. Regulation of the Other Genes by the formation of histone subunits, in HapC and HapE. Hap Complex Analysis of the sequence also indicated that HapC and HapE belong to the histone H2B and H2A families Hynes and his colleagues have shown that an respectively. As described above, the N-terminal region A. nidulans Hap complex designated AnCF regulates of NF-YA and the C-terminal region of NF-YC have the acetamidase gene (amdS), which is required for been found to be required for transcriptional activa- utilization of acetamide as a nitrogen and carbon tion.34–36) Although none of the Hap subunits contained source.48,49) AnCF is also involved in regulation of the obvious glutamine-rich domains in filamentous fungi, the -amino-butyrate transferase gene (gatA).39,49) When an function of transcriptional enhancement might be in- amdS–lacZ fusion gene was expressed in the hapC corporated in the non-conserved regions of the HapB/C/ strain, its expression level fell significantly. Further- E subunits, as in the case of NF-Y. In order to examine more, deletion of the CCAAT sequence led to a great this possibility, we constructed a series of truncated decrease in amdS–lacZ expression.48) The hapB or genes that encode the A. oryzae Hap subunits and hapE strain also expressed the amdS–lacZ gene at surveyed the regions in each subunit that are involved almost the same level as the hapC strain.40) All these in the formation of the CCAAT-binding complex, DNA- data confirmed the regulatory role of AnCF in amdS binding, and transcriptional enhancement of the fungal expression. genes. In vitro results indicated that only the conserved A. nidulans and P. chrysogenum are known to pro- core regions of the three Hap subunits were sufficient to duce penicillin. The penicillin biosynthesis enzymes form the DNA-binding complex (Tanoue et al., unpub- comprise -(L-a-aminoadipyl)-L-cysteinyl-D-valine syn- lished results). Furthermore, in vivo results indicated that thetase, , and acyl coenzyme A: the non-conserved N-terminal and C-terminal regions of isopenicillin N acyltransferase (IAT), encoded by the HapC and HapB respectively were required for in vivo genes acvA (pcbAB), ipnA (pcbC), and aat (penDE) function of the Hap complex-mediated transcriptional respectively. The genes acvA and ipnA are bidirection- enhancement, while the other non-conserved regions ally oriented and separated by an intergenic region of were dispensable. Further analyses revealed that the N- 872 bp.50) Brakhage and his colleagues found that a terminal region of HapC and the C-terminal region of CCAAT-binding factor, PENR1 (penicillin regulator 1), HapB were involved in the stabilization of HapC binds to a CCAAT-containing DNA motif (box I) in the (Tanoue et al., unpublished results) and nuclear local- intergenic region. Deletion of the CCAAT sequence led ization of HapB, respectively.53) to an eightfold increase in acvA expression and Since it was suggested that filamentous fungi might simultaneously to a reduction in ipnA expression to possess Hap4p homologs, we also examined a domain about 30%.51) In addition to box I, another CCAAT- comprising 26 amino acids that is conserved among containing DNA motif (box II) was identified by EMSA. HapE and its homologs in filamentous fungi and yeasts. Substitution for the CCAAT core sequence by GATCC This domain is almost identical to the so-called Hap4p- led to a fourfold reduction in expression of the aatA– recruiting-domain of Hap5.54) But deletion of the lacZ fusion gene, indicating that box II is functional and corresponding domain in A. oryzae HapE had no effect positively influences aatA expression.38,52) Thus peni- on transcription enhancement, suggesting that no homo- cillin biosynthesis is under the control of the CCAAT- log of Hap4p exists in Aspergillus species. This is also binding complex. supported by the fact that no Hap4p homologs have been In addition to the taa, amdS, gatA, aatA, ipnA, am, found except in some yeast strains.55,56) Recently, the and cbh2 genes described so far, the gdhA (glutamate genome sequences of A. nidulans (http://www.broad. dehydrogenase) and fmdS (formamidase) genes in mit.edu/annotation/fungi/aspergillus/) and A. oryzae A. nidulans as well as the xyn1 (xylanase) and xyn2 (Consortium for Genome Analysis of Aspergillus ory- (xylanase) genes in T. reesei have been shown to be zae, unpublished data) have been determined, but no controlled by Hap complexes.37) orthologs of the hap4 gene have been found by BLAST analysis of these data. VI. Domain Analysis of the Subunits of the These results do not exclude the possibility that a Hap Complex different Hap4p-like factor might exist in filamentous fungi. Previously, we successfully cloned a gene Three A. nidulans Hap subunits are sufficient for encoding a novel transcriptional activator, HapX, which sequence-specific binding,40) and this has also been interacted with the Hap complex.57) Nevertheless, hapX observed in the case of the A. oryzae CCAAT-binding deletion did not affect expression of either the taa or the complex.6) Each subunit of the Hap complex contains an eglA gene. It is possible that there are other factors that evolutionarily conserved domain. Conserved domains in interact with the Hap complex to enhance transcription HapB, HapC, and HapE comprise 53, 84, and 74 amino of subsets of Hap complex-dependent genes. CCAAT-Box Binding Protein in Filamentous Fungi 667 VII. Assembly and Nuclear Localization of Brakhage and his colleagues have investigated the the Aspergillus Hap Complex nuclear import of the CCAAT-binding complex subunits of A. nidulans in collaboration with our group.53) Using Formation of the trimeric CCAAT-binding complex various green fluorescent protein (GFP) constructs, a has been extensively studied using NFY/CBF and the nuclear localization signal sequence (NLS) of the HapB S. cerevisiae Hap complex. Two subunits, NF-YB/ protein was identified, outside the evolutionarily con- CBF-A/Hap3/HapC and NF-YC/CBF-C/Hap5/HapE, served domain. HapB-EGFP was transported into the contain the histone-fold motif in their conserved nucleus in both the hapC and the hapE strain, domains.58) The NF-YB/CBF-A/Hap3/HapC and NF- indicating that the NLS interacts with the import YC/CBF-C/Hap5/HapE subunits form a heterodimer machinery independent of the other Hap subunits. In through the histone-fold motifs. Subsequently, the dimer contrast, HapC-EGFP did not enter the nucleus in the interacts with the third subunit, NF-YA/CBF-B/Hap2/ absence of HapE or HapB. A similar observation was HapB, to form a heterotrimeric complex (Fig. 2). This made for HapE-EGFP, which did not localize to the heterotrimer can bind to the CCAAT sequences. The nucleus in the absence of HapC or HapB. Furthermore, assembly mechanism of the CCAAT-binding complex the addition of HapB-NLS to either HapC or HapE led appears to be the same. This assumption has been to nuclear localization of the respective protein fusions, supported by complementation of the hap2 deletion indicating that both HapC and HapE lack a functional mutant of S. cerevisiae with the NF-YA cDNA of NLS. These data strongly suggest that formation of a humans,59) and by an in vitro hybrid complex formation heterodimer of HapC and HapE is first required, and that that involves HapC of A. nidulans and Hap2 and Hap5 they can be transported only as a heterodimer via the of S. cerevisiae.3) The steps involved in the assembly of HapB protein into the nucleus. Therefore, the HapB the CCAAT-binding complex have been studied using subunit is the primary cargo of the import machinery, the CBF-A/B/C complex. First a CBF-A/C dimer that while HapC and HapE are transported to the nucleus is a homolog of HapC/E is formed, which allows CBF- only as a heterodimer and in complex with HapB via a B, a HapB homolog, to join to the complex. CBF-B piggyback mechanism. In order to extend these findings cannot interact with either CBF-A or CBF-C alone.60,61) to other filamentous fungi, we carried out a GFP study A study of the crystal structure of the human NF-YB/C with A. oryzae Hap subunits in collaboration with dimer found close interactions of the two proteins via Brakhage’s group. We generated GFP fusion constructs, histone fold motifs, which are also found in HapC and which were fused at the N-terminus of A. oryzae HapB, HapE, as described above.62) Recently, using in vitro HapC, and HapE, and were expressed in the hapB, translated subunits, we found a similar mode of hapC, and hapE strains, respectively (Goda et al. assembly of the HapB/C/E complex of Aspergillus unpublished results). GFP-derived fluorescence obser- species (Nakamura et al., unpublished results). vation confirmed the results, which were identical to those obtained for the A. nidulans subunits and strongly supported the ‘‘piggy back model,’’ i.e., the HapB subunit is the primary cargo of the nuclear import machinery, while HapC and HapE are transported to the nucleus only when they form a complex with HapB.53) It was shown that an NLS is located at the non-conserved region of the C-terminus (322PASQKRK328)inA. nidu- lans HapB. Although a computer program suggested another putative NLS (PNLS, 279RRPR282) in the conserved domain, it was found to be non-functional.53) The complete sequence of the A. oryzae HapB subunit was highly similar to that of A. nidulans,6) but the corresponding sequence from A. oryzae (320PSAQK- RK326) slightly differed from that of A. nidulans. Hence we examined whether the sequence corresponding to the A. oryzae HapB subunit, as well as the other PNLS (280RRPR283), is functional as an NLS. Initially, a Fig. 2. Assembly and Nuclear Localization of the Aspergillus Hap truncated derivative of GFP-AoHapB, designated GFP- Complex. AoHapBC309 (aa 1–309), which lacked the C-termi- The HapC and HapE subunits form a heterodimer through the nal PNLS (320–326), was constructed and examined. histone-fold motifs. Subsequently, the dimer interacts with the third Although the ratio of GFP-derived fluorescence in the subunit, HapB, to form a heterotrimeric complex. The HapC and nucleus to that in the cytoplasm was found to be low, HapE subunits, however, carry no localization signals. Hence HapC and HapE are transported to the nucleus only as a heterodimer and in obvious fluorescence was still detected in the nuclei, complex with HapB via a ‘‘piggy back mechanism’’. The stars indicating that the other NLS(s) exist in the HapB indicate the nuclear localization signals. subunit of A. oryzae. Hence we introduced site-directed 668 M. KATO mutations in the two PNLSs (280RRPR283 to AAAA and 323QKRK326 to AAAA). The introduction of these site-directed mutations in each PNLS led to a slight fluorescence in the cytoplasm, indicating that both mutations decreased nuclear localization ability, but the GPF-AoHapB derivatives were detected primarily in the nuclei. Simultaneous introduction of the mutations in the PNLSs resulted in complete loss of the nuclear localization ability of the GFP-AoHapB derivative. The results obtained with the C-terminal truncation mutant and site-directed mutants in the two PNLSs indicated that both the PNLSs of A. oryzae HapB were functional Fig. 3. A Working Model to Account for the Quantity Control during nuclear translocation, in contrast to the conclu- Mechanisms of the Hap Subunits. sion obtained from A. nidulans HapB. The reason for the HapE becomes insoluble in the absence of HapC. Subsequently, difference between these closely related species is HapE is degraded in vivo. This unstable property of HapE can lend unclear at present, but these results may suggest the itself to quantity control of Hap subunits. With regard to the third subunit HapB, the expression of the gene is negatively regulated by diversity of NLSs among the HapB/Hap2/NF-YAs of the Hap complex at the transcription level.64) various eukaryotes.

VIII. Quantity Control Mechanisms That that the transcript level of the hapB gene in the deletion Regulate Levels of the Hap Subunits strains of the Hap subunit-encoding gene was signifi- cantly higher than that in the wild type. The hapB gene The results of our electrophoretic mobility shift assays has been shown to be negatively regulated by the Hap (EMSAs) indicate that deletion of any of the hapB, complex.64) hapC,orhapE genes results in loss of CCAAT-binding The nuclear import of heterotrimeric complexes can activity.63) CCAAT-binding activity was restored by the also guarantee equimolar concentrations of the different addition of the recombinant HapB and HapE subunits to components in the nucleus. It has been found that only the extracts prepared from the hapB and hapE the HapB subunit contains NLSs. Neither the HapC strains respectively. On the other hand, the recombinant nor the HapE subunits carries an NLS, and both are HapC subunit did not reconstitute CCAAT-binding transported into the nucleus via a piggy-back mechanism activity with the hapC extract.63) A plausible explan- in complex with HapB. ation for these results is that the hapC strain lacks other Hap subunits as well as the HapC subunit. In order IX. Open Questions on the Hap Complex of to address this possibility, we carried out a reconstitution Filamentous Fungi study with the recombinant subunits and extracts prepared from the respective hap subunit deletion Increasing evidence suggests that the Hap complex mutants. Western blot analysis of the Hap subunits and enhances transcription of numerous fungal genes, but Northern blot analysis of the hap genes with the some important questions remain to be answered. First, respective deletion mutants were also performed. The how does the Hap complex enhance promoter activity? results suggest that the number of HapC molecules Transcription factor complexes of NF-Y/CBF have been adjusts to that of HapE molecules by forming stable implicated in chromatin remodeling because it has been heterodimers prior to the assembly of the Hap complex observed that formation of nucleosome-free regions (Fig. 3).63) Recently, we performed a reconstitution depends on the binding of CCAAT-binding complexes study with the recombinant subunits and S35-labeled in to several promoter regions. It has also been reported vitro translated subunits (Nakamura et al., unpublished that the Hap complex of A. nidulans affects chromatin results). Significant amounts of translated HapE were structure in vivo.65) Furthermore, it has been reported recovered in the insoluble fraction, while the other two that human NF-Y complex can interact with histone subunits were recovered in the soluble fraction. How- acetyltransferases P/CAF and p300, suggesting that NF- ever, HapE was recovered in the soluble fraction when Y plays a role in modifying the nucleosome positioning, recombinant HapC was added in the translation reaction. which enables the other transcription factors to recruit to These results indicate that the HapC subunit plays a role the corresponding promoter.66–69) Hence characteriza- in increasing the solubility of the HapE subunit. tion of the relationship between the Hap complex and Although many CCAAT complexes from various eu- histone remodeling activities, such as histone acetyl- karyotes have been characterized, there is little infor- transferase and histone deacetylase, are urgently re- mation regarding the stability of their subunits. These quired. Secondly, apart from histone modifying en- findings suggest that similar mechanisms controlling the zymes, what factors interact with the Hap complex? NF- quantity of the subunits might exist in other eukaryotes. Y was reported to interact with many factors such as With regard to the third subunit, HapB, it is also known TBP, TAFII100, TAFs with the histone fold motif, CCAAT-Box Binding Protein in Filamentous Fungi 669 coactivators (HSP-CBF for the heat shock proteins and amdS, and gatA promoters. Mol. Gen. Genet., 254, 119– RNF4 for GTP cyclohydrolase I), and some specific 126 (1997). transcriptional regulators.70–73) Histone H3/H4 dimer 3) Kato, M., Aoyama, A., Naruse, F., Tateyama, Y., has also been reported to interact with NF-Y.74) This is Hayashi, K., Miyazaki, M., Papagiannopoulos, P., Davis, reasonable, because NF-YA/HapC and NF-YC/HapE M. A., Hynes, M. J., Kobayashi, T., and Tsukagoshi, N., The Aspergillus nidulans CCAAT-binding factor AnCP/ show a weak similarity to histones H2B and H2A AnCF is a heteromeric protein analogous to the HAP respectively. In order to understand the function of the complex of Saccharomyces cerevisiae. Mol. Gen. Gen- Hap complex of filamentous fungi, we must survey all et., 257, 404–411 (1998). the factors that interact with the Hap complex. Third, 4) Nagata, O., Takashima, T., Tanaka, M., and Tsukagoshi, how is the function of the Hap complex regulated? N., Aspergillus nidulans nuclear proteins bind to a Recent studies have suggested that the NF-Y tran- CCAAT element and the adjacent upstream sequence in scription factor is involved in transcription repression of the promoter region of the starch-inducible Taka- the cell cycle-regulatory genes in response to amylase A gene. Mol. Gen. Genet., 237, 251–260 induction or DNA damage in mammals.75,76) The cdk2- (1993). dependent phosphorylation of NF-Y and its involvement 5) Tanaka, A., Kato, M., Hashimoto, H., Kamei, K., in transcription repression by the p53–p21 signaling Naruse, F., Papagiannopoulos, P., Davis, M. A., Hynes, M. J., Kobayashi, T., and Tsukagoshi, N., An Aspergillus pathway have been demonstrated.77) More recently, oryzae CCAAT-binding protein, AoCP, is involved in localization of NF-YC is regulated depending on the the high-level expression of the Taka-amylase A gene. 78) cell cycle. In filamentous fungi, no obvious change in Curr. Genet., 37, 380–387 (2000). the localization of the Hap subunits during the cell cycle 6) Tanaka, A., Kamei, K., Tanoue, S., Papagiannopoulos, has been observed, but it is probable that the function of P., Steidl, S., Brakhage, A. A., Davis, M. A., Hynes, M. the Hap complex is controlled by some stimuli. Down J., Kato, M., Kobayashi, T., and Tsukagoshi, N., regulation of many genes can be triggered. Finally, what AoHapB, AoHapC, and AoHapE, subunits of the types of genes does the Hap complex regulate? As Aspergillus oryzae CCAAT-binding complex, are func- discussed earlier, the yeast Hap complex mainly tionally interchangeable with the corresponding subunits regulates genes involved in respiration, while the in Aspergillus nidulans. Curr. Genet., 39, 175–182 Aspergillus Hap complex has been found to enhance (2001). 7) Tani, S., Kawaguchi, T., Kato, M., Kobayashi, T., and the expression of a broader spectrum of genes, such as Tsukagoshi, N., A novel nuclear factor, SREB, binds to a those coding for amylase, acetamidase, and penicillin cis-acting element, SRE, required for inducible expres- biosynthesis enzymes. It is still unclear, however, sion of the Aspergillus oryzae Taka-amylase A gene in whether the Hap-dependent genes share common fea- A. nidulans. Mol. Gen. Genet., 263, 232–238 (2000). tures in filamentous fungi. In order to address this 8) Tani, S., Katsuyama, Y., Hayashi, T., Suzuki, H., Kato, question, we are trying to identify all the Hap-dependent M., Gomi, K., Kobayashi, T., and Tsukagoshi, N., genes using DNA microarray technology. Characterization of the amyR gene encoding a transcrip- tional activator for the amylase genes in Aspergillus Acknowledgments nidulans. Curr. Genet., 39, 10–15 (2001). 9) Tani, S., Itoh, T., Kato, M., Kobayashi, T., and I thank Dr. N. Tsukagoshi and Dr. T. Kobayashi Tsukagoshi, N., In vivo and in vitro analyses of the AmyR binding site of the Aspergillus nidulans agdA for providing me the opportunity to carry out this work promoter; requirement of the CGG direct repeat for and for helpful discussion. I would also like to thank Dr. induction and high affinity binding of AmyR. Biosci. M. J. Hynes and Dr. A. A. Brakhage for fruitful discus- Biotechnol. Biochem., 65, 1568–1574 (2001). sion. 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