37NISR Research GRANT
2009 Research Grant

Analysis on the Transcriptional Regulation Mechanisms Controlled by the CCAAT-box Binding Complex as a Key Factor
in Filamentous Fungi
Masashi KATO
Graduate School of Bioagricultural Sciences, Nagoya University(Present address: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University)

Research aims
The CCAAT box is one of the most common cis-elements present in the promoter regions of many eukaryotic
genes. A statistical analysis of over 500 promoters revealed
that the CCAAT sequence is one of the widespread elements that is present in approximately 30% of the eukaryotic promoters.1,2)
The CCAAT sequence in filamentous fungi has beenshown to modulate the expression of many genes.3–5)
CCAAT-binding proteins, which are homologs of the yeast
Hap complex, have also been characterized.3–5) The hapCgene, which is a homolog of the hap3 gene of Saccharomyces cerevisiae, has been isolated from Aspergillus
nidulans by M. Hynes and colleagues. We analyzed thisfactor in cooperation with Hyne’s group. Disruption of thehapC gene resulted in the loss of binding between theCCAAT-binding protein and the CCAAT sequence, indicating that HapC is a component of the CCAAT-binding protein complex.5–7) We further confirmed the result by a supershift assay using anti-HapC antibodies.6) Furthermore, aDNA-binding protein complex has been successfully reconstituted using recombinant HapC together with the recombinant S. cerevisiae Hap2p and Hap5p subunits.6) The hapBand hapE genes, which are orthologs of the hap2 and hap5
genes of S. cerevisiae, have also been cloned from A. nidulans, and a DNA-binding complex has been reconstitutedwith recombinant HapC, HapE, and HapB.6) All these data
indicate that the CCAAT-binding protein complex of A.
nidulans is a counterpart of the S. cerevisiae Hap complex.
Therefore, we designated this factor as the A. nidulans Hapcomplex.
A yeast-two-hybrid screen suggested physical interactionof A. nidulans HapB with a protein of an as yet unknown
function, termed HapX.8) HapX displays no similarity to S.
cerevisiae Hap4p, besides an N-terminal 17-amino acidmotif, which has been shown to be essential for interactionof Hap4p with the S. cerevisiae Hap2p/Hap3p/Hap5p. It has
recently been shown that various iron-dependent pathways
are repressed under iron-deficient conditions via the interaction of HapX with the HapB/C/E complex.9) HapX harbors a basic domain from the bZip motif except for a
coiled-coil domain instead of leucine-zipper; this suggeststhat HapX possesses a DNA-binding ability that is different
from that of S. cerevisiae Hap4p. In this study, therefore,
we analyzed the interaction of the HapX-HapB/C/E complex with the promoter of the cytochrome c gene (cycA) ofA. nidulans, which is one of the HapX-dependent genes.
Electrophoretic mobility shift assays (EMSAs) and DNA
footprint analyses using recombinant HapX-HapB/C/E andvarious DNA probes revealed that both the CCAAT-box andan adjacent sequence of the cycA promoter are required forthe recognition by the HapX-HapB/C/E complex. The
DNA-binding ability of HapX presumably allows independent regulation by HapB/C/E and HapX-HapB/C/E.
Methods
The strains used in this study included A. nidulans BPU(genotype: biA1, pyrG89, wA3, pyroA4) as a wild-typestrain and A. nidulans hapXD (genotype: biA1, pyrG89,wA3, hapXD, pyroA4), for the b -galactosidase assay. pPYcycAp-lacZ, a reporter plasmid used for the analysis of thecycA promoter, was constructed as follows: a promoter region of the cycA gene was amplified with chromosomal
DNA from the A. nidulans wild-type strain; the promoter
fragment was fused to the lacZ gene and inserted to the integration vector pPY-7.
For the DNA binding experiments, a truncated HapX(1–200) was prepared and used. The recombinant protein
was produced in Escherichia coli and purified with a NiNTA agarose column. The subunit proteins of theHapB/C/E complex were also prepared as recombinant proteins, and reconstituted to form the Hap core complex.
Electrophoretic gel mobility shift assay (EMSA) andDNase I footprinting experiments were carried out as described previously.6)
Results
HapX interacts with a target promoter in the presence of the HapB/C/E complex: EMSAs were carried out
with the HapB/C/E complex and HapX in order to examine
the binding specificity for HapX-dependent (cytochrome cgene: cycA) and independent (amylase gene: taaG2) promoters, respectively (Fig. 1). HapX can bind to the cycA

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Fig. 1.

EMSAs using the HapB/C/E complex and HapX. HapX interacts with a target promoter in the presence of the HapB/C/E complex.

Fig. 2. Footprint analysis of the cycA promoter with HapX and theHapB/C/E complex. (A) Coding strand (B) Noncoding strand.

promoter in the presence of the HapB/C/E complex, while
HapX cannot bind to the taaG2 promoter despite the presence of the HapB/C/E complex, suggesting that the recruitment of HapX to the HapB/C/E complex is promoter sequence-dependent.
Footprint analysis of the cycA promoter withHapB/C/E/X complex: Footprint analyses of the cycA promoter with HapX and the HapB/C/E complex were performed (Fig. 2). When HapX was added to the HapB/C/E
complex, the protected region was extended, suggestingthat HapX may have a DNA binding activity. This is consistent with the fact that HapX possesses a basic domain ofthe bZip-like motif. It is reported that a bZip transcription
factor, AP-1, recognizes TGAGTCA sequence. In ourstudy, we found a similar sequence, TGATTCA, in the protected region.
The ATCA sequence in the cycA promoter is necessary for the recognition of HapX: To examine whether
the TGAATCA (633 to 627) sequence is involved in
the HapX binding, EMSAs were performed with mutant
cycA promoters (Fig. 3). When mutations were introducedin the ATCA (630 to 627) sequence, the intensities ofthe upper shift band were significantly decreased. The mutation in the A (631) residue also slightly decreased thebinding affinity of HapX.
The HapX-binding sequence is conserved among thecycA promoters of Aspergillus species (data not shown).
The ATCA sequence and its distance from the CCAAT box
are strictly conserved among the cycA promoters of Aspergillus species, as far as we examined. We also found adownstream conserved sequence. Although its function remains unknown at present, we speculate that it could be acting as the binding site for a positive regulator of the cycAgene.
The ATCA sequence is required for the repression of
the cycA promoter under the iron-depleted conditions:
To examine the in vivo function of the ATCA sequence ofthe cycA promoter, we constructed some lacZ fusion genes
under control of the wild-type and mutant cycA promoters(Fig. 4). Mutations were introduced into the ATCA (630)
and CCAAT (611) sequences, respectively. The b -gal activities of the extracts from mycelia were measured. In thewild-type background (hapX ), the b -gal activity underiron-deficient conditions (Fe) was decreased by approximately 50% compared with that under the iron-replete conditions (Fe). On the other hand, lacZ-fusion genes withmutations within the ATCA or CCAAT sequence exhibited
derepression under the iron-deficient conditions. Furthermore, in the hapXD background, even the wild-type pro-

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Fig. 5. A model of the transcriptional regulation by HapX andHapB/C/E complex.

Conclusion

Fig. 3. The ATCA sequence in the cycA promoter is necessary for therecognition of HapX.

In this study, the following new findings were revealed.
(i) HapX interacts with the target promoter in the presenceof the HapB/C/E complex. (ii) The ATCA sequence in thecycA promoter is necessary for the recognition of HapX.
(iii) The HapX-binding sequence is conserved among thecycA promoters of Aspergillus species. (iv) The ATCA sequence is required for the repression of the cycA promoter
under the iron-deficient conditions. Recently, we haveshown that various iron-dependent pathways are repressed
under iron-deficient conditions by interaction of HapX with
the HapB/C/E complex. A tentative model (Fig. 5) for transcriptional regulation by HapX and the HapB/C/E complex
is depicted, on the basis of the results presented here andsome speculations. Under the iron-deficient conditions,
HapX recognizes the HapB/C/E complex and the ATCA sequence in the cycA promoter region. HapX is thought to interact with the DNA sequence through the bZip-like domain. Under the ion-replete conditions, binding of the iron
ion to the C-terminal domain (cys-rich domain) would alter
the conformation of HapX, resulting in its decreased interaction with the HapB/C/E complex. This permits derepression of HapX-dependent genes.
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Fig. 4. Analysis of the lacZ fusion genes under the control of the wildtype and mutant cycA promoters. The ATCA sequence is required for therepression of the cycA promoter under the iron-deficient conditions.

2)

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moter exhibited derepression under the iron-deficient conditions. Taken together, our data led us to conclude that
HapX represses iron-dependent genes by interacting with
the ATCA sequence and the Hap complex.

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