Biosci. Biotechnol. Biochem., 66 (10), 2224–2227, 2002

Note Involvement of a CCAAT-binding Complex in the Expression of a Nitrogen--Speciˆc Gene, isp6＋, in Schizosaccharomyces pombe

Akio NAKASHIMA,1 Masaru UENO,1 Takashi USHIMARU,2 and Masahiro URITANI1,†

1Department of Chemistry and 2Department of Biology and Geoscience, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka 422-8529, Japan

Received February 26, 2002; Accepted May 16, 2002

The ˆssion yeast gene isp6＋ is needed in nitrogen- expression of isp6+ under good nutrient conditions starvation response but its transcriptional regulation has rather than to induce it during nitrogen starvation. been unclear. isp6＋ was repressed under nutrient Ste11 is a transcription factor that induces a varie- conditions, in which cAMP-dependent kinase ty of genes during nitrogen starvation, and the A, the stress-activated protein kinase cascade, and the ste11D mutant is defective in sexual development CCAAT-binding complex were concerned. The triggered by nitrogen starvation.7) In ste11D,theex- CCAAT-binding complex also was involved in the in- pression of isp6+ was controlled normally (Fig. 1C). duction of isp6＋ during nitrogen starvation. The Ste11-binding sequence, the TR-box, was absent in the upstream region of isp6+. Therefore, ste11+ Key words: CCAAT-binding complex; cAMP-depen- was not involved in the expression of isp6+. dent ; ˆssion yeast; nitro- The upstream region of isp6+ contained one gen starvation cAMP response element (CRE) and two CCAAT ele- ments (Fig. 2A). CRE is the binding site of the tran- The ˆssion yeast gene isp6+ encodes a possible scription complex comprising Atf1WGad7 and Pcr1, vacuolar protease, which is involved in sexual which is regulated by both PKA and Wis1 path- diŠerentiation triggered by nitrogen starvation. The way.8–11) The amount of mRNA of isp6+ was high in gene is repressed under nutrient conditions and is in- the atf1D mutant even under good nutrient condi- duced by nitrogen starvation by unknown mechan- tions and also during nitrogen starvation (Fig. 2B). ism.1) We examined the expression of isp6+ in The results may be explained by the previous conclu- mutants defective in their response to nitrogen star- sion that Atf1 activates the transcription of its target vation (Fig. 1, A and B). Pka1 is a catalytic subunit genes when it is phosphorylated by the Wis1 pathway of the cAMP-dependent protein kinase A (PKA), but it represses the genes when unphosphorylated.12) and the pka1D mutant expresses nitrogen-starvation- For example, basal expression of ctt1+ is derepressed speciˆc genes even with su‹cient nutrients availa- in atf1D but it is repressed in the mutant of the Wis1 ble.2) In pka1D, both mRNA and protein levels of pathway. Interestingly, the levels of the protein en- isp6+ were elevated in nutrient medium and they in- coded by isp6+ werethesameintheatf1D mutant creased further with nitrogen starvation. Cgs1 is a and the wild type (Fig. 2C). The results were regulatory subunit of PKA and the cgs1D mutant is reproducible but the reason for the discrepancy in defective in its nitrogen-starvation response.3) In transcription and translation was unclear. Expression cgs1D, isp6+ was induced during nitrogen starvation. of isp6+ may be regulated at the post-transcriptional Wis1 is a stress-activated protein kinase kinase and level also. the wis1D mutant is defective in response to nitrogen The CCAAT-binding complex recognizes the starvation.4,5) In wis1D, both repression and induc- CCAAT element. Php2 is a subunit of the complex, tion of isp6+ was normal in both transcription and and disruption of the php2+ gene completely abolish- translation. Similar data were obtained with the es CCAAT-binding ability.13) In the php2D mutant, wis1Dpka1D double mutant; the results were consis- isp6+ was slightly expressed even under good nutrient tent with previous ˆndings that the Wis1 pathway is conditions and its level did not increase further dur- needed for the expression of PKA-repressed genes ing nitrogen starvation (Fig. 2B). The amount was such as fbp1+.6) The results suggest that PKA and the much lower than those in the wild type. The results Wis1 pathway may function mainly to suppresses the were similar on the protein level (Fig. 2C), suggesting

† To whom correspondence should be addressed. Fax: +81-54-237-3384; E-mail: scmurit＠ipc.shizuoka.ac.jp Abbreviations: CBB, Coomassie Brilliant Blue R-250; CRE, cAMP response element; PKA, cAMP-dependent protein kinase A Transcriptional Regulation of isp6+ in Fission Yeast 2225

Fig. 1. Expression of isp6+ in Mutants with a Defective Nitrogen-Starvation Response. A, Northern hybridization of isp6+. Total RNA (25 mg) from cells in the mid-log phase (``log'') or after nitrogen starvation for 4 h (``-N'') was put in each lane and analyzed. Northern blotting with the entire coding region of isp6+ as a probe is shown in the upper panel. Ribosomal RNA stained with ethidium bromide is shown in the lower panel. Experimental procedures were as previously de- 17) scribed. The relative intensity of transcription is indicated under the top panel with the signal in WTWlog taken to be 1.0. WT denotes wild type. B, Immuno-blotting of Isp6. A protein extract (5 mg) was put in each lane and analyzed. Immuno-blotting with anti-Isp6 an- tibody is shown in the upper panel, and the gel stained with Coomassie Brilliant Blue R-250 (CBB) is shown in the lower panel. Ex- perimental procedures were as described previously.18) C, Northern hybridization of isp6+ in the ste11D mutant. Genotypes of the strains used: WT (JY476), h90 ade6-M210 leu1; wis1D (NS13), h90 his1-102 wis1::his1+; pka1D (NS11), h90 ura4-D18 pka1::ura4+; cgs1D (JZ858), h90 ade6-M216 leu1 ura4-D18 cgs1::ura4+; wis1Dpka1D (AN11), h„ his1-102 leu1 ura4-D18 wis1::his1+ pka1::ura4+; ste11D (JZ396), h90 ade6-M216 leu1 ura4-D18 ste11::ura4+. that Php2 is involved in both the induction of isp6+ may aŠect the expression of isp6+ in an indirect way. during nitrogen starvation and the repression of CCAAT-binding complex mutants have a growth isp6+ under good nutrient conditions. The fbp1+ defect when the carbon source is not fermentable be- gene is repressed by and transcriptionally cause of their defects in mitochondrial function. The activated by glucose starvation.14) Mutation of the isp6D mutant does not have such a phenotype but is CCAAT-binding complex reduces fbp1+ expression defective in the nitrogen-starvation response. Activi- under both repression and derepression conditions, ty of the mitochondrial nuclease Pnu1 is elevated which is consistent with the above suggestion. The during nitrogen starvation without increase in the Php2-dependent DNA-protein complex was detected amounts of protein, but the activation was not de- in the upstream region of isp6+ (Fig. 2D, lanes 2–7). tected in isp6D.16) php2D activated Pnu1 little if at all The expression of isp6+ could be regulated through during nitrogen starvation, in agreement with the the association of the CCAAT-complex with the up- ˆnding that the expression of isp6+ was low in the stream region. However, the CCAAT-binding com- mutant. plex regulates many genes.15) The CCAAT-complex 2226 A. NAKASHIMA et al.

Fig. 2. Involvement of a CCAAT-binding Complex in Induction of isp6+ during Nitrogen Starvation. A, Scheme of the upstream region of isp6+. Two CCAAT elements are indicated by open boxes and CRE is represented by a shaded box. Numbers indicate the positions of the extreme nucleotides of each element farthest from the transcription initiation site (+1). The line with two arrowheads denotes the extent of the probe („483 to „335) used in the gel retardation assay (see Fig. 2D). B, Northern hybridization in php2D and atf1D. Procedures were as described in the Fig. 1A legend. The relative intensity of transcription is shown under the top panel with the signal in WTWlog taken to be 1.0. WT denotes wild type. C, Immuno-blotting in php2D and atf1D.Proce- dures were as described in the Fig. 1B legend. D, Gel retardation assay of the upstream region of isp6+. Soluble fractions (``S-100'') were prepared from cells in the mid-log phase as previously described.18) The probe was obtained by polymerase chain reaction with a set of the following primers, 5?-TGAGTGGCCCTTACCAA-3? and 5?-CACGATATGTTCACTTGACG-3?, and was end-labeled with [g- 32 P]ATP (185 TBqWmmol) and T4 polynucleotide kinase. Each soluble fraction (10 mg of protein) was mixed with 0.2 mg of poly(dI:dC), 0.1 mg of poly(U), and 0.01 pmol of the labeled probe in a buŠer containing 20 mM Tris-HCl (pH 7.5), 4 mM KCl, 3 mM MgCl2,1mM EDTA, 10 mM 2-mercaptoethanol, 0.2z Nonidet P-40, 0.3z Triton X-100, and 5z . The Reaction was done in a total volume of 10 68ml for 30 min at room temperature, DNA-protein complexes were separated on an 8z polyacrylamide gel, and the gel was dried and autoradiographed. The position of the DNA-protein complexes is indicated on the left. Reactions with 50-fold and 100-fold excess of the unlabeled probe DNA were done. Lane 1 contains the control (the labeled probe without any soluble fraction). ``Free probe'' in- dicates the labeled probe without interaction with . Genotypes of the strains used: WT (JY333), h„ ade6-M216 leu1; php2D (AN12), h„ ade6-M216 leu1 ura4-D18 php2::ura4+;and atf1D (CN803), h„ ade6-M216 leu1 ura4-D18 atf1::ura4+. Transcriptional Regulation of isp6+ in Fission Yeast 2227 Acknowledgments encodes a phosphoprotein with a bZIP domain, which is required for proper G1 arrest and gene ex- We thank Dr. M. Yamamoto (University of pression under nitrogen starvation. Genes Cells, 1, Tokyo) for strains JY741, JY333, JZ858, and JZ396, 391–408 (1996). Dr N. Mutoh (Institute for Developmental Research) 10) Watanabe, Y., and Yamamoto, M., Schizosac- charomyces pombe pcr1+ encodes a CREB ATF for AN12 and CN803, and Dr. K. Okazaki (Kazusa W proteininvolvedinregulationofgeneexpressionfor DNA Institute) for NS11, NS13, and AN11. This sexual development. Mol. Cell. 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