Neuroscience Letters, 146 (1992) 25-28

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© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 09027

In situ DNA-protein binding: a novel method for detecting DNA-binding activity of transcription factor in brain Xiao-Bing Wang, Yasuhiro Watanabe, Takesi Osugi, Mitsushi Ikemoto, M a s a y u k i Hirata and N a o m a s a Miki Department of Pharmacology, Osaka University Medical School, Osaka (Japan) (Received 3 June 1992; Revised version received 22 July 1992; Accepted 23 July 1992)

Key words: Transcription factor; In situ DNA-protein binding; Methamphetamine; Brain A novel method, in situ DNA-protein binding (in situ DPB), was developed to detect the distribution and DNA-binding activity of AP-1 and Spl binding proteins in situ. The regional distribution of AP-1 binding protein in mouse brain was different from that of Spl. Antibody against the DNA-binding domain of Jun protein markedly reduced the AP-1 but not the Spl binding activity. The binding activity of AP-1 probe increased markedly in the brain after administration of methamphetamine. These results suggest that the in situ DPB is convenient and sensitive for detecting the distribution and the DNA-binding activity of transcription factors in situ.

Various stimuli induce gene expression in cells, resulting in changes of phenotypes in adaption to a new environment. The transcriptional activities of genes depend on transcription factors that interact with specific ciselements in the promoter region. Several transcription factors that are activated in response to intracellular second messengers have been reported as intermediary transcriptional regulators in signal transduction processes. The phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, stimulates transcription of several genes [2, 11, 12, 15] through a specific cis-acting sequence, the TPA response element (TRE) [3, 4, 16]. This element is present in the 5'-upstream regions of several genes and is recognized by a transcription factor AP-1 [4, 16] which has been identified as a complex of Fos and Jun in various cells. Expression offos and jun in specific brain nuclei has been characterized by the in situ hybridization method and the immunohistochemical method [5, 10, 17, 19], but these methods cannot detect the DNA-binding activity of Jun and Fos proteins. The DNA-binding activity of Jun and Fos has been also studied in vitro by gel shift assay [1, 7, 9]. However, this assay is not suitable for detecting the precise distribution of DNA-binding protein in the heterogeneous tissue such as the brain. Correspondence: N. Miki, Department of Pharmacology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. Fax: (81) 06-875-7369.

In this work, we developed a novel method to detect the DNA-binding activity of DNA-binding protein in situ with a specific DNA probe in thin sections of mouse brain. Synthetic 22 mer DNAs, CTAGTGATGAGTCAGCCGGATC and GATCGATCGGGGCGGGGC GATC, which contain a consensus sequence of AP-I (TGAGTCA) and Spl (GGGGCGGGGC) site, respectively, were used as probes. After annealing with their complementary DNAs, the probes were labeled with [35S]deoxy-CTP at 3'-terminal [6]. Thin sections of mouse brain were prepared at -20°C, freeze-dried and fixed with 0.2% paraformaldehyde for 30 min. After washing twice with HEPES buffer containing 10 mM HEPES, 40 mM NaC1, 10 mM MgC12, 1 mM DTT, 1 mM EDTA and 0.25% BSA (pH 7.4), the sections were treated with DNase I (0.1 mg/ml) dissolved in HEPES buffer at 30°C for 30 min and washed once with the same buffer except that MgCI 2 was replaced by 10 mM EDTA. Then they were washed twice with HEPES buffer. The DNA-binding reaction was performed by incubation of the sections with the labeled DNA probe (1 ng/ml) dissolved in HEPES buffer containing polydeoxyinosinicdeoxycytidylic acid (poly (dI-dC), 0.5 /~g/ml, Sigma) and 100 ng poly(dC) for 1 h at 30°C. Then the sections were washed 5 times with HEPES buffer (pH 7.4) for 45 min. After the last washing, the sections were dried and exposed to an Amersham hyperfilm. As shown in Fig. 1, the binding of DNA probes was clearly detectable in dis-

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Fig. 1. Specificityof AP-I binding in brain sections. A: AP-I binding proteins detected by gel shift assay in the nuclear extract of the brain. Nuclear extracts were prepared from mouse brains and reacted with [35S]-dCTP labeled AP-I and Spl probe at 30°C for 30 min. DNAproteins complexwere separated on 4% polyacrylamidegel (5 fig protein in each lane). The probe and competitors used in this experiment are diagrammed above the panel. 'jun Ab' represents the antibody against the DNA-binding domain of c-Jun protein. AP-I cold represents the 100-foldexcessof unlabeled AP-1 probe. The arrow indicates a complexof DNA-Spl binding protein. B: bindings of AP-I (a,b) and Spl (c,d) probes in the presence (b,d) and absence (a,c) of 100-fold excess of unlabeled AP-1 probe (AP-1 cold). C: binding of AP-1 (a,b) and Spl (c,d) probes in sections preincubated with (b,d) and without (a,c) antibody (Anti-jun, 50#g/ml) against the DNA-bindingdomain of Jun protein for 16 h at 4°C.

tinct regions of the mouse brain. We then examined the nature of the DNA-binding detected in brain sections. Pretreatment of the brain sections with protease K (5 mg/ml, 30 min) completely abolished binding of the D N A probes in the sections, indicating that the labeling detected in the section represented the binding of proteins with D N A probe. Next we investigated the stability of the DNA-binding proteins by fixing the sections with paraformaldehyde before the DNA-binding reaction. Treatment with 0.2% paraformaldehyde (25°C for 30 min) effectively fixed the proteins in the sections without affecting the DNA-binding activity, but 0.4% paraformaldehyde greatly reduced the DNA-binding activity, and fixation with more than 0.6% paraformaldehyde for

30 min resulted in a complete loss of DNA-binding activity. These results suggest the importance of undenatured proteins in thin sections for reaction with a D N A probe. To determine whether the labeling by the AP-t probe was specific to the AP-1 sequence, we carried out competition experiments with excess unlabeled AP-1 probe by gel shift assay and the in situ DPB method. The nuclear extracts of brain were incubated with the [35S]dCTP labeled AP-1 or Spl probe for 30 min at 30°C, and then the mixture was subjected to electrophoresis on 4% polyacrylamide gel [18]. One major retarded band was detected in the AP- 1 and Sp I binding (Fig. 1A, indicated by arrow). The presence of 100-fold excess of the unlabeled AP-1 probe specifically abolished the complex formation with 35S-labeled AP-1 probe (Fig. 1A) and also markedly reduced the AP-1 binding activity in the brain sections (Fig. 1B, b). However, even 300-fold excess of unlabeled AP-1 probe did not compete with the Spl binding activities in gel shift assay (Fig. 1A) and the in situ DPB method (Fig. 1B, d). The 100-fold unlabeled mutated AP- 1 probe (CTAGTGATAAATCAGCCGGATC) which differs in two bases from AP-1 site in the core DNA-binding sequence did not compete with the AP-I binding both in the gel shift and in situ DPB method. These results indicate sequence-specific labeling of DNA-binding proteins in situ. Pretreatment of thin sections of the brain with an antibody (50 pg/ml) against the DNA-binding domain of Jun (Oncogene Science, Inc. Cat, no. PC06) for 16 h at 4 ° C, blocked effectively the binding of AP-1 probe (Fig. 1C, b). The antibody also inhibited the formation of a DNAprotein complex in gel shift assay (Fig. IA), but did not inhibit formation of a complex with the Spl probe in the gel shift assay (Fig. IA) and binding of the Spl probe to brain sections (Fig. 1C, d). From these results, we concluded that the in situ DPB method allows to detect specified DNA-binding proteins that recognize unique D N A sequences in situ. AP-1 binding protein is widely distributed in the striatum, the medial septum, the ventral pallidum (Fig. 2b), the medial habenula, hypothalamus especially in the ventromedial nucleus, the lateral hypothalamus, and arcuate nucleus (Fig. 2c), some lower brain stem regions especially in the principle trigeminal nucleus, the dorsal tegmental nucleus. It was also found at high density in the layer IV of the cerebral cortex and the layer II of piriform cortex, but at low density in the amygdala complex (Fig. 2c). Conspicuous binding activity was seen in the glomerular, mitral and granular cell layers of main olfactory bulb (Fig. 2a), cerebellar cortex (Fig. 2d) and dentate gyrus of hippocampus (Fig. 2c,e). In the hippocampus, the pyramidal cell layers in the CA1 region of

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Ammon's horn were labeled more strongly than those in the other CA regions (Fig. 2e). The regional pattern of distribution was similar to that of Jun-like immunoreactivity and its mRNA detected with Jun antibody and in situ hybridization [10, 17, 19]. The labelings observed in the pia mater and plexus chorioideus even after competing with unlabeled probe or antibody against the DNAbinding domain of Jun seem to be non-specific binding, since a similar phenomenon is also observed in the in situ hybridization. We also carried out experiments with a Spl probe (Fig. 2f) which is recognized by a specific transcription factor [13, 14]. In some regions such as hippocampus (Fig. 2e,f), the Spl binding activity was evenly observed in the pyramidal cell layer throughout all regions of Ammon's horn, while the AP-1 binding was predominantly seen in CA1. In the dentate gyrus, the Spl probe showed less binding than the AP-1 probe (Fig. 2e,f). The Spl probe labeled the cerebral cortex diffusely, whereas the AP-1 probe mainly labeled the layer IV of the cerebral cortex (Fig. 2e,f). The different regional distributions of AP-1 and Spl binding proteins suggest that the two transcription factors play specific regulatory roles in different regions of the brain. Using a computer-assisted image-analyzing system (MICD, Imaging Research Inc, Canada), we compared

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the relative intensities (signal/noise ratio) of the AP-1 binding on the autoradiographic film obtained from the in situ DPB method and gel shift assay in several brain regions including hippocampus, caudate putamen, cerebral and cerebellar cortices, and found that the AP-1 binding detected in gel shift assay was parallel with that detected by the in situ DPB method. These data provided additional evidence that the labeling detected by the in situ DPB method represents the distribution of AP-1 binding proteins in the brain. Recent studies have shown that expression of Fos protein is enhanced in the striatum and nucleus accumbens on treatment with methamphetamine [8, 20] which is known to cause the dramatic change in neural activity. However, less is known about the effect of methamphetamine on the DNA-binding activity of AP-1 binding protein in the brain. Using the in situ DPB method, we examined the distribution and the DNA-binding activity of AP-1 and Spl binding proteins in the brain of methamphetamine-treated mouse. After administration with

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Fig. 2. Patterns of in situ DNA-protein binding (in situ DPB) of AP-1 and Spl elements in mouse brains. Dark-field autoradiograms in selected brain regions are shown by the in situ DPB method. Dense grains and bright areas represent high densities of AP-1 binding protein in sections of the olfactory region (a), forebrain (b), thalamus (c), cerebellum (d), hippocampus (e), and of the Spl binding protein in the hippocampus (f). The arrows in e and f indicate the difference in the AP-1 and Spl binding.

Fig. 3. Changes in AP-1 binding proteins induced by methamphetamine. An adult mouse was treated with methamphetamine (5 mg/ kg, s.c.). The brain was isolated rapidly 3 h later and sections were prepared. The in situ DNA-proteins binding reaction was carried out with AP-1 (a,b) and Spl (c,d) probe, a,c: sections prepared from mouse brain injected with saline, b,d: sections prepared from mouse brain treated with methamphetamine.

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methamphetamine (5 mg/kg, s.c., for 3 h), the AP-1 binding activity was markedly increased in some regions such as the cingulate cortex, dentate gyrus, the layer IV of the isocortex, the layer II of piriform cortex, habenula complex and some regions of thalamus and hypothalamus (Fig. 3). The Spl binding activity was also increased in the cingulate cortex, hippocampus, various regions of hypothalamus, layer II of the piriform cortex and the medial group of amygdala complex. These data suggest that the methamphetamine stimulation may result in changes in gene transcription by its effect on the transcription factors. The present data suggest that the in situ DPB is a useful tool for identifying the sites of gene expression by various stimuli and also for screening the drugs which affect gene expression in the brain. We thank Dr. Hiroshi Kiyama, Department of Anatomy, Osaka University Medical School, for his critical reading of this manuscript. 1 Abate, C., Patel, L., Rauscher III, F.J. and Curran, T., Redox regulation of Fos and Jun DNA-binding activity in vitro, Science, 249 (1990) 1157-1161. 2 Angel, P., Baumann, I., Stein, B., Delius, H., Rahmsdorf, H.J. and Herrlich, E, 12-o-Tetradeeanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5' flanking region, Mol. Cell. Biol., 7 (1987) 2256-2266. 3 Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R., Rahmsdorf, H.J., Jonat, C., Herrlich, E and Karin, M., Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor, Cell, 49 (1987) 729-739. 4 Chiu, R., Imagawa, M., Imbra, R.J., Bockoven, J.R. and Karin, M., Multiple cis- and trans-acting elements mediate the transcriptional response to phorbol ester, Nature, 329 (1987) 648-651. 5 Cole, A.J., Abu-Shakara, S., Saffen, D.W., Baraban, J.M. and Worley, P.F., Rapid rise in transcription factor mRNAs in rat brain after electroshock-induced seizures, J. Neurochem., 55 (1990) 1920 1927. 6 Deng, G. and Wu, R., Terminal transferase: use in the tailing of DNA and for in vitro mutagenesis, Methods Enzymol., 100 (1983) 96-116.

7 Gentz, R., Rauscher IIl, F.J., Abate, C. and Curran, T., Parallel association of Fos and Jun leucine zippers juxtaposes DNA binding domains, Science, 243 (1989) 1695 1699. 8 Graybiel, A.M., Moratalla, R. and Robertson, H.A., Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striosome-matrix compartments and limbic subdivisions of the striatum, Proc. Natl. Acad. Sci. USA, 87 (1990) 6912 6916. 9 Halazonetis, T.D., Georgopoulos, K., Greenberg, M.E. and Leder, P., c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities, Cell, 55 (1988) 917-924. 10 Herdegen, T., Leah, J.D., Manisali, A., Bravo, R. and Zimmermann, M,, c-Jun-like immunoreactivity in the CNS of the adult rat: basal and transsynaptically induced expression of an immediateearly gene, Neuroscience, 41 (1991) 643-654. 11 Imbra, R.J. and Karin, M., Phorbol ester induces the transcriptional stimulatory activity of the SV40 enhancer, Nature, 323 (1986) 555 558. 12 Imbra, R.J. and Karin, M., Methallothionin gene expression is regulated by serum factors and activators of protein kinase C, Mol. Cell. Biol., 7 (1987) 1358 1363. 13 Kadonaga, J.T., Carner, K.R., Masiarz, F.R. and Tjian, R., Isolation of cDNA encoding transcription factor Spl and functional analysis of the DNA binding domain, Cell, 51 (1987) 1079 1090. 14 Kadonaga, J.T., Courey, A.J., Ladika, J. and Tjian, R.. Distinct regions of Spl modulate DNA binding and transcriptional activation, Science, 242 (1988) 1566-1570, 15 Lee, W., Haslinger, A., Karin, M. and Tjian, R., Activation of transcription by two factors that bind promoter region and enhancer sequence of the human methallothionein and SV40, Nature, 325 (1987) 368 372. 16 Lee, E, Mitchell, P.J. and Tjian, R., Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements, Cell, 49 (1987) 741 752. 17 Mellstrom, B., Achaval, M., Montero, D., Naranjo, J.R. and Sassone-Corsi, E, Differential expression of thejun family members in rat brain, Oncogene, 6 (1991) 1959-1964. 18 Osugi, T., Taniura, H., Ikemoto, M. and Miki, M., Effects of chronic exposure of NG108-15 cells to morphine or ethanol on binding of nuclear factors to cAMP-response element, Biochem. Biophys. Res. Commun., 174 (1991) 25-31. 19 Smeyne, R.J., Schilling, K., Robertson, L., Luk, D., Oberdick, J., Curran, T. and Morgan, J.I., Fos-lacZ transgenic mice: mapping sites of gene induction in the central nervous system, Neuron, 8 (1992) 13-23. 20 Young, S.T., Porrino, L.J. and Iadarola, M.J., Cocaine induces striatal c-Fos immunoreactive proteins via dopaminergic Dt receptors, Proc. Natl. Acad. Sci. USA, 88 (1991) 1291-1295.

In situ DNA-protein binding: a novel method for detecting DNA-binding activity of transcription factor in brain.

A novel method, in situ DNA-protein binding (in situ DPB), was developed to detect the distribution and DNA-binding activity of AP-1 and Sp1 binding p...
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