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Developmentally and regionally regulated alterations of octamer- and GC-box-binding activities during the postnatal development of mouse cerebellum
Developmental Brain Research, 61 (1991) 161-168 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0165-3806/91/$03.50 ADONIS 016538069151295A
161
BRESD 51295
Developmentally and regionally regulated alterations of octamer- and GC-box-binding activities during the postnatal development of mouse cerebellum Hiroaki Sakurai 1, Kaoru Kikuchi 1, Tomofusa Tsuchiya 1, Hiroshi Kanazawa 2 and Masaaki Tsuda 1 1Department of Microbiology, Facultyof PharmaceuticalSciences and 2Department of Applied Biology, Faculty of Technology, Okayama University, Tsushima-naka, Okayama 700 (Japan) (Accepted 2 April 1991)
Key words: Cerebellum; Cerebral cortex; Transcription factor; Gel mobility analysis; Octamer-binding protein; GC-box-binding protein
Gel-mobility analyses using a DNA probe containing the SV40 enhancer-promoter revealed that the DNA-binding activities of cerebellar extracts changed drastically during the postnatal development of mouse brain. Three major DNA-protein complexes formed on the 72-bp enhancer (complexes E-I, E-II and E-III) were detected in the extracts prepared from the 2- and 10-day-old mouse cerebellum, but two of them (complexes E-II and E-III) were not detected in 3- and 7-week-old mice. In contrast, the formation of the complexes E-II and E-III, but not E-I, was observed in the cerebral cortex extracts throughout postnatal development until at least 7 weeks after birth. The addition of the octamer motif as a competitor abolished the formation of these three complexes. On the other hand, the formation of DNA-protein complexes on the 21-bp promoter by the cerebellar extracts increased after birth, while this increase in the formation of the complexes was not detected in the cerebral cortex extracts. This complex formation was found to be dependent upon the GC-box and to be stimulated by the addition of Zn 2÷, indicating that the protein binding to the 21-bp promoter may contain intramolecular zinc fingers. Thus, the expression of the octamer- and the GC-box-binding proteins are developmentally and regionally controlled in the cerebellum during the postnatal development of the mouse brain.
INTRODUCTION The development of the vertebrate brain proceeds in a regionally controlled fashion, and results in the formation of many brain regions, whose morphologies and functions are distinct from each other. Recently, the expression of some transcriptional factors containing the h o m e o d o m a i n or zinc fingers were found to be segmentally regulated in the rat and in the mouse hindbrain by in situ hybridization 11'15'16. It has also been reported that a large family of regulatory proteins containing the P O U - d o m a i n , which is known to be shared by the octamer-binding proteins Oct-1 and Oct-2, the pituitary transcription factor Pit-1 and the nematode homeotic unc-86 gene product, were shown to be expressed throughout the development of rat brain 6. On the other hand, several lines of evidence obtained from studies of Drosophila melanogaster have suggested that some transcriptional factors containing the P O U - d o m a i n or zinc fingers may play a key role in determining neuronal cell
differentiation 7'1°. Such neuronal cell differentiation is thought to be differentially regulated in each brain region. In order to understand the developmental program of formation of the mammalian central nervous system (CNS), therefore, it is important to investigate the expression of transcriptional factors during the development of the brain in relation to the regionality of the brain. The development of the rodent cerebellum proceeds postnatally, and the formation of neuronal circuits between neurons in the cerebellum continues after birth 1. During this postnatal development of rodent cerebellum, a number of genes responsible for the formation of the cerebellum may be expressed and their expression regulated by several kinds of transcriptional factors. To survey possible alterations in the expression of transcriptional factors during the development of the mouse cerebellum, we have used the gel-mobility assay because of its convenience for detecting changes in specific DNA-binding activities of transcriptional factors.
Correspondence: M. Tsuda, Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama 700, Japan. Fax: (81) (862) 55 7456.
162 MATERIALS
AND METHODS
DNA fragments as probes The Pvull-NcoI DNA fragments (233 bp) containing the SV40 enhancer and promoter were cut out from pSV2CAp plasmid DNA. Oligonucleotides (E72, P21, PI, P2, P3, P4, OCT, TRE, P3m1, P3m2, P3m3 and NS; see Fig. 1) were synthesized by a DNA synthesizer (MilliGen/Biosearch), purified by polyacrylamide gel electrophoresis and annealed with the counter oligonucleotides, respectively. DNA probes were end-lab&led with [32P]dCIY by DNA polymerase I (Klenow fragment), and purified by Sephadex G-50 column chromatography. Preparation of nuclear extractsfrom brain The nuclear extracts were prepared from cerebral cortexes and cerebella of mice according to the procedure of Dignam et al.3 with a slight modification. Brains were removed from mice 2, IO,21 and 49 days after birth, and the cerebral cortexes and cerebella were cut out from the brain. After washing them with phosphate-buffered saline (PBS), the tissue was homogenized for 10 strokes in a Dounce B-type homogenizer with 1 ml Buffer A containing 10 mM Tris-HCl (pH 7.9), 1.5 mM MgCl,, 10 mM KCI, 0.5 mM dithiothreitol (DTI’). The cells were collected by the centrifugation at 2000 rpm for 10 min, suspended in 5 ~01s. of Buffer A and allowed to stand on ice for 15 min. After centrifugation, the pellets were again suspended with 2 vols. of Buffer A and homogenized for 10 strokes. After washing the pellets with 2 ~01s. of Buffer A twice, the nuclei were collected by centrifugation at 15,000 rpm for 20 min at 4 “C, suspended in an equal volume of Buffer B containing 20 mM Tris-HC1 (pH 7.9), 25% glycerol, 420 mM KCl, 1.5 mM M&h, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM phenyhnethylsulfonyl fluoride (PMSF) and homogenized by 20 strokes. The suspension was kept on ice for 1 h, followed by centrifugation at 15,000 rpm for 20 min. The supematant was dialyzed against the Buffer C containing 20
mM Tris-HCl (pH 7.9), 20% glycerol, 100 mM KCI, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF for 12 h. After centrifugation at 15,000 rpm for 15 min at 4 “C, the supernatant was stored at -80 “C Transfectionexperiments in cultured brain cells Cerebral cortexes or cerebella of l-week-old mice were minced by scissors and treated with a solution containing 0.125% trypsin (Gibco) and 1 mM EDTA for 20 min at 37 “C. After culturing for 2 days in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, transfection with 8 yg of the pSV2CAT or pAlOCAT plasmid DNA per m-mm culture dish (Corning) was carried out by the calcium-phosphate co-precipitation method as described by Gorman5. The pSV2CAT plasmid contains both the 72-bp repeats and the 21-bp repeats, but the pAlOCAT plasmid only the 21-bp repeats’. After transfection for about 16 h and prolonged culture for 2 days, cytoplasmic extracts were prepared and their CAT activities were measured by the method described by German’. Gel-mobiliry assay DNA-protein binding was performed in the reaction mixture (20 ~1) containing 20 mM Tris-HCl (pH 7.5), 1 mM D’IT, 1 mM EDTA, 100 mM NaCl, 50 mM KCI, 2pg poly[dI-dC] (Sigma), 10% glycerol, 0.1-l ng 32P-labelled DNA probe and nucfear extract (3-10 pg protein) for 30 min at 20 “C. When the effect of Zn2’ on the DNA-protein complex formation was observed, an incubation in the presence of 1 mM ZnCl, for 15 min on ice was carried out and the DNA-protein binding reaction was started by the addition of the DNA probe. The DNA-protein complexes were resolved on a 4% polyacrylamide gel (3O:l cross-linking ratio) containing 67 mM Tris-HCI (pH 7.5), 33 mM sodiumacetate, 2.5% glycerol and 1 mM EDTA. Electrophoresis was carried out at 11 V/cm for about 2 h in the cold room. After electrophoresis, the gel was f?xed with 10% acetic acid and 10% methanol, dried and autoradiogaphed. A gel-mobility competition assay was performed in the presence of competitor DNAs at approximately 400-fold excess. DNase I protection analysis The DNA binding reaction was performed in the reaction mixture (50~1) containing 20 mM Tris-HCl (pH 7.5), 6.5 mM MgCh, 50 mM KCl, 0.5 mM DlT, 0.4 mM EDTA, 10% glycerol, 0.5% polyethyleneglycol, 1 ,ug poly[dI-dC], 0.8 pg 32P-labelled DNA probe and nuclear extract (approximately 25 pg). After the incubation for 15 min at room temperature, 2 U of DNase I (Sigma) were added with 50 ~1 of 10 mM MgCI, and 5 mM CaC&, and the mixture was incubated for 1 min at room temperature. After extraction of DNA with phenol-chloroform and precipitation of DNA with ethanol, DNA was subjected to denaturing polyacrylamide gel electrophoresis.
RESULTS
OCT P3ml P3m2 P3m3 TRE NS
Fig. 1. Organization of the SV40 enhancer-promoter and several motifs used as probes and competitors for the gel-mobility assay. The top line shows the structure of the SV40 early promoter containing the 72-bp and the 21-bp repeats. The black boxes indicate the original DNA sequences included in the SV40 enhancer-promoter used as probes or competitors (EP.233, E72, P21, Pl, P2, P3, P4, OCT, P3m1, P3n12, P3m3 and TRE). The open boxes included in the DNA fragments indicate the non-specific DNA sequences (NS). The locations of the several DNA motifs on the SV40 enhancer-promoter which were already investigated’2*‘3 are shown. TRE means the TPA (12-0-tetradecanoyl phorbol-13acetate) responsive element.
Gel-mobility patterns of SV40 enhancer-promoter Since the SV40 enhancer-promoter has several DNA-
protein binding motifs 12,13(Fig. l), we expected to detect changes in specific DNA-binding activities if the expression of DNA-binding proteins related to the SV40 enhancer (72-bp repeats) -promoter (21-bp repeats) were postnatally altered in mouse brain. Tram&&ion experiments of pSV2CAT and pAlOCAT plasmid DNAs using cultured primary cerebeILar and cerebral cortex cells prepared from l-week-old mouse brain (Fig. 2) revealed that the 72-bp enhancer can enhance transcriptional efficiency in both the cultured cerebral cortex (lanes 1 and 3) and cerebellar (lanes 2 and 4) cells, suggesting the
163
MAc-CM
CM-~
1
2
pSV2CAT
3
4
pAl 0CAT
Fig. 2. Transfection experiments of the cultured cerebellar and cerebral cortex cells. The procedure of transfection is described in Materials and Methods. pSV2CAT (lanes 1 and 2) or pA10CAT (lanes 3 and 4) plasmid DNAs were transfected to the cultured cerebral cortex (lanes 1 and 3) or the cerebeUar (lanes 2 and 4) cells. The positions of monoacetylated [14C]chloramphenicol and [14C]chloramphenicol on the thin-layer chromatography are shown as MAc-CM and CM, respectively.
presence of proteins which are able to bind to the SV40 enhancer in these cells. Using the EP233 probe (233 bp) containing both the SV40 e n h a n c e r - p r o m o t e r (Fig. 1), we performed gelmobility experiments using the cerebellar and cerebral
cortex extracts which were prepared from 2- and 10-day-, and 3- and 7-week-old mice. As shown in Fig. 3, several D N A - p r o t e i n complexes (complexes EP-I - EP-VI) were distinctly recognized according to the differences in their gel mobilities. From the extracts prepared from the cerebral cortexes (Fig. 3, lanes 1-4), the complexes EP-IV, EP-V and E P - V I were clearly detected, and their gel-mobility patterns were not altered from 2 days to 7 weeks after birth. In contrast, the gel-mobility patterns of the cerebellar extracts (Fig. 3, lanes 5-8) drastically changed during postnatal development, in which the complex EP-IV, EP-V and E P - V I were detected until 10 days but not at 3 or 7 weeks after birth, while the complexes EP-I and E P - I I were clearly detected 3 and 7 weeks after birth. The complex EP-III, which was not detected in the cerebral cortex extract, appeared to be present throughout the development of the cerebellum from 2 days to 7 weeks after birth. To determine whether the complexes could be formed depending upon the 72-bp enhancer or the 21-bp promoter of the SV40 D N A , we carried out gel-mobility experiments in the presence of the 72-bp or 21-bp D N A fragments as competitors. W h e n the 72-bp enhancer D N A fragment (E72) was added as a competitor (Fig. 4, lane 2), the complexes EP-III, EP-IV, EP-V and EP-VI
Cerebral cortex Age :
2d
10d
3w
Cerebellum 7w
2d
10d
3w
7w
5
6
7
8
EP -I
EP-II EP-III
]b.
~,~
E P - I V ~,~ E P - v ~,-
EP-VI ~,-
lb.
1
2
3
4 Probe : EP233
Fig. 3. Gel-mobility experiments using the PvuII-NcoI DNA fragment (233 bp) as a probe. Nuclear extracts prepared from the cerebral cortexes (lanes 1-4) and cerebella (lanes 5-8) of 2-day- (lanes 1 and 5), 10-day- (lanes 2 and 6), 3-week- (lanes 3 and 7) and 7-week-old (lanes 4 and 8) mice were added to the reaction mixture at the amount of 5 (lanes 1 and 5), 6 (lanes 2 and 6), 9 (lanes 3 and 7) and 9 (lanes 4 and 8) /zg protein, respectively. EP233 shown in Fig. 1 was used as a probe. The age of the mice after birth (2 and 10 days, 3 and 7 weeks), which were used for preparation of the extracts, are shown at the top of the figure, designated as 2d, 10d, 3w and 7w, respectively.
164 were competed, but when the 21-bp promoter D N A fragment (P21) was added as a competitor, the complexes EP-I and EP-II were competed (lane 3). These results indicate that the complexes EP-I and EP-I1 were formed on the 21-bp promoter, and the complexes EP-III, EP-IV, EP-V and EP-VI on the 72-bp enhancer.
Gel-mobility patterns of the 72-bp enhancer Using the 72-bp enhancer D N A fragment (E72) as a probe, we compared the gel-mobility patterns of the extracts prepared from the cerebral cortex and the cerebellum (Fig. 5). The complex E-I was detected only in the cerebellar extracts throughout the development until 7 weeks after birth (Fig. 5, lanes 5-8), but not in the cerebral cortex extracts (lanes 1-4). The complexes E-II and E-III were detected in the cerebral cortex extracts throughout the development (lanes 1-4), while in the cerebellar extracts they were detected only until 10 days after birth but not from 3 weeks (lanes 5-8).
Competitors:
EP-I
~,~
EP-II EP-III
~b. ~,~
--
E72
P21
The complex formation is competed by the octamer motif To identify what motifs on the 72-bp enhancer contribute to the formation of the complexes E-I, E-II and E-III shown in Fig. 5, we performed competition experiments using several D N A sequences included in the 72 bp enhancer as competitors (Fig. 6). As shown in Fig. 6A, addition of the P4 D N A fragment competed the formation of the E-I, E-II and E-III complexes most severely (lane 4). Addition of the P2 or P3 D N A fragment showed a weaker competition (lanes 3 and 5) but addition of the P1 D N A fragment did not show any competition (lane 2). Since the P2 and P4 D N A fragments contain the octamer motif, we examined competition effects of the addition of OCT D N A fragments on the complex formation. As shown in Fig. 6B, the formation of the complexes E-I, E-II and E-III was abolished in the presence of the OCT competitors (compare lane 11 with lane 10). When the P4 probe was used (Fig. 6C), complexes P4-I, P4-II and P4-III were detected (lanes 12 and 15), but the addition of the OCT D N A fragments as competitors abolished the formation of these complexes (lanes 13 and 16). These results indicate that all the complex formation on the 72-bp enhancer depend upon the octamer motif. The complex P4-I was formed with a higher efficiency in the cerebellar extract than in the cerebral cortex extract, while the complexes P4-II and
Cerebral cortex
Cerebellum
Age: 2d lOd 3w 7w
2d
lOd
3w
6
7
7w
EP-IV E-
EP-V
E-I E-II
E P-VI
1
2
3 1
Probe :
2
3
4
5
8
EP233
Fig. 4. Competition experiments by the 72-bp or 21-bp DNA sequences for the gel-mobility experiment using the EP233 probe. The gel-mobility experiment was carried out using the total brain extracts of 2-week-old mice and the PvuII-NcoI 233-bp DNA fragment (EP233) as a probe. No competitors (lane 1), and a 400-fold excess of the 72-bp (E72; lane 2) or the 21-bp (P'21; lane 3) DNA fragments as competitors were added with the EP233 probe.
Probe : E72
Fig. 5. Gel-mobility experiments using the E72 probe. Nuclear extracts prepared from the cerebral cortexes (lanes 1-4) and the cere~lla (lanes 5-8) were added at the amount of 5 (lanes I and 5), 6 (lanes 2 and 6), 9 (lanes 3 and 7) and 9 (lanes 4 and 8)/~g protein. The 72-bp DNA sequence (E72) was used as a probe. The age of the mice from which the extracts were prepared are shown at the top of the figure. The non-specific bands are shown as ns.
165 C Cerebral cortex Competitors :
-
P1
P2
P4
P3 P3ml P3m2 P3m3 NS
--
OCT
-
Cerebellum
OCT NS
--
OCT NS
P 4 - [ ~'E-I~"
P4- I I ~,P 4 - I I I ~,-
E -I~"
E-II~ E-III I,-
E-Ill-
E-III J,nsiP-
ns~
F~-
F=,, 1
2
Probes :
3
4
5
6
7
8
9
F 10
E72
11
12
13
14
E72
15
16
17
P4
Fig. 6. Competition experiments using several DNA sequences included in the 72-bp enhancer. A and B. Gel-mobility experiments using the total brain extract prepared from 2-week-old mice and the 72-bp DNA sequences (E72) as a probe were carried out in the absence of competitors (lanes 1 and 10) or in the presence of a 400-fold excess of the several competitors, i.e. P1 (lane 2), P2 (lane 3), P4 (lane 4), P3 (lane 5), P3ml (lane 6), P3m2 (lane 7), P3m3 (lane 8), NS (lane 9) and OCT (lane 11), shown in Fig. 1. C. Gel-mobility experiments using cerebral cortex (lanes 12-14) and cerebellar (lanes 15-17) extracts prepared from 3-week-old mice and the P4 probe were carried out in the absence of competitors (lanes 12 and 15) or in the presence of OCT (lanes 13 and 16) or NS (lanes 14 and 17).
P 4 - I I I were strongly d e t e c t e d in the c e r e b r a l cortex extract (Fig. 6C). Since b o t h extracts were p r e p a r e d f r o m the b r a i n s of 3 - w e e k - o l d mice, the c o m p l e x e s P4-I, P4-II a n d P 4 - I I I s h o u l d c o r r e s p o n d to the c o m p l e x e s E - I , E - I I a n d E - I l l , respectively, given b y the E 7 2 p r o b e .
the P3 D N A f r a g m e n t (Fig. 1) s h o w e d a slight c o m p e tition with the c o m p l e x f o r m a t i o n (Fig. 6 A , lanes 7 a n d 8). T h e s e results i n d i c a t e that the 3" s e q u e n c e s a d j a c e n t to t h e o c t a m e r m o t i f c o n t r i b u t e , at least in part, to the f o r m a t i o n of the c o m p l e x e s o n the 72-bp e n h a n c e r .
W h e n the P 3 m l D N A f r a g m e n t whose m u t a t i o n s w e r e l o c a t e d 5 n u c l e o t i d e s f r o m the 5" e n d of the P3 f r a g m e n t (Fig. 1), was a d d e d as a c o m p e t i t o r , n o c o m p e t i t i o n was o b s e r v e d (Fig. 6 A , l a n e 6), while a d d i t i o n of the P 3 m 2 a n d P 3 m 3 w h o s e m u t a t i o n s were l o c a t e d f u r t h e r inside
Zinc effect on the complex formation promoter
on the SV40
Since the f o r m a t i o n of the c o m p l e x e s E P - I a n d E P - I I was a b o l i s h e d by the 21-bp p r o m o t e r as a c o m p e t i t o r
TABLE I
DNA-protein complexes detected by gel-mobility assays Detection of the bands obtained by the gel-mobility experiments using E72 (Fig. 5), P4 (Fig. 6C), P21 (Fig. 8) and TRE (Fig. 9) as probes were summarized as follows: - , not detected; +, detected; + + and + + +, strongly detected.
Motifs
Probes
Complexes
Cerebral cortex 2d
Octamer
E72
P4
GC-box
P21
TRE
TRE
E-I E-II E-Ill P4-I P4-II P4-III P-I P-II T-I
. + +
+ + +
Cerebellum
lOd .
. + +
+ + +
3w
7w
2d
lOd
3w
7w
+ +
+ +
+ + +
+ + +
+ _ _
+ _
.
-
+
+ + + + ++
_ _ +++ +++ ++
+ + +
+ + +
++ ++ +
_
++ ++ +
166 C Zn 2+ .
4-
+
--
--
+
+
-
EDTA
-
+
+
--
--
+
4"
Extract
fl 1
Probes •
2
3
4
5
6
EP233
7
P21
8
9
10
P21
Fig. 7. Effects of Zn 2+ on the DNA-protein complex formation on the 21-bp DNA sequences (P21). A and B. Gel-mobility experiments using EP233 (lanes 1-4) or P21 (lanes 5-8) as a probe were carried out in the absence of Zn 2+ and 1 mM EDTA (lanes 1 and 5) or in the presence of Zn 2+ (lanes 2 and 6), Zn 2+ plus 1 mM EDTA (lanes 3 and 7) and 1 mM EDTA (lanes 4 and 8). C. DNase 1-protection analysis using the P21 probe was carried out in the absence (lane 9) or the presence (lane 10) of the extract. The eerebellar extract prepared from 3-week-old mice was used for all experiments.
Cerebral cortex Age:
2d
10d
3w
Cerebellum
7w
2d
10d
3w
7w
4
5
6
7
8
p.
P-I
1
2
3
Probe : P21
Fig. 8. Gel-mobility experiments using the P21 probe. The gelmobility experiments using the P21 probe with the cerebral cortex (lanes 1-4) and the eerebellar (lanes 5-8) extracts were carried out in the presence of Zn 2+. The ages of the mice from which the extracts were prepared are shown at the top of the figure.
(Fig. 4, lane 3), we examined whether the formation of the complexes EP-I and E P - I I requires Z~n2+ because Spl DNA-binding proteins which involve zinc fingers intramolecularly are known to bind to the GC-box of the SV40 promoter s. W h e n Zn 2+ was added: to the D N A protein binding reaction mixture, the formation of the complexes EP-I and E P - I I in the cerebellar extract was greatly stimulated (Fig. 7A, compare lane 2 with lane 1). A simultaneous addition of E D T A with Zn 2+ suppressed the stimulation of the complex formation (lane 3) and an addition of E D T A alone did not affect the complex formation (lane 4), indicating that the efficiency of the formation of the complexes EP-I and E P - I I depends upon the presence of Zn 2+ in the reaction mixture. The stimulation of the complex formation by Zn 2+ was also supported by the experiment using the P21 probe for the gel-mobility assay (Fig. 7B). Two major complexes P-I and P-II were stimulated in the presence of Zn 2+ (compare lane 6 with lane 5), but suppressed by the addition of E D T A (lane 7). DNase-I-protection analysis of the 21-bp p r o m o t e r sequences in the cerebellar extract revealed the presence of proteins preferentially binding to the 3" half GC-box (Fig. 7C, compare lane 10 with lane
9).
167 Cerebral cortex Age:
2d
lOd
3w
Cerebellum 7w
2d
lOd
3w
7w
5
6
7
8
}1..-
1
2
3
4 Probe : TRE
Fig. 9. Gel-mobility experiments using the TRE probe. Gel-mobility experiments using the TRE probe with the cerebral cortex (lanes 1-4) and the cerebellar (lanes 5-8) extracts were carried out.
Developmental increase of the complex formation on the 21-bp promoter To confirm the alteration of the DNA-binding activities to the 21-bp promoter during the postnatal development of the cerebellum as shown in Fig. 3, we performed gel-mobility experiments of the 21-bp promoter sequences in the presence of Zn 2+. As shown in Fig. 8, the formation of the complexes P-I and P-II in the cerebellar extracts increased after birth (lanes 5-8), while such an increase in the formation of the complexes after birth was not observed in the cerebral cortex extracts (lanes 1-4).
Gel-mobility patterns of TRE As shown in Fig. 9, the gel-mobility patterns of TRE were similar between the cerebral cortex and the cerebellum, although the increase of the TRE binding activities was observed with the extracts prepared from the 3-week-old mice. This result indicates that the expression of the TRE-binding proteins could be similarly regulated in both brain regions and different from those of the octamer- and the GC-box-binding proteins. DISCUSSION
Using gel-mobility assays, we have demonstrated that the octamer and the GC-box binding activities expressed in the cerebellum changed in a different manner from those in the cerebral cortex during the postnatal development of the mouse brain (Table I). We have found that 3 major DNA-protein complexes (E-I, E-II and E-Ill, see Figs. 5 and 6) were formed by the brain nuclear
extracts depending upon the octamer motif. Since the complex E-I was also detected by the extracts prepared from liver and kidney of 2-week-old mice (data not shown), the proteins responsible for the formation of the complex E-I could be the Oct-1 protein because Oct-1 is believed to be ubiquitously expressed in several tissues ~4. The E-II and E-III complexes were not formed by the extracts prepared from liver and kidney (data not shown), suggesting that the proteins responsible for the formation of E-II and E-III are expressed in a brainspecific manner. Recently, using in situ hybridization, He et al. 6 demonstrated that several proteins containing the POU-domain, which is widely accepted as a domain responsible for specific DNA-binding, were found to be expressed in rat brain in a spatially and temporally regulated fashion. Thus we can speculate that the proteins responsible for the formation of the complexes E-II and E-III might correspond to at least some of them. Some DNA-binding proteins other than the octamerbinding proteins could, at least in part, contribute to the formation of the complexes E-I, E-II and E-III, because the sequences downstream from the octamer motif were slightly competitive to the formation of the E-I, E-II and E-Ill complexes (Fig. 6A). So it is possible that some interactions between the octamer- and its 3" adjoining region-binding proteins could regulate the formation of the E-I, E-II and E-III complexes. Two major complexes, P-I and P-II, were found to be formed on the 21-bp promoter of SV40 DNA in the cerebellar extracts (Fig. 7). The formation of these complexes increased after birth, while such an increase in the complex formation was not detected in the cerebral cortex extracts (Fig. 8). Since the addition of Zn 2÷ stimulated the formation of the complexes P-I and P-II (Fig. 7), the proteins responsible for their formation should contain intramolecular zinc fingers. The Spl protein is known to be the GC-box-specific binding protein z'4 and to involve such zinc fingers 8. In addition, Spl is known to consist of 2 polypeptides of 105 and 95 kDa, both of which have the ability to bind to the GC-box z, suggesting that the complexes P-I and P-II were formed by these Spl-like proteins. Recently, evidence has been accumulating that the transcriptional factors intramolecularly containing the POU-domain or the zinc fingers are expressed in the mammalian brain 6ALAS,16 or account for the differentiation of certain neuronal cell-types7'1°. Thus, it can be speculated that the expression of the Oct- and Spl-like proteins in the cerebellum may participate in the neuronal cell differentiation in the cerebellum. Identification of the cell types expressing the responsible genes by using the in situ hybridization may clarify the morphological aspects of the phenomena described in this study.
168 Acknowledgements. We thank Dr. Y. Suzuki for helpful comments on the manuscript. This research was supported in part by a
Grant-in-Aid for Special Project Research from the Naito Foundation, Tokyo.
REFERENCES
9 Laimins, L.A., Khoury, G., Gorman, C., Howard, B. and Gruss, P., Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 6453-6457. 10 Moses, K., Ellis, M.C. and Rubin, G.M., The glass gene encodes a zinc-finger protein required by Drosophila photoreccptor cells, Nature, 340 (1989) 531-536. 11 Murphy, P., Davidson, D.R. and Hill, R.E,, Segment-specific expression of a homeobox-containing gene in the mouse hindbrain, Nature, 341 (1989) 156-159. 12 Nomiyama, H., Fromental, C., Xiao, J.H. and Chambon, P., Cell-specific activity of the constituent elements of the simian virus 40 enhancer, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7881-7885. 13 Rosales, R., Vigneron, R., Macchi, M., Davidson, I., Xiao, J.H. and Chambon, P., In vitro binding of cell-specifiC and ubiquitous nuclear proteins to the octamer motif in the SV40 enhancer and related motifs present in other promoters and enhancers, EMBO J., 6 (1987) 3015-3025. 14 Strum, R.A., Das, G. and Herr, W., The ubiquitous octamerbinding protein Oct-I contains a POU domain with a homeobox subdomain, Genes Dev., 2 (1988) 1582-1599. 15 Wilkinson, D.G., Bhatt, S., Chavrier, P., Bravao, R. and Charnay, P., Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse, Nature, 337 (1989) 461-464. 16 Wilkinson, D.G., Bhatt, S., Cook, M., BoncineUi, E. and Krumlauf, R., Segmental expression of Hox-2 homeoboxcontaining genes in the developing mouse hindbrain, Nature, 341 (1989) 405-409.
1 Altman, J., Morphological development of the rat cerebellum and some of its mechanisms. In S.L. Palay and V. Chan-palay (Eds.), Experimental Brain Research Supplementum 6, The Cerebellum New Vistas, Springer, Berlin, 1982, pp. 8-50. 2 Briggs, M.R., Kadonaga, J.T., Bell, S.P. and Tjian, R., Purification and biochemical characterization of the promoterspecific transcription factor, Spl, Science, 234 (1986) 47-52. 3 Dignam, J.D., Lebobitz, R.M. and Roeder, R.G., Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei, Nucleic Acids Res., 11 (1983) 1475-1489. 4 Dynan, W.S. and Tjian, R., The promoter-specific transcription factor Spl binds to upstream sequences in the SV40 early promoter, Cell, 35 (1983) 79-89. 5 Gorman, C., High efficiency gene transfer into mammalian cells. In D.M. Glover (Ed.), DNA Cloning- A Practical Approach. Vol. I, IRL Press, Oxford, 1982, pp. 143-190. 6 He, X., Treacy, M.N., Simmons, D.M., Ingraham, H.A., Swanson, L.W. and Rosenfeld, M.G., Expression of a large family of POU-domain regulatory genes in mammalian brain development, Nature, 340 (1989) 35-42. 7 Johnson, W.A. and Hirsh, J., Binding of a Drosophila POUdomain protein to a sequence element regulating gene expression in specific dopaminergic neurons, Nature, 343 (1990) 467-470. 8 Kadonaga, J.T., Carner, K.R., Masiartz, 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.
161
BRESD 51295
Developmentally and regionally regulated alterations of octamer- and GC-box-binding activities during the postnatal development of mouse cerebellum Hiroaki Sakurai 1, Kaoru Kikuchi 1, Tomofusa Tsuchiya 1, Hiroshi Kanazawa 2 and Masaaki Tsuda 1 1Department of Microbiology, Facultyof PharmaceuticalSciences and 2Department of Applied Biology, Faculty of Technology, Okayama University, Tsushima-naka, Okayama 700 (Japan) (Accepted 2 April 1991)
Key words: Cerebellum; Cerebral cortex; Transcription factor; Gel mobility analysis; Octamer-binding protein; GC-box-binding protein
Gel-mobility analyses using a DNA probe containing the SV40 enhancer-promoter revealed that the DNA-binding activities of cerebellar extracts changed drastically during the postnatal development of mouse brain. Three major DNA-protein complexes formed on the 72-bp enhancer (complexes E-I, E-II and E-III) were detected in the extracts prepared from the 2- and 10-day-old mouse cerebellum, but two of them (complexes E-II and E-III) were not detected in 3- and 7-week-old mice. In contrast, the formation of the complexes E-II and E-III, but not E-I, was observed in the cerebral cortex extracts throughout postnatal development until at least 7 weeks after birth. The addition of the octamer motif as a competitor abolished the formation of these three complexes. On the other hand, the formation of DNA-protein complexes on the 21-bp promoter by the cerebellar extracts increased after birth, while this increase in the formation of the complexes was not detected in the cerebral cortex extracts. This complex formation was found to be dependent upon the GC-box and to be stimulated by the addition of Zn 2÷, indicating that the protein binding to the 21-bp promoter may contain intramolecular zinc fingers. Thus, the expression of the octamer- and the GC-box-binding proteins are developmentally and regionally controlled in the cerebellum during the postnatal development of the mouse brain.
INTRODUCTION The development of the vertebrate brain proceeds in a regionally controlled fashion, and results in the formation of many brain regions, whose morphologies and functions are distinct from each other. Recently, the expression of some transcriptional factors containing the h o m e o d o m a i n or zinc fingers were found to be segmentally regulated in the rat and in the mouse hindbrain by in situ hybridization 11'15'16. It has also been reported that a large family of regulatory proteins containing the P O U - d o m a i n , which is known to be shared by the octamer-binding proteins Oct-1 and Oct-2, the pituitary transcription factor Pit-1 and the nematode homeotic unc-86 gene product, were shown to be expressed throughout the development of rat brain 6. On the other hand, several lines of evidence obtained from studies of Drosophila melanogaster have suggested that some transcriptional factors containing the P O U - d o m a i n or zinc fingers may play a key role in determining neuronal cell
differentiation 7'1°. Such neuronal cell differentiation is thought to be differentially regulated in each brain region. In order to understand the developmental program of formation of the mammalian central nervous system (CNS), therefore, it is important to investigate the expression of transcriptional factors during the development of the brain in relation to the regionality of the brain. The development of the rodent cerebellum proceeds postnatally, and the formation of neuronal circuits between neurons in the cerebellum continues after birth 1. During this postnatal development of rodent cerebellum, a number of genes responsible for the formation of the cerebellum may be expressed and their expression regulated by several kinds of transcriptional factors. To survey possible alterations in the expression of transcriptional factors during the development of the mouse cerebellum, we have used the gel-mobility assay because of its convenience for detecting changes in specific DNA-binding activities of transcriptional factors.
Correspondence: M. Tsuda, Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama 700, Japan. Fax: (81) (862) 55 7456.
162 MATERIALS
AND METHODS
DNA fragments as probes The Pvull-NcoI DNA fragments (233 bp) containing the SV40 enhancer and promoter were cut out from pSV2CAp plasmid DNA. Oligonucleotides (E72, P21, PI, P2, P3, P4, OCT, TRE, P3m1, P3m2, P3m3 and NS; see Fig. 1) were synthesized by a DNA synthesizer (MilliGen/Biosearch), purified by polyacrylamide gel electrophoresis and annealed with the counter oligonucleotides, respectively. DNA probes were end-lab&led with [32P]dCIY by DNA polymerase I (Klenow fragment), and purified by Sephadex G-50 column chromatography. Preparation of nuclear extractsfrom brain The nuclear extracts were prepared from cerebral cortexes and cerebella of mice according to the procedure of Dignam et al.3 with a slight modification. Brains were removed from mice 2, IO,21 and 49 days after birth, and the cerebral cortexes and cerebella were cut out from the brain. After washing them with phosphate-buffered saline (PBS), the tissue was homogenized for 10 strokes in a Dounce B-type homogenizer with 1 ml Buffer A containing 10 mM Tris-HCl (pH 7.9), 1.5 mM MgCl,, 10 mM KCI, 0.5 mM dithiothreitol (DTI’). The cells were collected by the centrifugation at 2000 rpm for 10 min, suspended in 5 ~01s. of Buffer A and allowed to stand on ice for 15 min. After centrifugation, the pellets were again suspended with 2 vols. of Buffer A and homogenized for 10 strokes. After washing the pellets with 2 ~01s. of Buffer A twice, the nuclei were collected by centrifugation at 15,000 rpm for 20 min at 4 “C, suspended in an equal volume of Buffer B containing 20 mM Tris-HC1 (pH 7.9), 25% glycerol, 420 mM KCl, 1.5 mM M&h, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM phenyhnethylsulfonyl fluoride (PMSF) and homogenized by 20 strokes. The suspension was kept on ice for 1 h, followed by centrifugation at 15,000 rpm for 20 min. The supematant was dialyzed against the Buffer C containing 20
mM Tris-HCl (pH 7.9), 20% glycerol, 100 mM KCI, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF for 12 h. After centrifugation at 15,000 rpm for 15 min at 4 “C, the supernatant was stored at -80 “C Transfectionexperiments in cultured brain cells Cerebral cortexes or cerebella of l-week-old mice were minced by scissors and treated with a solution containing 0.125% trypsin (Gibco) and 1 mM EDTA for 20 min at 37 “C. After culturing for 2 days in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, transfection with 8 yg of the pSV2CAT or pAlOCAT plasmid DNA per m-mm culture dish (Corning) was carried out by the calcium-phosphate co-precipitation method as described by Gorman5. The pSV2CAT plasmid contains both the 72-bp repeats and the 21-bp repeats, but the pAlOCAT plasmid only the 21-bp repeats’. After transfection for about 16 h and prolonged culture for 2 days, cytoplasmic extracts were prepared and their CAT activities were measured by the method described by German’. Gel-mobiliry assay DNA-protein binding was performed in the reaction mixture (20 ~1) containing 20 mM Tris-HCl (pH 7.5), 1 mM D’IT, 1 mM EDTA, 100 mM NaCl, 50 mM KCI, 2pg poly[dI-dC] (Sigma), 10% glycerol, 0.1-l ng 32P-labelled DNA probe and nucfear extract (3-10 pg protein) for 30 min at 20 “C. When the effect of Zn2’ on the DNA-protein complex formation was observed, an incubation in the presence of 1 mM ZnCl, for 15 min on ice was carried out and the DNA-protein binding reaction was started by the addition of the DNA probe. The DNA-protein complexes were resolved on a 4% polyacrylamide gel (3O:l cross-linking ratio) containing 67 mM Tris-HCI (pH 7.5), 33 mM sodiumacetate, 2.5% glycerol and 1 mM EDTA. Electrophoresis was carried out at 11 V/cm for about 2 h in the cold room. After electrophoresis, the gel was f?xed with 10% acetic acid and 10% methanol, dried and autoradiogaphed. A gel-mobility competition assay was performed in the presence of competitor DNAs at approximately 400-fold excess. DNase I protection analysis The DNA binding reaction was performed in the reaction mixture (50~1) containing 20 mM Tris-HCl (pH 7.5), 6.5 mM MgCh, 50 mM KCl, 0.5 mM DlT, 0.4 mM EDTA, 10% glycerol, 0.5% polyethyleneglycol, 1 ,ug poly[dI-dC], 0.8 pg 32P-labelled DNA probe and nuclear extract (approximately 25 pg). After the incubation for 15 min at room temperature, 2 U of DNase I (Sigma) were added with 50 ~1 of 10 mM MgCI, and 5 mM CaC&, and the mixture was incubated for 1 min at room temperature. After extraction of DNA with phenol-chloroform and precipitation of DNA with ethanol, DNA was subjected to denaturing polyacrylamide gel electrophoresis.
RESULTS
OCT P3ml P3m2 P3m3 TRE NS
Fig. 1. Organization of the SV40 enhancer-promoter and several motifs used as probes and competitors for the gel-mobility assay. The top line shows the structure of the SV40 early promoter containing the 72-bp and the 21-bp repeats. The black boxes indicate the original DNA sequences included in the SV40 enhancer-promoter used as probes or competitors (EP.233, E72, P21, Pl, P2, P3, P4, OCT, P3m1, P3n12, P3m3 and TRE). The open boxes included in the DNA fragments indicate the non-specific DNA sequences (NS). The locations of the several DNA motifs on the SV40 enhancer-promoter which were already investigated’2*‘3 are shown. TRE means the TPA (12-0-tetradecanoyl phorbol-13acetate) responsive element.
Gel-mobility patterns of SV40 enhancer-promoter Since the SV40 enhancer-promoter has several DNA-
protein binding motifs 12,13(Fig. l), we expected to detect changes in specific DNA-binding activities if the expression of DNA-binding proteins related to the SV40 enhancer (72-bp repeats) -promoter (21-bp repeats) were postnatally altered in mouse brain. Tram&&ion experiments of pSV2CAT and pAlOCAT plasmid DNAs using cultured primary cerebeILar and cerebral cortex cells prepared from l-week-old mouse brain (Fig. 2) revealed that the 72-bp enhancer can enhance transcriptional efficiency in both the cultured cerebral cortex (lanes 1 and 3) and cerebellar (lanes 2 and 4) cells, suggesting the
163
MAc-CM
CM-~
1
2
pSV2CAT
3
4
pAl 0CAT
Fig. 2. Transfection experiments of the cultured cerebellar and cerebral cortex cells. The procedure of transfection is described in Materials and Methods. pSV2CAT (lanes 1 and 2) or pA10CAT (lanes 3 and 4) plasmid DNAs were transfected to the cultured cerebral cortex (lanes 1 and 3) or the cerebeUar (lanes 2 and 4) cells. The positions of monoacetylated [14C]chloramphenicol and [14C]chloramphenicol on the thin-layer chromatography are shown as MAc-CM and CM, respectively.
presence of proteins which are able to bind to the SV40 enhancer in these cells. Using the EP233 probe (233 bp) containing both the SV40 e n h a n c e r - p r o m o t e r (Fig. 1), we performed gelmobility experiments using the cerebellar and cerebral
cortex extracts which were prepared from 2- and 10-day-, and 3- and 7-week-old mice. As shown in Fig. 3, several D N A - p r o t e i n complexes (complexes EP-I - EP-VI) were distinctly recognized according to the differences in their gel mobilities. From the extracts prepared from the cerebral cortexes (Fig. 3, lanes 1-4), the complexes EP-IV, EP-V and E P - V I were clearly detected, and their gel-mobility patterns were not altered from 2 days to 7 weeks after birth. In contrast, the gel-mobility patterns of the cerebellar extracts (Fig. 3, lanes 5-8) drastically changed during postnatal development, in which the complex EP-IV, EP-V and E P - V I were detected until 10 days but not at 3 or 7 weeks after birth, while the complexes EP-I and E P - I I were clearly detected 3 and 7 weeks after birth. The complex EP-III, which was not detected in the cerebral cortex extract, appeared to be present throughout the development of the cerebellum from 2 days to 7 weeks after birth. To determine whether the complexes could be formed depending upon the 72-bp enhancer or the 21-bp promoter of the SV40 D N A , we carried out gel-mobility experiments in the presence of the 72-bp or 21-bp D N A fragments as competitors. W h e n the 72-bp enhancer D N A fragment (E72) was added as a competitor (Fig. 4, lane 2), the complexes EP-III, EP-IV, EP-V and EP-VI
Cerebral cortex Age :
2d
10d
3w
Cerebellum 7w
2d
10d
3w
7w
5
6
7
8
EP -I
EP-II EP-III
]b.
~,~
E P - I V ~,~ E P - v ~,-
EP-VI ~,-
lb.
1
2
3
4 Probe : EP233
Fig. 3. Gel-mobility experiments using the PvuII-NcoI DNA fragment (233 bp) as a probe. Nuclear extracts prepared from the cerebral cortexes (lanes 1-4) and cerebella (lanes 5-8) of 2-day- (lanes 1 and 5), 10-day- (lanes 2 and 6), 3-week- (lanes 3 and 7) and 7-week-old (lanes 4 and 8) mice were added to the reaction mixture at the amount of 5 (lanes 1 and 5), 6 (lanes 2 and 6), 9 (lanes 3 and 7) and 9 (lanes 4 and 8) /zg protein, respectively. EP233 shown in Fig. 1 was used as a probe. The age of the mice after birth (2 and 10 days, 3 and 7 weeks), which were used for preparation of the extracts, are shown at the top of the figure, designated as 2d, 10d, 3w and 7w, respectively.
164 were competed, but when the 21-bp promoter D N A fragment (P21) was added as a competitor, the complexes EP-I and EP-II were competed (lane 3). These results indicate that the complexes EP-I and EP-I1 were formed on the 21-bp promoter, and the complexes EP-III, EP-IV, EP-V and EP-VI on the 72-bp enhancer.
Gel-mobility patterns of the 72-bp enhancer Using the 72-bp enhancer D N A fragment (E72) as a probe, we compared the gel-mobility patterns of the extracts prepared from the cerebral cortex and the cerebellum (Fig. 5). The complex E-I was detected only in the cerebellar extracts throughout the development until 7 weeks after birth (Fig. 5, lanes 5-8), but not in the cerebral cortex extracts (lanes 1-4). The complexes E-II and E-III were detected in the cerebral cortex extracts throughout the development (lanes 1-4), while in the cerebellar extracts they were detected only until 10 days after birth but not from 3 weeks (lanes 5-8).
Competitors:
EP-I
~,~
EP-II EP-III
~b. ~,~
--
E72
P21
The complex formation is competed by the octamer motif To identify what motifs on the 72-bp enhancer contribute to the formation of the complexes E-I, E-II and E-III shown in Fig. 5, we performed competition experiments using several D N A sequences included in the 72 bp enhancer as competitors (Fig. 6). As shown in Fig. 6A, addition of the P4 D N A fragment competed the formation of the E-I, E-II and E-III complexes most severely (lane 4). Addition of the P2 or P3 D N A fragment showed a weaker competition (lanes 3 and 5) but addition of the P1 D N A fragment did not show any competition (lane 2). Since the P2 and P4 D N A fragments contain the octamer motif, we examined competition effects of the addition of OCT D N A fragments on the complex formation. As shown in Fig. 6B, the formation of the complexes E-I, E-II and E-III was abolished in the presence of the OCT competitors (compare lane 11 with lane 10). When the P4 probe was used (Fig. 6C), complexes P4-I, P4-II and P4-III were detected (lanes 12 and 15), but the addition of the OCT D N A fragments as competitors abolished the formation of these complexes (lanes 13 and 16). These results indicate that all the complex formation on the 72-bp enhancer depend upon the octamer motif. The complex P4-I was formed with a higher efficiency in the cerebellar extract than in the cerebral cortex extract, while the complexes P4-II and
Cerebral cortex
Cerebellum
Age: 2d lOd 3w 7w
2d
lOd
3w
6
7
7w
EP-IV E-
EP-V
E-I E-II
E P-VI
1
2
3 1
Probe :
2
3
4
5
8
EP233
Fig. 4. Competition experiments by the 72-bp or 21-bp DNA sequences for the gel-mobility experiment using the EP233 probe. The gel-mobility experiment was carried out using the total brain extracts of 2-week-old mice and the PvuII-NcoI 233-bp DNA fragment (EP233) as a probe. No competitors (lane 1), and a 400-fold excess of the 72-bp (E72; lane 2) or the 21-bp (P'21; lane 3) DNA fragments as competitors were added with the EP233 probe.
Probe : E72
Fig. 5. Gel-mobility experiments using the E72 probe. Nuclear extracts prepared from the cerebral cortexes (lanes 1-4) and the cere~lla (lanes 5-8) were added at the amount of 5 (lanes I and 5), 6 (lanes 2 and 6), 9 (lanes 3 and 7) and 9 (lanes 4 and 8)/~g protein. The 72-bp DNA sequence (E72) was used as a probe. The age of the mice from which the extracts were prepared are shown at the top of the figure. The non-specific bands are shown as ns.
165 C Cerebral cortex Competitors :
-
P1
P2
P4
P3 P3ml P3m2 P3m3 NS
--
OCT
-
Cerebellum
OCT NS
--
OCT NS
P 4 - [ ~'E-I~"
P4- I I ~,P 4 - I I I ~,-
E -I~"
E-II~ E-III I,-
E-Ill-
E-III J,nsiP-
ns~
F~-
F=,, 1
2
Probes :
3
4
5
6
7
8
9
F 10
E72
11
12
13
14
E72
15
16
17
P4
Fig. 6. Competition experiments using several DNA sequences included in the 72-bp enhancer. A and B. Gel-mobility experiments using the total brain extract prepared from 2-week-old mice and the 72-bp DNA sequences (E72) as a probe were carried out in the absence of competitors (lanes 1 and 10) or in the presence of a 400-fold excess of the several competitors, i.e. P1 (lane 2), P2 (lane 3), P4 (lane 4), P3 (lane 5), P3ml (lane 6), P3m2 (lane 7), P3m3 (lane 8), NS (lane 9) and OCT (lane 11), shown in Fig. 1. C. Gel-mobility experiments using cerebral cortex (lanes 12-14) and cerebellar (lanes 15-17) extracts prepared from 3-week-old mice and the P4 probe were carried out in the absence of competitors (lanes 12 and 15) or in the presence of OCT (lanes 13 and 16) or NS (lanes 14 and 17).
P 4 - I I I were strongly d e t e c t e d in the c e r e b r a l cortex extract (Fig. 6C). Since b o t h extracts were p r e p a r e d f r o m the b r a i n s of 3 - w e e k - o l d mice, the c o m p l e x e s P4-I, P4-II a n d P 4 - I I I s h o u l d c o r r e s p o n d to the c o m p l e x e s E - I , E - I I a n d E - I l l , respectively, given b y the E 7 2 p r o b e .
the P3 D N A f r a g m e n t (Fig. 1) s h o w e d a slight c o m p e tition with the c o m p l e x f o r m a t i o n (Fig. 6 A , lanes 7 a n d 8). T h e s e results i n d i c a t e that the 3" s e q u e n c e s a d j a c e n t to t h e o c t a m e r m o t i f c o n t r i b u t e , at least in part, to the f o r m a t i o n of the c o m p l e x e s o n the 72-bp e n h a n c e r .
W h e n the P 3 m l D N A f r a g m e n t whose m u t a t i o n s w e r e l o c a t e d 5 n u c l e o t i d e s f r o m the 5" e n d of the P3 f r a g m e n t (Fig. 1), was a d d e d as a c o m p e t i t o r , n o c o m p e t i t i o n was o b s e r v e d (Fig. 6 A , l a n e 6), while a d d i t i o n of the P 3 m 2 a n d P 3 m 3 w h o s e m u t a t i o n s were l o c a t e d f u r t h e r inside
Zinc effect on the complex formation promoter
on the SV40
Since the f o r m a t i o n of the c o m p l e x e s E P - I a n d E P - I I was a b o l i s h e d by the 21-bp p r o m o t e r as a c o m p e t i t o r
TABLE I
DNA-protein complexes detected by gel-mobility assays Detection of the bands obtained by the gel-mobility experiments using E72 (Fig. 5), P4 (Fig. 6C), P21 (Fig. 8) and TRE (Fig. 9) as probes were summarized as follows: - , not detected; +, detected; + + and + + +, strongly detected.
Motifs
Probes
Complexes
Cerebral cortex 2d
Octamer
E72
P4
GC-box
P21
TRE
TRE
E-I E-II E-Ill P4-I P4-II P4-III P-I P-II T-I
. + +
+ + +
Cerebellum
lOd .
. + +
+ + +
3w
7w
2d
lOd
3w
7w
+ +
+ +
+ + +
+ + +
+ _ _
+ _
.
-
+
+ + + + ++
_ _ +++ +++ ++
+ + +
+ + +
++ ++ +
_
++ ++ +
166 C Zn 2+ .
4-
+
--
--
+
+
-
EDTA
-
+
+
--
--
+
4"
Extract
fl 1
Probes •
2
3
4
5
6
EP233
7
P21
8
9
10
P21
Fig. 7. Effects of Zn 2+ on the DNA-protein complex formation on the 21-bp DNA sequences (P21). A and B. Gel-mobility experiments using EP233 (lanes 1-4) or P21 (lanes 5-8) as a probe were carried out in the absence of Zn 2+ and 1 mM EDTA (lanes 1 and 5) or in the presence of Zn 2+ (lanes 2 and 6), Zn 2+ plus 1 mM EDTA (lanes 3 and 7) and 1 mM EDTA (lanes 4 and 8). C. DNase 1-protection analysis using the P21 probe was carried out in the absence (lane 9) or the presence (lane 10) of the extract. The eerebellar extract prepared from 3-week-old mice was used for all experiments.
Cerebral cortex Age:
2d
10d
3w
Cerebellum
7w
2d
10d
3w
7w
4
5
6
7
8
p.
P-I
1
2
3
Probe : P21
Fig. 8. Gel-mobility experiments using the P21 probe. The gelmobility experiments using the P21 probe with the cerebral cortex (lanes 1-4) and the eerebellar (lanes 5-8) extracts were carried out in the presence of Zn 2+. The ages of the mice from which the extracts were prepared are shown at the top of the figure.
(Fig. 4, lane 3), we examined whether the formation of the complexes EP-I and E P - I I requires Z~n2+ because Spl DNA-binding proteins which involve zinc fingers intramolecularly are known to bind to the GC-box of the SV40 promoter s. W h e n Zn 2+ was added: to the D N A protein binding reaction mixture, the formation of the complexes EP-I and E P - I I in the cerebellar extract was greatly stimulated (Fig. 7A, compare lane 2 with lane 1). A simultaneous addition of E D T A with Zn 2+ suppressed the stimulation of the complex formation (lane 3) and an addition of E D T A alone did not affect the complex formation (lane 4), indicating that the efficiency of the formation of the complexes EP-I and E P - I I depends upon the presence of Zn 2+ in the reaction mixture. The stimulation of the complex formation by Zn 2+ was also supported by the experiment using the P21 probe for the gel-mobility assay (Fig. 7B). Two major complexes P-I and P-II were stimulated in the presence of Zn 2+ (compare lane 6 with lane 5), but suppressed by the addition of E D T A (lane 7). DNase-I-protection analysis of the 21-bp p r o m o t e r sequences in the cerebellar extract revealed the presence of proteins preferentially binding to the 3" half GC-box (Fig. 7C, compare lane 10 with lane
9).
167 Cerebral cortex Age:
2d
lOd
3w
Cerebellum 7w
2d
lOd
3w
7w
5
6
7
8
}1..-
1
2
3
4 Probe : TRE
Fig. 9. Gel-mobility experiments using the TRE probe. Gel-mobility experiments using the TRE probe with the cerebral cortex (lanes 1-4) and the cerebellar (lanes 5-8) extracts were carried out.
Developmental increase of the complex formation on the 21-bp promoter To confirm the alteration of the DNA-binding activities to the 21-bp promoter during the postnatal development of the cerebellum as shown in Fig. 3, we performed gel-mobility experiments of the 21-bp promoter sequences in the presence of Zn 2+. As shown in Fig. 8, the formation of the complexes P-I and P-II in the cerebellar extracts increased after birth (lanes 5-8), while such an increase in the formation of the complexes after birth was not observed in the cerebral cortex extracts (lanes 1-4).
Gel-mobility patterns of TRE As shown in Fig. 9, the gel-mobility patterns of TRE were similar between the cerebral cortex and the cerebellum, although the increase of the TRE binding activities was observed with the extracts prepared from the 3-week-old mice. This result indicates that the expression of the TRE-binding proteins could be similarly regulated in both brain regions and different from those of the octamer- and the GC-box-binding proteins. DISCUSSION
Using gel-mobility assays, we have demonstrated that the octamer and the GC-box binding activities expressed in the cerebellum changed in a different manner from those in the cerebral cortex during the postnatal development of the mouse brain (Table I). We have found that 3 major DNA-protein complexes (E-I, E-II and E-Ill, see Figs. 5 and 6) were formed by the brain nuclear
extracts depending upon the octamer motif. Since the complex E-I was also detected by the extracts prepared from liver and kidney of 2-week-old mice (data not shown), the proteins responsible for the formation of the complex E-I could be the Oct-1 protein because Oct-1 is believed to be ubiquitously expressed in several tissues ~4. The E-II and E-III complexes were not formed by the extracts prepared from liver and kidney (data not shown), suggesting that the proteins responsible for the formation of E-II and E-III are expressed in a brainspecific manner. Recently, using in situ hybridization, He et al. 6 demonstrated that several proteins containing the POU-domain, which is widely accepted as a domain responsible for specific DNA-binding, were found to be expressed in rat brain in a spatially and temporally regulated fashion. Thus we can speculate that the proteins responsible for the formation of the complexes E-II and E-III might correspond to at least some of them. Some DNA-binding proteins other than the octamerbinding proteins could, at least in part, contribute to the formation of the complexes E-I, E-II and E-III, because the sequences downstream from the octamer motif were slightly competitive to the formation of the E-I, E-II and E-Ill complexes (Fig. 6A). So it is possible that some interactions between the octamer- and its 3" adjoining region-binding proteins could regulate the formation of the E-I, E-II and E-III complexes. Two major complexes, P-I and P-II, were found to be formed on the 21-bp promoter of SV40 DNA in the cerebellar extracts (Fig. 7). The formation of these complexes increased after birth, while such an increase in the complex formation was not detected in the cerebral cortex extracts (Fig. 8). Since the addition of Zn 2÷ stimulated the formation of the complexes P-I and P-II (Fig. 7), the proteins responsible for their formation should contain intramolecular zinc fingers. The Spl protein is known to be the GC-box-specific binding protein z'4 and to involve such zinc fingers 8. In addition, Spl is known to consist of 2 polypeptides of 105 and 95 kDa, both of which have the ability to bind to the GC-box z, suggesting that the complexes P-I and P-II were formed by these Spl-like proteins. Recently, evidence has been accumulating that the transcriptional factors intramolecularly containing the POU-domain or the zinc fingers are expressed in the mammalian brain 6ALAS,16 or account for the differentiation of certain neuronal cell-types7'1°. Thus, it can be speculated that the expression of the Oct- and Spl-like proteins in the cerebellum may participate in the neuronal cell differentiation in the cerebellum. Identification of the cell types expressing the responsible genes by using the in situ hybridization may clarify the morphological aspects of the phenomena described in this study.
168 Acknowledgements. We thank Dr. Y. Suzuki for helpful comments on the manuscript. This research was supported in part by a
Grant-in-Aid for Special Project Research from the Naito Foundation, Tokyo.
REFERENCES
9 Laimins, L.A., Khoury, G., Gorman, C., Howard, B. and Gruss, P., Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 6453-6457. 10 Moses, K., Ellis, M.C. and Rubin, G.M., The glass gene encodes a zinc-finger protein required by Drosophila photoreccptor cells, Nature, 340 (1989) 531-536. 11 Murphy, P., Davidson, D.R. and Hill, R.E,, Segment-specific expression of a homeobox-containing gene in the mouse hindbrain, Nature, 341 (1989) 156-159. 12 Nomiyama, H., Fromental, C., Xiao, J.H. and Chambon, P., Cell-specific activity of the constituent elements of the simian virus 40 enhancer, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7881-7885. 13 Rosales, R., Vigneron, R., Macchi, M., Davidson, I., Xiao, J.H. and Chambon, P., In vitro binding of cell-specifiC and ubiquitous nuclear proteins to the octamer motif in the SV40 enhancer and related motifs present in other promoters and enhancers, EMBO J., 6 (1987) 3015-3025. 14 Strum, R.A., Das, G. and Herr, W., The ubiquitous octamerbinding protein Oct-I contains a POU domain with a homeobox subdomain, Genes Dev., 2 (1988) 1582-1599. 15 Wilkinson, D.G., Bhatt, S., Chavrier, P., Bravao, R. and Charnay, P., Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse, Nature, 337 (1989) 461-464. 16 Wilkinson, D.G., Bhatt, S., Cook, M., BoncineUi, E. and Krumlauf, R., Segmental expression of Hox-2 homeoboxcontaining genes in the developing mouse hindbrain, Nature, 341 (1989) 405-409.
1 Altman, J., Morphological development of the rat cerebellum and some of its mechanisms. In S.L. Palay and V. Chan-palay (Eds.), Experimental Brain Research Supplementum 6, The Cerebellum New Vistas, Springer, Berlin, 1982, pp. 8-50. 2 Briggs, M.R., Kadonaga, J.T., Bell, S.P. and Tjian, R., Purification and biochemical characterization of the promoterspecific transcription factor, Spl, Science, 234 (1986) 47-52. 3 Dignam, J.D., Lebobitz, R.M. and Roeder, R.G., Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei, Nucleic Acids Res., 11 (1983) 1475-1489. 4 Dynan, W.S. and Tjian, R., The promoter-specific transcription factor Spl binds to upstream sequences in the SV40 early promoter, Cell, 35 (1983) 79-89. 5 Gorman, C., High efficiency gene transfer into mammalian cells. In D.M. Glover (Ed.), DNA Cloning- A Practical Approach. Vol. I, IRL Press, Oxford, 1982, pp. 143-190. 6 He, X., Treacy, M.N., Simmons, D.M., Ingraham, H.A., Swanson, L.W. and Rosenfeld, M.G., Expression of a large family of POU-domain regulatory genes in mammalian brain development, Nature, 340 (1989) 35-42. 7 Johnson, W.A. and Hirsh, J., Binding of a Drosophila POUdomain protein to a sequence element regulating gene expression in specific dopaminergic neurons, Nature, 343 (1990) 467-470. 8 Kadonaga, J.T., Carner, K.R., Masiartz, 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.