Descripción de especies dominantes identificadas

The difference between Oct-2.5 and other Oct-2 isoforms is mainly that the Oct-2.5 molecule lacks the strong C-terminal activation domain present in other isoforms of Oct- 2(Wirth et al., 1991; chapter 1.3.2). It is interesting to test the ability o f other Oct-2 forms to interfere with transactivation by activation domains. In particular the effect o f Oct-2.2 on activation by the VP 16 acidic domain in BHK-21 cells was tested. Oct-2.2 was able to significantly repress transactivation by the VP 16 acidic domain(Figure III.6), even though it was less effective than Oct-2.5(compare Figure III.2 with Figure III.6). The region(s) required for transcriptional inhibition by Oct-2.2 was examined by applying a series of plasmids expressing N and C-terminal deleted derivatives of Oct-2.2( Figure III.5, Muller-Immergluck et al 1990, Gerster et al., 1990). As illustrated in figure III.6 progressive deletion of the N-terminus of Oct-2.2 leaving the C-terminus intact resulted in a progressive reduction in the ability to inhibit transactivation by the VP 16 acidic domain until a construct lacking the first 161 amino acids of Oct-2.2 was unable to inhibit activation. Interestingly however, progressive deletion of the C-terminus o f Oct-2.2 removing the activation domain resulted in an increase in the ability to inhibit transactivation by the VP 16 acidic activation domain. Thus constructs C370 and C357 containing the entire N-terminus but with almost the entire C-terminus deleted showed a stronger ability to inhibit transactivation than the full length Oct-2.2 construct. As

OCT-2.2 X55 X99 POU 99 X136 C460 C427 C392 C370 C344 N154C376 LEU-Z _P479

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•** -4. 460 427 392 370 357 344 376

Figure III.5. Schem atic d iag ram of a series of Oct-2.2 deleted d erivativ es(M uller- Im m erg lu ck et al., 1990, G e rster et al., 1990).

The glutamine-rich activation domain, the repressor domain, the POU DNA-binding domain the leucine-zipper domain and the proline-rich activation domain o f Oct-2.2 are indicated.

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250-1 2 0 0- 150- 1 0 0- 5 0 -

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Figure III.6. Inhibition of Gal4-VP16 acidic activation domain fusion protein mediated trans-activation by the deleted derivatives of Oct-2.2.

CAT assay results of co-transfecting BHK cells with 10 pg of 03G5E1BCAT plasmid and 5 pg Gal4-VP16 plasmid in the presence of 10 pg of the empty expression vector pJ7 or each o f the deleted derivatives of Oct-2.2(as indicated in figure IIL5). All values are represented relative to the degree of trans-activation by Gal4-VP16 acidic activation domain fusion protein in the presence of the empty vector pJ7(set at 100%). The results are expressed as the mean ± SD of at least two independent transfection experiments after normalization.

expected the ability to inhibit was lost upon further C-terminal truncation to amino acid 344 since this truncation disrupts the POU domain and would interfere with DNA binding. A construct called N154C376, containing the minimal repressor domain defined in previous studies(Lillycrop et al., 1994a) and POU domain showed strong inhibition(Figure III.6 and Figure III.7). The results indicated that the Oct-2 repressor domain(located between 142-181 amino acids) would be responsible for inhibition of transactivation in the absence of the C-terminal activation domain.

To map the region(s) responsible for the repression by Oct-2.5, a number o f constructs expressing truncated Oct-2 were assayed for their inhibition potential. The same experimental protocol was used to test the inhibitory effects in BHK cells o f a construct called Oct2.2(2-357), encoding the N-terminal region of Oct-2 and the central POU domain and lacking any C-terminal sequences(derived fi-om Oct-2.2, Muller-Immergluck et al., 1990), a construct called N154C376, containing the minimal repressor domain and POU domain(Gerster et al., 1990) and a construct called Oct2POU, expressing only the POU domain of Oct-2(Lillycrop et al 1994a)(see figure III.l). As illustrated in figure III.7, the isolated POU domain showed no inhibitory effect on transactivation by the VPljS activation domain whereas the minimal repressor domain linked to the POU domain as well as Oct-2.5 and the entire N-terminus of Oct-2 linked to the POU domain showed strong inhibition. These findings suggested that the Oct-2 repressor domain would be responsible for inhibition of transactivation by Oct-2.5.

Next, the ability of the N-terminus of Oct-2 to interfere with transactivation by various activation domains was tested. In these experiments(figure III.8), the construct Oct2.2(2- 357) was able to strongly inhibit transactivation by acidic domains as well as by the HOB motif of c-Fos parallelling the ability of Oct-2.5 to do this. Interestingly, especially in the case of the acidic domain from VP 16 the effect of the N-terminus was much stronger than that of Oct-2.5, resulting in almost total repression of transactivation. The N-terminus significantly repressed transactivation mediated by proline-rich activation domains of CTF/NFl and AP2 as well as by the glutamine rich activation domain of SPI (Figure

G al4(l-147 Gal4-VP16 PJ7 + PJ7 + Oct-2.5 + POU + Oct-2.2 (2-357) + N154C357

Figure III.7. The N -term inal rep ressor dom ain is responsible for inhibition by Oct- 2.5.

A representative CAT assay o f BHK cells transfected with the 03G 5E1B C A T reporter with the Gal4-VPl 6 construct in the presence o f the empty expression vector pJ7, Oct-2.5 expression vector, the isolated POU domain expression plasmid, the Oct-2.2(2-357) construct and the N154C357 construct(as indicated in figure III.l). The Gal4-VP16 acidic activation domain fusion protein can strongly activate the reporter compared to the Gal4 DNA-binding domain alone. Oct-2.5, the N-terminus linked to the POU domain(Oct-2.2 2-357) and the minimal repressor domain linked to the POU domain(N 154C376) but not the isolated POU domain showed strong inhibition o f trans-activation by Gal4-VP16.

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200 n 1 5 0 - 10 0- 50- ■ OCT2.2(2-357) H OCT2-POU

Figure III.8. Inhibition o f trans-activation in BHK cells by the N-terminus of Oct-2 or the isolated Oct-2 POU domain.

CAT assay results of co-transfecting BHK cells with 10 }ig of 03G5E1BCAT plasmid and 10 pg of each of GaJ4-activation domain fusion constructs(as indicated in figure IIL1.)( in the case of Gal4-VP16 only 5 pg plasmid was used) in the presence of 10 pg of the empty expression vector pJ7 or the Oct-2.2(2-357) expression vector or the isolated Oct-2 POU domain(as indicated in figure III.l). In each case the degree o f trans- activation of the 03G5E1BCAT construct is compared in the presence of Oct-2.2(2-3 57) expression vector or the POU expression vector to that observed in the presence of the empty vector pJ7(set at K00%). The results are expressed as the mean ± SD of at least two independent transfection experiments after normalization.

III.8). In all cases only very weak or no inhibition was observed with Oct2POU indicating that the inhibitory effects are dependent upon the N-terminus of Oct-2. Hence the entire N-terminus is able to inhibit activation by a variety of activation domains of different classes, although some of these effects are masked within intact Oct-2.5.

111.2.3. The N-terminus of Oct-2 is not able to inhibit its own C-terminal activation domain

Interestingly however, the N-terminus of Oct-2 was not able to inhibit transactivation by its own C-terminal activation domain when the two regions are delivered to the DNA via separate DNA binding domains(Figure III.8). To investigate this effect further, a construct, in which the N and C-terminus of Oct-2.2 were fused to the Gal4 DNA binding domain so that both termini were located continuously on a single molecule not separated by the DNA binding domain(Figure III.9 Friedl and Matthias 1995), was used in this study. When this construct was co-transfected with a reporter plasmid containing five Gal4 binding sites cloned at position -105 relative to the transcriptional start site of the HSV thymidine kinase gene promoter(Madden et al., 1993), 6 fold transactivation was observed(figure III. 10). Hence the C-terminal activation domain can function in the presence of the N-terminus when both are linked in cis to the Gal4 DNA binding domain. In contrast much weaker activation was observed using reporter constructs in which the Gal4 sites had been removed to either -770 or +1000 relative to the transcriptional start site(Figure III. 10). This is in agreement with the weak stimulation of transcription produced when the Oct-2 C-terminal activation domain is bound at the enhancer position(Muller-Immergluck et al., 1990, Tanaka and Herr 1990) and the ability of the Oct-2 repressor domain to inhibit all three of these reporter constructs(Lillycrop et ah,

1994a).

111.2.4. The minimal N-terminal Oct-2 repressor domain is able to inhibit transactivation mediated by different classes of activation domains

OCT-2.2 EXPRESSION PLASMIDS: GAL0CT2N-C GAL4(l-93) GAL4(l-93) GAL4(l-93) POU REPORTER PLASMIDS: GAL-TK-CAT TK 5XGAL GAL+IOOO-TK-CAT CAT 5XGAL GAL-770-TK-CAT CAT TK -f-1000 5XGAL

Figure III.9. Effector and reporter gene constructs.

A schematic diagram of Oct-2.2 and the fusion protein in which the N and C terminus of Oct-2.2 (Friedl et ah, 1996)are fused to the Gal4 DNA-binding domain . The N-terminal glutamine-rich and the C-terminal proline-rich activation domains together with the POU DNA-binding domain are indicated. Three TK-CAT reporter plasmids with the Gal4 binding sites cloned at different positions are shown(Madden et ah, 1993).

u UJ u 1000 8 ’ 50 tu > 500 > 250

Figure III.IO. Trans-activation of the TK promoter by the Gal4-Oct-2N-Oct-2C fusion protein in BHK cells.

CAT assay results of co-transfecting BHK cells with 10 pg of each of the reporter constructs(Gal-TK-CAT, Gal-770-TK-CAT, Gal+lOOO-TK-CAT) and 10 pg o f Gal4- Oct-2N-Oct-2C expression plasmid or 10 pg of the Gal4 DNA-binding domain expression plasmid. Values are expressed relative to the degree of trans-activation in the presence of the Gal4 DNA-binding domain(set at 100%). The results are represented as the mean ± SD of at least two independent transfection experiments after normalization.

As described above, the repressor domain was able to inhibit transactivation by the VP 16 acidic activation domain on the reporter plasmid 03G5E1BCAT. Similar experiments were carried out to test the ability of the repressor domain to inhibit transactivation by other different activation domains. As shown in figure III. 11 and III. 12, the minimal repressor domain linked to its own DNA binding domain was able to significantly repress all the activation domains tested. In contrast to the entire N-terminus, this minimal N- terminal repressor domain even had some effect on the C-terminal activation domain of Oct-2. The inhibitory effect of the repressor domain on the thymidine kinase gene promoter, which contains targets for endogenous transcriptional activators(Jones et al., 1985), was also tested. The construct N154C376 was cotransfected with a construct in which the Oct-2 high affinity binding site (AT GOT AAT G AG AT) had been cloned upstream of the thymidine kinase promoter(Morris et al., 1994). A clear reduction in promoter activity was observed(Figure III. 13, III. 14). In addition, no inhibitory effect was observed on the promoter without the Oct-2 DNA-binding site(Figure III. 14).

III.2.5. The basal core promoter elements influence repression of the RNA

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