La configuración de la red ciclista

Figure 3.3 - interference of activation mediated by the Oct-2 C- terminai activation domain by the three Oct-2 N-terminal repressor domains in neuronal ND7 cells.

Data p resen ted of CAT assay results from co -tra n sfe ctin g ND7 cells w ith 5 |ig pT 7 G 5 -T A T A -C A T reporter plasm id, 5pg pG al4 -O ct-2C activa to r and 5 |ig o f one o f the three cloned O ct-2 N -term inal repressor dom ains. V = pTet"^ ve cto r w ith no N -term inal re p re sso r dom ain inserted. R D 1 , RD2 and RD3 = re le va n t Oct-2 N-term inal repressor dom ain cloned in fram e into pTet^ vector. All C A T activity is expresse d relative to the deg re e of reporte r fran s-activa tion observed w ith pG al4 -O ct-2C and the em pty pTet'^ vector (set at 100%). Data are the result o f three separate transfection expe rim e nts and are presented as the m ean ± SD.

Co-transfection experiments into BHK cells were performed exactly as described above for ND7 cells. Figure 3.4 shows that when co-transfected with the C-terminal activation domain, RD1 exhibits very weak but significant repression of the activation of the reporter gene construct

(f=9.79, p<0.0005). This is in broad agreement with the very weak

interference of activation by the C-terminal of Oct-2 previously reported (Liu et al. 1996). The RD2 and RD3 regions were still unable to interfere with the activation mediated by the Oct-2 C-terminal activation domain.

Finally, in order to rule out the possibility that the pTet^ - repression domain constructs themselves are responsible for the activation of the reporter construct these co-transfection experiments were repeated, substituting the pGal4 (1-93) construct, which expresses the Gal4 DNA binding domain alone (amino acids 1-93), for the pGal4-0ct-2C construct. These experiments revealed no activation what so ever of the reporter gene construct in either ND7 or BHK cell lines (data not shown).

These findings therefore suggest that neither the RD2 nor RD3 inhibitory domains are able to repress gene activation by the Oct-2 C-terminal

activation domain when delivered to the DNA in trans. This effect has

been demonstrated in both neuronal and non-neuronal cell types. In

agreement with previously reported findings the RDI inhibitory domain is able to repress the activation of the Oct-2 C-terminal inhibitory domain in a

cell specific manner when delivered to the DNA in trans (Liu et al. 1996).

These results raise the possibility that the three inhibitory domains function

by different mechanisms. These results obtained for RDI are in

agreement with previous findings where the RDI construct has been shown to interfere with the C-terminal Oct-2 activation domain in a cell specific manner (Liu et al. 1996).

o 300

< 200

EXPRESSED REPRESSOR DOMAIN

Figure 3.4 - Interference of activation mediated by the Oct-2 C- terminal activation domain by the three Oct-2 N-terminal repressor domains in BHK fibroblast cells.

Data presented of C AT assay results from co -tra n sfe ctin g BH K cells w ith 5|ag p T 7 G 5 -T A T A -C A T reporter plasm id, 5pg pG al4 -O ct-2C activa to r and 5 |ig o f one o f the three cloned O ct-2 N -term inal repressor dom ains. V = pTet^ ve cto r w ith no N -term inal re p re sso r dom ain inserted. RD1, RD2 and RD3 = releva nt O ct-2 N -term inal re pressor dom ain cloned in fram e into pTet^ vector. All C A T activity is expressed relative to the deg re e o f reporter fran s-activa tion observed w ith pG al4 -O ct-2C and the em pty pTet^ vector (set at 100%). Data are the result o f three separate tran sfectio n expe rim e nts and are presented as the m ean ± SD.

This cell specificity argues for the presence of a cell-specific factor in

neuronal cells. Such a factor may take the form of a co-repressor

molecule, which along with the RD1 region is able to disrupt the interaction of the C-terminal activation domain with the basal transcription complex. Alternatively the cell specific factor may directly interact with the basal transcription complex, for example with a specific TAP, blocking the interaction of the activation domain. Interestingly the cell-specific factor

may be an isoform of Oct-2 itself. It has been reported that the

predominantly expressed neuronal isoforms, Oct-2.4 and Oct-2.5 that lack the C-terminal activation domain, are able to functionally interact with Oct- 2.1 neutralising its activation ability (Lillycrop et al. 1994b). Such an effect

is mediated in trans where the activation and repression domains are

found on separate molecules. RDI has been shown to repress the C-

terminal Oct-2 activation domain in trans. It is likely that the repression of

the C-terminal activation domain by RD2 reported for in cis delivery to the

DNA (FriedI and Matthias, 1995) may be the result of a conformational arrangement which only occurs whilst both domains are on the same molecule. When the two domains are on separate proteins and delivery to

the DNA is in trans, no repression occurs.

3.2.4 - Investigation into the effect of the three Oct-2 N-terminal repressor domains on activation by other proline rich activation domains.

The C-terminal activation domain of Oct-2 belongs to the family of proline- rich activation domains. Other members of this family include AP2, the oncogene product Jun and the C-terminus of the CTF/NF1 factor (for review see Mitchell and Tjian, 1989). The CTF/NF1 factor is named for its

two separate cellular activities. The first activity allows the factor to

activate transcription of genes containing a CCAAT box (CTF = ÇCAAT

box transcription factor). Secondly these proteins can also activate

adenovirus DNA replication and have been show to be identical to nuclear factor 1 (NF 1). Hence the factor is referred to as CTF/NF1 (Jones et al.

1987).

Liu et al (1996) found that the activation by the C-terminal activation domain of CTF/NF1 was repressed to approximately 75% of control activation by full length Oct-2.5 in both ND7 and BHK cells. Likewise using RDI alone it was demonstrated that GTF/NF1 activation could be reduced to 50% of control activity in BHK cells (Liu et al. 1996). As described above (see 1.2.3), the basal transcription complex is assembled from a number of general transcription factors (GTFs) with whom the specific transcription factors interact to direct transcription. Amongst the GTFs, specific members of the IB P associated factors (TAFs) have been shown to interact directly with the different types of activation domain

(Chen et al. 1994). The mechanism of action of the RDI region is

proposed to be through interference of the activation domain's interactions with the basal transcription complex. In order to determine whether the two newly cloned Oct-2 N-terminal repressor domains (RD2 and RD3) are able to interfere with activation mediated by the proline-rich activation domain of CTF/NF1 transient transfection experiments were performed in BHK fibroblast cells. Thus the pT7G5-TATA-CAT reporter construct was co-transfected with a selected repressor domain expression construct and a pGal4-CTF/NF1 expression construct. The transiently transfected BHK cells were incubated for 72 hours and reporter gene activity was assayed as described (see 2.7.5).

Figure 3.5 shows that the transient transfection experiments performed with the RDI construct are not in agreement with the previously reported repression of the C-terminal of CTF/NF1 in BHK cells (Liu et al. 1996). In these experiments RDI significantly increases the activation mediated by CTF/NF1 approximately 1.5 fold (f=11.70, p<0.0005), whereas Liu et al. originally reported a 2 fold repression. The other two repressor domains tested in this assay against the proline-rich activation domain of CTF/NF1 both act as activators allowing the activation mediated by the CTF/NF1 activation domain to be increased to between two and three fold by the addition of either RD2 or RD3. Thus although all three tested repressor

300 H

EXPRESSED REPRESSOR DOMAIN

Figure 3.5 - Interference by the three Oct-2 amino-terminal repressor domains in BHK cells of activation mediated by the proline-rich CTF/NF1 C-terminal activation domain.

D ata p resen ted of C AT assay results from co -tra n sfe ctin g BH K cells w ith 5}ig T 7 G 5 -T A T A -C A T reporter plasm id, 5pg o f one o f the thre e cloned O ct-2 N -term inal repressor dom ains and 5pg pG al4-C TF/N F1 activator. V = pTet*^ ve cto r alone. RD1, RD2 and RD3 = relevant O ct-2 N -term inal re p re sso r dom ain cloned in fram e into pTet^ vector. All C A T activity is expressed relative to the degree o f re p o rte r fran s-activa tion observed w ith pG al4-C TF/N F1 and the em pty pTet^ ve cto r (set at 100% ). Data are the result o f thre e separate transfection expe rim e nts and are presented as the m ean ± SD.

Two other major families of activation domains have been identified, those rich in acidic residues and those rich in glutamine residues.

3.2.5 - Investigation into the effect of the three Oct-2 N-terminal repressor domains on activation by acidic activation domains.

Acidic activation domains have also been identified in a number of

different proteins. For example in yeast the galactose-dependant

activator protein Gal4 (Ma and Ptashne, 1987), and the GCN4 protein, which activates genes encoding enzymes for amino acid synthesis (Hope and Struhl, 1986). The domain is also found in the HSV VP16 co-activator

protein as well as in nuclear factor kappa B (NFkB), which was originally

identified activating transcription of immunoglobulins of the kappa class in B cells (Sen and Baltimore, 1986). Studies of these factors indicated that a high proportion of negatively charged amino acids, arranged into so- called 'acid blobs', were responsible for the domains activating ability (Reviewed by Hahn, 1993).

The HSV protein VP16 is a co-regulator of the ubiquitously expressed Oct- 1 transcription factor. In HSV infected cells Oct-1 and VP 16 associate to activate transcription from the HSV immediate early promoters. VP16 has been shown to alter the activity of Oct-1 by two different mechanisms. Firstly, VP16 by associating with Oct-1 through its POU domain stabilises

Oct-1 on VP16 responsive TAATGARAT elements. Secondly, VP16

binding has been shown to change the preference of Oct-1 from snRNA promoters to mRNA promoters. The C-terminal domain of VP16 has been shown to be a strong and independent frans-activator although it has no associated DMA-binding domain (Sadowski et al. 1988). The interaction

between VP 16 and Oct-1 is protein-protein in nature. Once the two

proteins have associated the Oct-1 protein is able to bind to the TAATGARAT motif where the VP16 activation domain activates transcription (Goding and O’Hare, 1989).

The RD1 region has been shown to interfere with activation mediated by

either of the acidic C-terminal activation domains of VP16 or NFkB (Liu et

al. 1996). In order to determine whether the mechanism of interference exhibited by RDI is also exhibited by either RD2 or RD3 transient transfection experiments were performed. The reporter construct T7G5- TATA-CAT was co-transfected into BHK cells along with either the pTet*^ vector alone or a selected repressor domain clone. Additionally either the pGal4-VP16 or pGal4-NpKB activation domain expression constructs were also co-transfected. Transiently transfected cells were incubated for 72 hours and reporter gene activity was assayed as described (see 2.7.5).

Figure 3.6 shows that the Oct-2 N-terminal repressor domain RDI is able to interfere with the transcriptional activation of the reporter construct mediated by the C-terminal activation domain of the transcription factor NFkB. This finding is in agreement with the previously reported findings

using the same in trans assay system (Liu et al. 1996). However neither

of the other two newly cloned Oct-2 N-terminal repressor domain regions proved able to interfere with the activation effect of the NFkB C-terminal

activation domain. Repressor RD2 appears to strongly activate

transcription up to 3 fold higher than that observed for the NFkB activation domain co-transfected with the empty pTet^ vector. Likewise repressor RD3 is unable to mediate repression but is able to enhance the activation of the NFkB activation domain in a non-significant manner (^=3.48, p=0.0127).

Likewise Figure 3.7 shows a similar finding with the second selected acidic

activation domain, the C-terminal region of VP-16. RDI is able to

effectively interfere with the activation mediated by the C-terminal activation domain of VP16. As a result activation is repressed 2 fold. In agreement with previous findings (Liu et al. 1996) this interference causes less repression less than that seen by RDI against activation by the acidic

activation domain of NFkB transcription factor (Figure 3.6). RD2 and

< 200

EXPRESSED REPRESSOR DOMAIN

Figure 3.6 - Interference by the three Oct-2 amino-terminal repressor domains in BHK cells of activation mediated by the acidic NFkB C- terminal activation domain.

Data p resen ted of CAT assay results from co -tra n sfe ctin g BHK cells w ith 5 |ig pT 7 G 5 -T A T A -C A T reporter plasm id, 5pg o f one of the three cloned O ct-2 N -term inal repressor dom ains and 5pg pG al4-N pKB activator. V = pTet^ ve cto r alone. RD1, RD2 and RD3 = relevant O ct-2 N -term inal repressor dom ain cloned in fram e into pTet^ vector. All C A T activity is expresse d relative to the degree o f re p o rte r fran s-activa tion observed w ith a pG al4-N pKB and the em pty pTet^ ve cto r (set at 100% ). Data are the result o f thre e separate transfection expe rim e nts and are presented as the m ean ± SD.

EXPRESSED REPRESSOR DOMAIN

Figure 3.7 - Interference by the three Oct-2 amino-terminal repressor domains in BHK cells of activation mediated by the acidic VP16 C- terminal activation domain.

Data presented of CAT assay results from co-transfecting BHK cells with 5pg pT7G5-TATA-CAT reporter plasmid, 5pg of one of the three cloned Oct-2 N-terminal repressor domains and 5pg pGal4-VP16 activator. V = pTet'^ vector alone. RD1, RD2 and RD3 = relevant Oct-2 N-terminal repressor domain cloned in frame into pTet^ vector. All CAT activity is expressed relative to the degree of reporter frans-activation observed with a pGal4-VP16 and the empty pTet^ vector (set at 100%). Data are the result of three separate transfection experiments and are presented as the mean ± SD.

RD3 are both unable to mediate repression and both actually enhance the activation of the activation domain around 2 fold. These finding show that the mechanism of interference with activation by acidic activation domains utilised by RD1 is not utilised by either RD2 or RD3 (see Figures 3.6 and 3.7).

In this case repression was recorded for the RD1 region for both VP16 and NFkB. As previously reported (Liu et al. 1996) the relative activation recorded was lower for the NFkB activation domain when compared to that recorded for the VP 16 activation domain. Experiments performed with the VP16 activation domain revealed that neither the RD2 nor the RD3 domains were able to repress activation. Results obtained using the NFkB activation domain showed that the RD2 region could in fact enhance activation by some three-fold, whilst the RD3 region had little effect. These results therefore show that the Oct-2 putative N-terminal repressor domains, both found N-terminal to the glutamine-rich activation domain, are unable to interfere with activation by acidic activation domains when

delivered to the DNA in trans. The RDI N-terminal repressor domain,

found C-terminal to the glutamine rich activation domain is able to disrupt the interaction of these acidic activation domains with components of the basal transcriptional complex and inhibit activation of transcription.

3.2.6 - Investigation into the effect of the three Oct-2 N-terminal repressor domains on activation by glutamine rich activation domains.

Glutamine-rich activation domains have been identified in the Drosophila

transcription factor Antennapedia (Laughon et al. 1986), and the mammalian transcription factors Spi (Courey and Tjian, 1988), Oct-1 and Oct-2. The glutamine rich activation domain identified in Oct-2 is located at the amino terminal of the molecule and is distinct from the C-terminal proline rich domain used above (see Figure 3.1 and 3.2.3). The different glutamine rich activation domains show little sequence homology and their overall glutamine-rich nature is thought to be responsible for their activity.

Liu et al (1996) demonstrated that the RDI region of Oct-2 was able to interfere with activation by a Spi C-terminal activation domain when

delivered to DNA in trans. The ability of the RD2 and RD3 regions to alter

activation in this system was therefore investigated. BHK cells were co­ transfected with the reporter pT7G5-TATA-CAT construct along with a selected repressor domain construct and the pGal4-Sp1 expression vector. Transiently transfected cells were incubated for 72 hours and reporter gene activity was assayed as described (see 2.7.5).

Figure 3.8 shows that neither RD2 nor RD3 were able to repress activation

by the C-terminal activation domain of Spi when delivered to the DNA in

trans. As expected the RDI domain was able to interfere with activation by Spi reducing activation approximately 2 fold.

Finally in order to ensure that the activation quantitated in all the above experiments was indeed due to the activation domain used in each transfection set, repeat transfections were performed using either the Gal4 (1-147) or Gal4 (1-93) DNA binding domains, which lack any activation domain, along with each of the Oct-2 N-terminal repressor domains and the T7G5-TATA-CAT reporter gene construct. In all cases no activation of the reporter gene was recorded (data not shown).

EXPRESSED REPRESSOR DOMAIN

Figure 3.8 - Interference by the three Oct-2 amino-terminal repressor domains in BHK cells of activation mediated by the glutamine rich C- terminal activation domain of Sp1.

Data presented of CAT assay results from co-transfecting BHK cells with 5|ig pT7G5-TATA-CAT reporter plasmid, 5|Lig of one of the three cloned Oct-2 N-terminal repressor domains and 5pg pGal4-Sp1 activator. V =

pTet^ vector alone. RD1, RD2 and RD3 = relevant Oct-2 N-terminal

repressor domain cloned in frame into pTet^ vector. All CAT activity is expressed relative to the degree of reporter frans-activation observed with pGal4-Sp1 and the empty pTet^ vector (set at 100%). Data are the result